Rhythm training is an integral part of sports. The purposes of the study were to analyze the effects of rhythm training on tennis performance and rhytmic competence of tennis players, to compare the improvement levels of tennis specific and general rhythm training and to examine the effects of shorter and longer tempos on rhythmic competence. Thirty university students whose mean score of International Tennis Number (ITN) was 7.3 (±0.9) were divided randomly into three sub-groups: Tennis Group, General Rhythm Training Group and Tennis-Specific Rhythm Training Group. The experimental procedure lasted 8 weeks. During this period, all groups had the same tennis training twice a week. The Tennis Group had regular tennis training sessions. In addition to regular tennis training sessions, the General Rhythm Training Group followed the general rhythm training sessions and the Tennis-Specific Rhythm Training Group had tennis-specific rhythm training. The measurement instruments were ITN, Rhythmic Competence Analysis Test and Untimed Consecutive Rally Test. The results indicated that participation in tennis-specific or general rhythm training resulted in progress in tennis playing levels, forehand consistency performance and rhythmic competence of the participants. On the other hand, attendance to the regular 8-week tennis training was enough to solely increase the tennis playing level but not sufficient to develop forehand consistency performance and rhythmic competence. Although the participants in the TRTG had better improvement scores than the ones in the GRTG, no significant difference was found between the rhythm training groups. The results also revealed that participants exhibited higher rhythmic competence scores on fast tempo compared to slow tempo. Rhythm is the dynamic grouping, structuring and accentuation of sequential elements of a process, of which arrangement is determined by a required and/or personally selected temporal scheme . PreviouA growing body of literature also indicates the role and importance of rhythm in tennis. According to This study was triggered by the interesting findings of several experiments that have been conducted in the last decade. The first one  investigIn this study, 30 university students, with a 7.3 (±0.9) mean score of International Tennis Number were divided randomly into three subgroups: Tennis Group (TG), General Rhythm Training Group (GRTG) and Tennis-Specific Rhythm Training Group (TRTG). The GRTG refers to training geared toward the development of fundamental locomotor and nonlocomotor skills aiming at synchronization of these skills with an external stimulus (metronome beats); whereas the TRTG means the rhythm training aiming at synchronization between movements that are specific to tennis and an external stimulus (metronome beats). The experimental procedure lasted 8 weeks. During this period, all groups had the same tennis training twice per week. The TG attended only regular tennis training sessions. In addition to regular tennis training sessions, the GRTG followed the general rhythm training sessions and the TRTG had tennis-specific rhythm training. The measurement instruments were ITN, Rhythmic Competence Analysis Test and Untimed Consecutive Rally Test.Although previous studies emphasized the existence of rhythm, and thus the importance of rhythm training, in sport skills, there was no sufficient explanation or exercise prescription with regard to sport-specific rhythm training. This experimental study attempted to provide data in this field by testing the effects of rhythm training in tennis. In addition to its theoretical value, the present study also is expected to have practical significance; it attempts to achieve a new rhythm training approach by developing a new training system that can be used by coaches, physical education teachers and other tennis related staff. It was hypothesized that (1) participation in rhythm training  would provide higher improvement scores on tennis performance and rhythmic competence than participation in regular tennis training, (2) participation in tennis-specific rhythm training would create higher improvement scores on tennis performance and rhythmic competence than participation in the general rhythm training, and (3) shorter and longer time intervals of tempo were expected to differentiate the rhythmic competence scores of the participants. The purpose of the study was to analyze the effects of rhythm training on tennis performance and rhytmic competence of tennis players, to compare the improvement levels of tennis specific and general rhythm training and to examine the effects of shorter and longer tempos on rhythmic competence.Thirty university students  whose tennis training histories varied between 2 and 42 months with a mean length of 12.8 months (±12.3), volunteered for this study. The mean age was of 23.1 (±2.3). Their mean International Tennis Number (ITN) was 7.3 (±0.9). Players at this level are fairly consistent while hitting medium paced shots, but are not yet comfortable with all strokes . All parITN was used to determine the tennis playing level of the participants. Under this system, players are rated from ITN 1 to ITN 10. ITN 1 represents a high level play and ITN 10 indicates a player who is new to the game . The parThe High/Scope Rhythmic Competence Analysis Test (RCAT)  was usedTwo observers independently scored videotaped tests for each movement. They used a 1–3 scale where 3 was assigned to movements that have accurate synchronization, 2 to nearly synchronized movements, and 1 to nonsynchronized movements. In order to evaluate the intra-observer agreement, two observers analyzed the videotaped RCAT performances of 10 participants for each tempo. Furthermore, to assess the inter-observer agreement, the same observers analyzed videotaped RCAT performance of 10 participants twice at two different times. Observer agreement analysis for RCAT was calculated according to the following formula : PercentThe Untimed Consecutive Rally Test (UCRT)  was usedIn the UCRT, each participant was allowed one warm-up trial. All participants in each group were made to take their warm-up trial prior to the beginning of the first test trial. Each participant attempted to achieve the greatest number of consecutive rallies into the target in each trial. In starting the ball for the rally, the participant dropped the ball and hit it on the first bounce into the target area. All balls were to be contacted on or prior to the first bounce throughout the consecutive rally. Each participant had a total of three recorded trials. All participants, regardless of the group, were to finish the first trial before the second trial was taken. Failure to accomplish the following ended the consecutive rally: 1) To rally or volley the ball into the designated target area, 2) To contact the ball on the first bounce when starting the rally, 3) To contact the ball on the first bounce or prior to the bounce throughout the consecutive rally, and 4) To have at least one foot behind the restraining line. The score for each trial was the number of consecutive good rallies. The final score for the test was the mean of three trials. Two observers standing at different angles recorded the number of hits to the target area separately, keeping their own record sheets. Their record sheets were subsequently compared.The participants were tested on a one by one basis and all tests comprised of practice trials. Each participant was pre-tested on three tests in the following order: ITN, RCAT and UCRT. The participants were randomly assigned into one of the experimental groups provided that there was an equal number of males and females in each group: Tennis Group (TG), General Rhythm Training Group (GRTG), and Tennis-Specific Rhythm Training Group (TRTG). Each group consisted of 5 male and 5 female participants. The experimental procedure lasted 8 weeks. During this period, all groups performed the same tennis training program two times per week. The TG continued only regular tennis training sessions. The GRTG followed the general rhythm training sessions in addition to regular tennis training, two times per week for 15 minutes at the beginning of their training sessions. The TRTG had additional tennis-specific rhythm training two times per week for 15 minutes at the beginning of the regular tennis training sessions. For diagnostic purposes, a mid-test was applied at the end of the fourth week. The final measurements were performed after the 8-week training program was completed.All groups had the same tennis training program two times per week for 75 minutes. Each session started with a general and sport-specific warm-up and continued with a practice of basic tennis strokes. A pilot test was administered to determine the appropriate tempos of rhythmic movements for the rhythm trainings. It was observed that synchronization between rhythmic movements and the steady beat of the metronome became easier when the tempo was higher. Therefore, the range of tempo for rhythm trainings decreased gradually from faster to slower. Additionally, because of the nature of such exercises as bouncing the tennis ball with hand and racket, the tennis-specific rhythm training was performed with two different ranges of tempo: fast and slow. The slow tempos were between 45 and 55 bpm and the fast ones were between 80 and 120 bpm. These ranges of tempos were also set for the rhythmic movements for general rhythm training as parallel to tennis-specific rhythm training. All training sessions started with slow movements and continued with fast movements. The programs of the GRTG and TRTG were designed taking into consideration the previous studies . The eigThe tennis-specific rhythm training consisted of several tennis-specific movements , which wParametric tests were initially supposed to be used in the analysis of the research data. However, non-parametric tests were conducted for the rest of the statistical process, since the Homogeneity of Variance assumption of One-Way ANOVA was violated. Thus, the Kruskal-Wallis Test was used to calculate the possible differences between initial scores and also to compare the improvement scores of groups. Afterwards, the Wilcoxon Test was used to examine the differences between initial, mid, and final test scores within each group. Finally, the Mann-Whitney U Test was conducted to determine the pairwise comparisons of groups for improvement scores and to analyze the rhythmic competence scores of participants for different tempos.The Kruskal – Wallis Test results of groups for improvement scores were presented in p<.05) between the GRTG and the TG. Participants in the GRTG had higher improvement scores in the slow tempo rhythm test than the participants in the TG. There was no significant difference in the other parameters between groups. The results also demonstrated that there were significant differences in forehand consistency test (3m) scores  and slow tempo rhythm test scores  between the TRTG and the TG. The participants in the TRTG had higher improvement scores in the forehand consistency test (3m) and in the slow tempo rhythm test than the participants in the TG. The groups did not differ significantly in the other parameters. Although the participants in the TRTG had better improvement scores on forehand consistency, rhythmic competence, and ITN than the ones in the GRTG, no significant difference was found between rhythm training groups. The Mann-Whitney U Test results of the pre-test rhythmic competence scores of participants for the tempos 50 (bpm) and 100 (bpm) was z=−2.99, p<.01, which was significant. According to these results, synchronization of participants’ movements to the external stimulus was more precise at fast tempo than at slow tempo.The Mann-Whitney U Test results indicated that there was a significant difference in slow tempo rhythm test scores . The duration of the training period might be a restrictive factor for improvement. If the participants had been exposed to rhythm training for more than eight weeks, there could have been significant differences between groups who had rhythm training and the TG. The results also showed that the participants in the TRTG improved their UCRT (3m) performance significantly more than the performance of the participants in the TG. According to The results revealed that the participants in rhythm groups (GRTG and TRTG) improved their UCRT (2m) performance significantly after the training program. No significant difference was found between the pre-test and post-test results of the participants in the TG. Although no significant differences were found among groups, the participants in the TRTG improved their performance more than the participants in the TG at a very close significance level (According to The results indicated that the participants in the rhythm groups improved their RCAT (50 bpm) performance significantly more than the performance of the participants in the TG. In other words, participation in either the general or the sport-specific rhythmic activities yielded the development of rhythmic competence performance. The results of the study were in line with the findings of The results showed that the participants in rhythm groups improved their RCAT (100 bpm) performance significantly after the training period. There was no significant difference between the pre-test and post-test results of the participants in the TG. Although the participants in rhythm groups had better scores than the participants in the TG, no significant difference was found between rhythm groups and the TG for the improvement scores. In other words, participation in the general or the sport-specific rhythm training could not bring about statistical improvement of rhythmic competence performance for the fast tempo. Additionally, there was no significant difference between the two rhythm groups. This finding might be explained with the similarity of time intervals between metronome beats for the fast tempo test (600 msec) and preferred tempo, which was determined by previous investigations as approximately 600 msec . Baruch p = .05), no significant difference was found among groups. Moreover, there was no statistical difference between ITN scores of participants in the rhythm groups. Participation in regular tennis training enhanced the tennis playing level of all participants regardless of the groups.The results revealed that the mean ITN scores of each group increased significantly after the training program. Although the participants in the TRTG improved their performance more than the participants in the TG at a very close significance level  and 2.2 (±0.5) for the fast tempo. Because the interval of preferred tempo was in correspondence with the interval of the fast tempo test, the participants performed better at fast tempo than at slow tempo. This result is in accordance with the findings of previous investigations .In conclusion, this is the first study that developed tennis specific rhythm training and assessed its effects on tennis performance. This experimental study focused on the importance of tennis-specific rhythm training. The effects of regular tennis training, general rhythm training and tennis-specific rhythm training on rhythm and tennis performance were investigated. At this point, the present study seems to be a leading study that developed a tennis specific rhythm training and assessed its effects on tennis performance. It can be concluded that tennis-specific rhythm training is a more effective method in order to enhance these parameters than general rhythm training. Since the generalization of the results of this study is limited to tennis, it is strongly recommended that future studies explore new approaches to sport-specific rhythm training


In anatomical and surgical pathology, the customary method of manual observation and measurement of immunohistochemically stained markers from microscopic images is tedious, expensive and time consuming. There is great demand for automated procedures for analyzing digital images (DIs) of these markers  given thWe previously developed an automated macro that allows quantification of several nuclear markers in various neoplasic tissues . In an aHistological sections of lymphomas and breast cancer tissues, previously immunohistochemically stained with standardized protocols ,9, were Stained tissue sections were viewed using brightfield illumination under a Leica DM LB2 upright light microscope  with a 40x plane-apochromatic objective. One hundred digital images were captured with a Leica DFC320 digital camera connected to a computer and controlled with the Leica IM50 v4.0 program. TIFF format DIs, with a resolution of 1392 x 1040 pixels (1.4 Mpixels) in RGB 24 true-color format, were selected on the basis of the presence or absence of the spurious stain deposits of the background, otherwise ensuring a variety of concentrations and distributions of stained nuclei . We had previously developed an automated macro to quantify stained nuclei in images without background using an RGB color model and iterative morphological segmentation . This maWe therefore developed a multistep procedure that discriminates DIs as a function of the presence or absence of background and that enables the more appropriate of the three macros to be applied directly. First, a mask overlaps objects with negative and light positive-intensity pixels Figure  so that The mean of two manual counts made by two trained observers was taken as the reference value (gold standard) to validate the results obtained with the old macro and the newly proposed procedure. The comparisons made were: manual 1 versus manual 2 readings; mean of manual readings versus old macro reading; mean of manual readings versus new procedure reading; and the old macro versus the new procedure readings. The extents of agreement between the manual and automatic results were evaluated with Bland-Altman and Kaplan-Meier analyses with their corresponding graphs. Bland-Altman graphs illustrate the differences between the compared methods with respect to the mean of each paired count. Kaplan-Meier curves portray the conditional probability of observing differences between results obtained from the methods compared. All statistical analyses were carried out with SPSS 19.0.Overall, the Bland-Altman graph Figure  indicateAs previously demonstrated, image complexity relative to the number of positively stained nuclei may affect the automated nuclear quantification . In the However, when images were grouped by background and complexity Figure , the difA general point regarding the proposed method is that, for high-complexity DIs with background Figure , the quaDespite specific and careful preparation of tissues and the use of blocking buffers, strong background staining can sometimes mask the detection of the target antigen during automated analysis. The results of the method presented in this paper are promising since the selective identification of brown color ranges and morphological parameters of selected objects in DIs enables the background to be discriminated during the automated localization and quantification of specific stained nuclei. The principle of this approach is applicable to all quantitative nuclear signals and should prove useful in a variety of tumor specimens, irrespective of the immunohistochemical techniques employed.DIs: digital images; ERs: estrogen receptors; FOXP3: forkhead box protein 3; PRs: progesterone receptors; TIFF: Tagged Image File FormatThe authors declare that they have no competing interests.ML, CL and JJ conceived the study. CL designed and implemented the algorithms. JB and AK provided IT support. RB and JJ carried out the manual quantification, discussed the methods and modified them. VG, BT and CC realized the automated quantification with the two automated methods and designed the database. AR analyzed the data. ML, CL and AK drafted the manuscript. All authors revised the manuscript and approved the final version.


Osteonecrosis of the jaw is a serious side effect in patients receiving nitrogen-containing bisphosphonates intravenously due to malignant diseases. Albeit far less frequently, osteonecrosis of the jaw has also been reported to occur due to the oral administration of nitrogen-containing bisphosphonates due to osteoporosis. Annual infusions of zoledronic acid have been recommended in order to improve patient compliance, to optimize therapeutic effects and to minimize side effects. To date, osteonecrosis of the jaw has not been linked to the annual administration of bisphosphonates.We report the case of a 65-year-old Caucasian woman suffering from osteoporosis who developed early stage osteonecrosis of the jaw in two locations, with chronic infections, after two months of oral bisphosphonate treatment and three annual administrations of zoledronic acid. Our patient was treated by fluorescence-guided resection of the necrotic jaw bone areas; local inflammation was treated by removal of a wisdom tooth and repeat root resections. Histopathology revealed typical hallmarks of osteonecrosis of the jaw.Osteonecrosis of the jaw may occur as a consequence of annual administrations of zoledronic acid. It is conceivable that, due to the pharmacological properties of bisphosphonates, a jaw bone that encounters frequent local inflammations is more likely to develop osteonecrosis. Osteoporosis can be managed effectively with bisphosphonates. These antiresorptive drugs significantly prevent skeletal complications, particularly fractures. Side effects of bisphosphonate therapy are rare but potentially serious as exemplified in the bisphosphonate-related osteonecrosis of the jaw (ONJ). First described in 2003,  ONJ is dRecent studies have revealed that annual intravenous administration of zoledronic acid decreases bone turnover and increases bone density in postmenopausal women with osteoporosis, thereby reducing the risk of vertebral, hip and other fractures. This bisphosphonate regime is generally well tolerated and has a favorable safety profile. Indeed, to date, no reports of bisphosphonate-related ONJ have emerged .This case described in this report suggests that annual infusions of zoledronic acid may lead to bisphosphonate-related ONJ and offers further insights into the pathomechanisms of ONJ.A 65-year-old female Caucasian patient, suffering from intraoral purulent discharge in her left mandibular angle and the front of her left upper jaw, was referred to our hospital by her dentist. Her medical history revealed that she had suffered from postmenopausal osteoporosis, which was initially treated with two months of alendronate (70 mg once weekly) administered orally, followed by three annual infusions of zoledronic acid (5 mg intravenously). In addition, our patient was allergic to penicillin and was treated for diabetes mellitus type II with metformin.Intraoral examination revealed the presence of a fistula formation in her left mandibular angle in region 38 Figure  communicA fluorescence-guided removal of necrotic bone parts in her left mandibular angle was performed as previously described  and her Histological evaluation revealed the typical hallmarks of an early ONJ lesion, including areas of necrotic bone coinciding with signs of infections as well as areas with increased bone turnover Figure .Osteoporosis, a health threat of major public concern, is effectively managed with the oral administration of bisphosphonates. They significantly prevent skeletal complications, particularly fractures . AlthougRecent studies have proclaimed that the annual intravenous administration of zoledronic acid for osteoporosis therapy is safe, particularly regarding the development of ONJ . In the Despite the large number of patients included in the HORIZON trial, the follow-up period was relatively short (limited to 36 months following the commencement of the study and 12 months after the third and last infusion of zoledronic acid) . Acidic Detailed regular intraoral examinations are therefore imperative in order to treat dentoalveolar inflammations and detect early stages of ONJ lesions. If diagnosed timely, the outcomes of ONJ therapy are good; surgical approaches or conservative treatment strategies result in favorable outcomes in over 80% or 60%, respectively .All patients receiving yearly infusions of bisphosphonates for osteoporosis should be adequately informed concerning the risk of ONJ. In addition, oral examinations and (where appropriate) preventive measures are called for in order to eliminate local inflammations--thereby minimizing the risk of ONJ manifestation.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.SO, CP and ME analyzed and interpreted the patient data regarding the disease and wrote the manuscript. KS performed the histological examination and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.


The species-area relationship (SAR) has proven to be one of the few strong generalities in ecology. The temporal analog of the SAR, the species-time relationship (STR), has received considerably less attention. Recent work primarily from the temperate zone has aimed to merge the SAR and the STR into a synthetic and unified species-time-area relationship (STAR) as originally envisioned by Preston (1960). Here we test this framework using two tropical tree communities and extend it by deriving a phylogenetic-time-area relationship (PTAR). The work finds some support for Preston's prediction that diversity-time relationships, both species and phylogenetic, are sensitive to the spatial scale of the sampling. Contrary to the Preston's predictions we find a decoupling of diversity-area and diversity-time relationships in both forests as the time period used to quantify the diversity-area relationship changes. In particular, diversity-area and diversity-time relationships are positively correlated using the initial census to quantify the diversity-area relationship, but weakly or even negatively correlated when using the most recent census. Thus, diversity-area relationships could forecast the temporal accumulation of biodiversity of the forests, but they failed to “back-cast” the temporal accumulation of biodiversity suggesting a decoupling of space and time. Predicting and quantifying the distribution of biodiversity through space has been a central goal of ecologists. Patterns of species diversity along ecological gradients and at multiple spatial scales are routinely quantified and mechanistic hypotheses are tested. A few generalities or laws have emerged from this work – most predominant among them is perhaps the species-area relationship (SAR). The SAR describes the increase in species richness with the area sampled. The SAR has attracted the attention of ecologists for decades providing the foundation for large basic and applied literatures  can provide complementary or novel information critical to our understanding of the structure of communities Faith . TherefoZ, stands for phylogenetic. Recent work by Morlon et al.  located in the Luquillo Experimental Forest in Puerto Rico  were sequenced and used to make a DNA supermatrix. The supermatrix was generated by globally aligning the matK and rbcL data and aligning the trnH-psbA sequence within families. The supermatrix and maximum likelihood were then used to infer a community phylogeny for each plot. Detailed methods regarding the extraction, sequencing, alignment, phylogenetic inference, and calibration can be found in Kress et al.  were calculated using three different spatial scales – 25 mPD Faith  instead ZS) and phylogenetic-area (ZP) relationships were higher in the BCI forest plot than that in the Luquillo forest plot  with the values from all other censuses with regression slopes typically around unity  across censuses, but nearly all of the slopes of the regressions were above unity suggesting an increase in spatial heterogeneity in the species and phylogenetic composition in Luquillo through time  and phylogenetic-time (WP) relationships. The variation in WS and WP values decreased as the spatial scale increased from 25 m2 to 625 m2 in both forest plots studied  and species-time (WS) relationships and the correlation between phylogenetic-area (ZP) and phylogenetic-time (WP) relationships. If spatial and temporal heterogeneity in the species and phylogenetic composition of tropical tree communities are positively associated, then the ZS − WS and ZP − WP relationships should be positive  from multiple angles. In the BCI forest plot, the ZS and ZP values calculated from using the first and the second censuses were positively related with the WS and WP values, but these correlations were weak when using the largest spatial scale to calculate WS or WP (ZS and ZP values calculated from later censuses were weakly positively correlated or, in some cases, negatively correlated with the WS and WP values.We quantified these correlations using WS or WP  and 2. TZS and WS and ZP and WP from the Luquillo forest plot were weaker than those found at BCI (ZS values from the two most recent censuses to the WS values from those subplots. This suggests spatial homogeneity in later censuses occurring in areas that experience high species turnover or vice versa (The correlations calculated between d at BCI  and 2. Fce versa .The importance and utility of the interaction between space and time when investigating patterns of species diversity was first highlighted by Preston . PrestonAs in previous studies, we also examined the influence of spatial scale on the STR, and now the PTR. In support of Preston's predictions and previous work from a temperate zone grassland (Adler and Laurenroth We also tested to determine whether the slope of the SAR or PAR changed through time in the two tree communities studied. In the relatively older growth forest of BCI we found that the SAR slopes were generally consistent through time. Conversely, the recently disturbed Luquillo tree community had SAR and PAR slopes that significantly increased through time. This indicates that the forest became more spatially heterogeneous both in species and phylogenetic composition with time since disturbance. Previous work has suggested that priority effects may generate the reverse trend in temperate forests (Christensen and Peet The consistency of the forest-wide SAR and PAR slopes in the BCI forest plot suggest that there may be no temporal decay, at least within the time frame of this study. Indeed the SAR and PAR slopes from each census were highly correlated, but the strength of the correlation did decrease with the time interval between censuses  and 4. IThe final goal of this work was to determine whether the species or phylogenetic compositional heterogeneity in space is predictive of the temporal heterogeneity as proposed by Preston . This prThe significant positive correlation between the SAR and PAR slopes of the initial census and the slope of the STR and the PTR provides some support for Preston's original prediction. However we again note that the strength of the relationship is not great. Interestingly, the positive correlation weakened or even reversed as later censuses were utilized to calculate the SAR or PAR  and 2. TThe space-time interaction in the species and phylogenetic compositions of the two tropical forests studied are more complex than originally envisioned by Preston . The posThis work aimed to conceptually and empirically expand the growing literature on the STAR by considering the PTAR and by providing the first analyses of the STAR or PTAR in diverse tropical tree communities. We also aimed to test the original predictions of Preston  regardinThis work also highlighted areas where a space for time substitution in the tropical tree communities studied was more complex than that envisioned by Preston  and that


Legionella spp, Mycobacteria spp and fungi, all able to cause life-threatening infections especially in immunocompromised patients. In this study, the suitability of new point-of-use water filters was evaluated in a clinical setting.Hospital water systems have regularly been shown to serve as a reservoir for waterborne pathogens such as Legionella spp. was detected. To protect the patients from exposure to Legionella, shower heads were replaced by point-of-use shower filters . The efficacy of 4 shower filters was tested during 5 weeks. Water samples were taken with and without use of the shower filter and were analyzed for the presence of Legionella, heterotrophic plate count (HPC), and fungi. After 5 weeks of use, shower filters were examined using scanning electron microscopy (SEM).During a routine control of hospital water, contamination with Legionella whereas all filtered water samples were Legionella free. Although other bacteria were present in the filtered water, the HPC was significantly reduced compared to the unfiltered water. Fusarium oxysporum and Fusarium solani were regularly detected in samples taken without shower filter but were never observed in filtered samples. SEM analysis showed a variety of structures on the inner side of the filter membranes while the outside of the membrane did not show any changes compared to an unused control filter.96% of the samples taken without the filter were positive for Legionella and Fusarium species from the showering water. The effects were seen over the complete 5 week study period.The point-of-use filters proved to be highly effective in eliminating potentially pathogenic None declared.


Introgression of Sahel livestock genes southwards in West Africa may be favoured by human activity and the increase of the duration of the dry seasons since the 1970’s. The aim of this study is to assess the gene flow patterns in Burkina Faso goat and to ascertain the most likely factors influencing geographic patterns of genetic variation in the Burkina Faso goat population.http://dx.doi.org/10.5061/dryad.41h46j37. Although overall differentiation is poor (FST = 0.067 ± 0.003), the goat population of Burkina Faso is far from being homogeneous. Barrier analysis pointed out the existence of: a) genetic discontinuities in the Central and Southeast Burkina Faso; and b) genetic differences within the goat sampled in the Sahel or the Sudan areas of Burkina Faso. Principal component analysis and admixture proportion scores were computed for each population sampled and used to construct interpolation maps. Furthermore, Population Graph analysis revealed that the Sahel and the Sudan environmental areas of Burkina Faso were connected through a significant number of extended edges, which would be consistent with the hypothesis of long-distance dispersal. Genetic variation of Burkina Faso goat followed a geographic-related pattern. This pattern of variation is likely to be related to the presence of vectors of African animal trypanosomosis. Partial Mantel test identified the present Northern limit of trypanosome vectors as the most significant landscape boundary influencing the genetic variability of Burkina Faso goat (p = 0.008). The contribution of Sahel goat genes to the goat populations in the Northern and Eastern parts of the Sudan-Sahel area of Burkina Faso was substantial. The presence of perennial streams explains the existence of trypanosome vectors. The South half of the Nakambé river (Southern Ouagadougou) and the Mouhoun river loop determined, respectively, the Eastern and Northern limits for the expansion of Sahelian goat genes. Furthermore, results from partial Mantel test suggest that the introgression of Sahelian goat genes into Djallonké goat using human-influenced genetic corridors has a limited influence when compared to the biological boundary defined by the northern limits for the distribution of the tsetse fly. However, the genetic differences found between the goat sampled in Bobo Dioulasso and the other populations located in the Sudan area of Burkina Faso may be explained by the broad goat trade favoured by the main road of the country.A total of 520 goat were sampled in 23 different locations of Burkina Faso and genotyped for a set of 19 microsatellites. Data deposited in the Dryad repository: The current analysis clearly suggests that genetic variation in Burkina Faso goat: a) follows a North to South clinal; and b) is affected by the distribution of the tsetse fly that imposes a limit to the Sahelian goat expansion due to their trypanosusceptibility. Here we show how extensive surveys on livestock populations can be useful to indirectly assess the consequences of climate change and human action in developing countries. Desertification limits the presence of vectors of trypanosomosis and, therefore, favours the introgression of Sahelian genes into southern trypanotolerant livestock populations in West Africa -3. This A detailed description of the geography, environmental areas and goat populations of Burkina Faso is given as Supplementary Background. Burkina Faso is a flat country with no clear geographic barriers limiting the spreading of animal populations. Only the Mouhoun river (formerly known as Black Volta) has perennial streams  the Each of the environmental areas of Burkina Faso is assumed to be the habitat of a different goat population ,10: the Glossina spp.) 25] LEA. http://www.esri.com/software/arcview/. The Inverse Distance Weighted (IDW) option with a power of two was selected for the interpolation of the surface. IDW assumes that each input point has a local influence that diminishes with distance. The area of sampling of each population was used as geographic coordinates, and the six nearest neighbors were used for the calculation. Interpolation surfaces were divided into seven equal classes.The PCA, LEADMIX and LEA scores obtained for each population were used to construct interpolation maps drawn using the Spatial Analyst Extension of ArcView, available at: Population Graph analysis ,44 was pi) the present limit of the Sahel area; ii) the present limit of the Sudan area; iii) the present Northern tsetse limit in Burkina Faso [iv) like boundary iii but including in the tsetse free area those populations sampled on the road from Ouagadougou to Bobo Dioulasso . These four analyses are expected to characterise if differentiation between the Sahelian, Mossi and Djallonké goat has genetic support (matrices i and ii), the genetic differentiation due to the presence or absence of trypanosome vectors (matrix iii) and the combined effect of the presence of trypanosome vectors and the human action (matrix iv). In all cases, p-values were obtained using 10,000 permutations. Statistical significance level of the four hypothesis tested on our dataset was assessed at α = 0.05 after Bonferroni correction.Partial Mantel test ,47 was cina Faso ; and iv)This work results from the collaboration between INERA and SERIDA, starting in 2007, on the characterisation of the Burkina Faso livestock populations and the assessment of the impact of the progressive desertization of the country. AT, HHT and FG conceived the project; AT, YZ, AK, GO-S, and HHT planned and performed the sampling. AT and YZ extracted DNA; IA and AT performed most laboratory analyses; LP-P contributed to laboratory analyses; IF and FG made statistical analyses; AT, IF and FG wrote the paper. All authors read and approved the final manuscript.Supplementary Background. Detailed description of the geography, environmental areas and goat populations of Burkina Faso.Click here for fileTable S1. Between-populations molecular coancestry and Reynolds’ distance matrices.Click here for fileFigure S1. Bidimensional scaling plots constructed using genetic distance matrices computed after removal of the five smallest populations .Click here for fileTable S2. Dummy matrices created to characterise the presence of landscape boundaries in the Burkina Faso territory.Click here for file


Chemotherapy-induced leukopenia has been shown to be associated with the outcomes of several types of cancer, but the association with childhood acute lymphoblastic leukemia (ALL) remains unknown. To elucidate the association of chemotherapy-induced leukopenia with the clinical outcome of childhood ALL, retrospective analysis was performed on 19 child patients with ALL treated according to the ALL-BFM 95 high-risk (HR) protocol. The mean minimum leukocyte count over the first three courses of the consolidation phase was used as the measure of hematological toxicity and ranged between 200 and 1,167/μl. The risk of relapse was significantly higher in patients with a mean minimum leukocyte count above the median of 433/μl . In conclusion, chemotherapy-induced leukopenia was found to correlate with relapse-free survival in childhood HR ALL. Dose escalation based on hematologic toxicity must be prospectively studied. Treatment outcome of childhood acute lymphoblastic leukemia (ALL) has evidently improved, but the prognosis of high-risk (HR) ALL remains unsatisfactory . RefinemHematological toxicity is the most frequent dose-limiting side effect of combination chemotherapy in the treatment of childhood ALL. The severity of each case of acute hematological toxicity is highly variable despite use of the same regimen. Chemotherapy-induced leukopenia may be a biological measure of drug activities and disease control ,5. The rThe association between less chemotherapy-induced leukopenia and poor clinical outcome has been previously reported for several malignancies, including lung cancer, breast cancer, osteosarcoma and Hodgkin lymphoma –12. ThisConventional treatment for ALL consists of induction, consolidation, reinduction and maintenance elements. Cytotoxic agents, dose levels and severity of myelosuppression are significantly different between treatment courses. This makes it difficult to define the measure of hematological toxicity compared with the treatment and to evaluate the prognostic significance of chemotherapy-induced leukopenia. In the ALL-BFM 95 HR protocol for childhood ALL, the consolidation phase consists of a series of intensive treatment courses (block therapy), with an interval of three to four weeks between blocks . This reIn total, 19 patients  consecutively diagnosed with ALL between November 2003 and September 2010 were studied and uniformly treated according to the ALL-BFM 95 HR protocol following induction therapy at the University of Tokyo Hospital , Saitama Children’s Medical Center  and Gunma Children’s Medical Center .Children diagnosed with ALL were enrolled in the Tokyo Children’s Cancer Study Group (TCCSG) L99-1502 study between November 2003 and January 2005, and in the L04-16 study between May 2005 and September 2010 . The patThe data regarding chemotherapeutic dosage, dates of administration and leukocyte counts were retrieved from the electronic patient databases of the hospitals involved. The parents of all patients provided written informed consent for the treatment. The current study was approved by the Ethics Committee of the University of Tokyo Hospital.An outline of the treatment regimens is shown in Granulocyte colony-stimulating factor (G-CSF) was administered in certain patients with febrile neutropenia and occasionally used prophylactically when severe and prolonged neutropenia was predicted.Blood examination was routinely performed several times a week. The minimum leukocyte count during each course of chemotherapy was recorded. The minimum leukocyte count was averaged over the first three courses of the consolidation phase. The mean was used as the measure of hematological toxicity for each patient. The leukocyte count during the induction phase was excluded from the analysis, since disease status markedly affected the leukocyte count until remission was achieved.To assess the correlation between leukocyte nadir and disease control, relapse-free survival (RFS) from the initiation of chemotherapy was selected as the endpoint.RFS curves were calculated by the Kaplan-Meier method and were compared by means of the log-rank test in a univariate analysis. The minimum leukocyte count in treatment courses with or without the use of G-CSF was compared with the Mann-Whitney U test to assess the effect of G-CSF on leukocyte nadir., Vienna, Austria).All statistical tests were two-tailed and P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using R software  and eight (42%) patients were female. All patients were treated with the same dose per body surface area in the consolidation phase with the exception of L-asparaginase, which was not administered to two patients in the third course due to anaphylaxis. Detailed patient characteristics are shown in Of the 19 patients, five suffered a relapse: Two during consolidation, one during reinduction and two during maintenance therapy. The median follow-up period of relapse-free patients was 51.5 months . The mean minimum leukocyte count was calculated for the first three courses of the consolidation phase, with the exception of two patients who relapsed during the third course of the consolidation phase; their mean minimum leukocyte count was calculated for the first two courses of the consolidation phase. The median of the mean minimum leukocyte count was 433/μl .Of the 19 patients, 13 received G-CSF at least once during treatment. The minimum leukocyte count was not significantly different between the courses with and without the use of G-CSF .RFS curves were compared by means of the log-rank test in a univariate analysis . VariablThe severity of acute hematological toxicity varies considerably in childhood ALL despite the use of the same chemotherapy. The current study analyzed patients with ALL in the same risk group and showed that patients with low hematological toxicity during chemotherapy exhibited a higher rate of relapse. HR of relapse was identified by low hematotoxicity in the first half of the consolidation phase. Early identification of the HR population enables us to intensify treatment in these patients.et al showed that a leukocyte nadir of >1,200/μl in induction chemotherapy is associated with poor overall survival in adult patients with acute myeloid leukemia (AML), although, no statistically significant difference was identified (Low hematological toxicity has been reported to be associated with a poorer outcome of other malignancies –12. Thisentified . This isentified ,16. The A false association between leukopenia and treatment outcome may have been established, since more severe leukopenia was predicted, as the patients had prolonged survival and received more treatment courses. In the present cohort, 17 of the 19 patients completed all three courses of the first half of the consolidation phase. The remaining two patients who relapsed in the third course also received two out of three courses. Low hematological toxicity could not be fully explained by a reduced number of chemotherapy courses.The results of the present study indicated that leukopenia may be used as a biomarker for effective chemotherapy dose, supporting the theory of individualizing chemotherapy dosage based on hematological toxicity . PatientThe current study was unable to evaluate the influence of other possible prognostic factors by multivariate analysis, as the number of patients was too small. However, patients in the present cohort were stratified into the same risk group and were roughly adjusted for the conventional factors, including age, leukocyte count at diagnosis, immunophenotypes of leukemic blasts and early treatment response. This may be one of the reasons why these factors were not associated with relapse. In addition, chemotherapy-induced leukopenia is unlike the conventional risk factors, since it reflects the response of normal hematopoietic cells, but not tumor cells. Leukocyte nadir is thus predicted to be an independent prognostic factor. Further investigation in a larger cohort is required to assess this possibility.In conclusion, the degree of chemotherapy-induced leukopenia was found to correlate with RFS in child patients with ALL. Trials exploring intrapatient dose escalation are warranted.


A method for effective development of solid-phase immunoassays on a glasssurface and for optimization of related protocols by highly sensitivequantitative monitoring of each assay step has been proposed and experimentallyimplemented. The method is based on the spectral correlation interferometry(SCI) that allows real-time measuring of the thickness of a biomolecular layerbound to the recognition molecular receptors on the sensor chip surface. Themethod is realized with compact 3-channel SCI-biosensors that employ as thesensor chips standard cover glass slips without deposition of any additionalfilms. Different schemes for antibody immobilization on a glass surface havebeen experimentally compared and optimized toward a higher sorption capacity ofthe sensor chips. Comparative characterization of the kinetics of eachimmunoassay stage has been implemented with the optimized protocols: i)covalent immobilization of antibody on an epoxylated surface and ii)biotinylated antibody sorption on a biotinylated surface via a high-affinitybiotin-streptavidin bond. We have shown that magnetic nanoparticles employed aslabels with model detection of cardiac troponin I further amplify the SCIsignal, resulting in 100-fold improvement of the detection limit. The developedprotocols can also be used with the alternative immunoassay platforms,including the label methods based on registration of only the final assayresult, which is the quantity of bound labels. In recent years much attention has been focused on studies of methods for theidentification of the protein markers of diseases in complex biological fluidssuch as blood, serum, saliva and others. The development of immunoassay methodsfor detection of these substances is of high importance for clinical  and emerThe key characteristics of immunoassays  depend on the selected antibodies, mode oftheir immobilization on the solid phase, square of the solid phase surface,incubation time and concentration of immunoreagents, as well as on thecomposition of the buffer and stabilizing solutions . If one Earlier, we proposed the original methods of spectral phase , 26and This work was aimed at developing a method for optimization of sandwich-typeimmunoassays using realtime monitoring of each assay stage with the spectralcorrelation interferometry. Optimization of the magnetic immunoassay , 34,35 ReagentsA complex of subunits of the cardiac troponins I, T and C, monoclonalantibodies to cardiac troponin I (clones 19C7 and 16A11), conjugates ofmonoclonal antibodies to cardiac troponin I (clones 19C7 and 16A11) with biotinwere kindly provided by prof. A.G. Katrukha . (3-Aminopropyl)triethoxysilane, (3-glycidyloxypropyl)trimethoxysilane, and biotin N-hydroxysuccinimide ester were purchased fromSigma Aldrich (USA); commercially available nanoparticles of ~50 nm composed ofseveral crystals of ferric oxide and covered with a polymeric coating withcovalently conjugated recognition biomolecules of streptavidin or monoclonalrat anti-mouse antibodies to isotype IgG1 were obtained from Miltenyi Biotec(Germany) . Other reagents were at least of analytical grade.Characterization of magnetic nanoparticles (MNP)Microphotographs of MNP were made using a JEOL JEM-2100 transmission electronmicroscope with an accelerating voltage of 200 kV.The method of spectral correlation interferometry (SCI)The spectral correlation interferometry (SCI) method described in detail inRefs. -29 emploFig. 1A). Duringthe biochemical reaction under study, biomolecules from the solution (ligands)bind to the receptor molecules on the sensor chip, thus increasing the opticalpath of the probe beam reflected from the “liquidbiolayer”interface. The result of interference of these two beams depends on thebiological layer thickness, whose variations during the reaction are calculatedusing the changes in the phase of the correlation signal while scanning thebase of the first interferometer.The SCI method uses the interference between a reference beam reflected fromthe lower surface of the cover slip and a probe beam reflected from the upperglass surface with biorecognition molecules, which allows real-time registration of the dynamicsof the molecular reactions on the surface of the cover slips with the picometerresolution of thickness averaged over the observation spot . Thehigh sensitivity and wide dynamic range make the developed biosensor anattractive instrument with affordable consumables (disposable sensor chips) forreal-time immunoassays in disease diagnostics, detection of pathogens in food,and environmental monitoring. Further studies will aim at estimating theefficiency of cTnI detection in real biological samples and validating thecorrelation between the obtained results and the data received by conventionalmethods.Beside the dignostic significance of the developed immunoassay achieved due tothe amplification of the SCI signal, our results may be of special interest forthe investigation and kinetic characterization of the interactions ofnanoparticles with molecules. We should note that to date the most widespreadlabel-free biosensors that allow estimation of the kinetic parameters ofnanoparticles are those based on the surface plasmon resonance (SPR), 15.TheThe dependence of the signal amplification efficacy on the MNP size deserves aseparate study. On the one hand, bigger magnetic particles  can better amplify the signal. However, bigger particles are proneto gravity sedimentation and may cause a high nonspecific signal. Neithersedimentation nor nonspecific interaction with the surface was observed in thiswork while using 50 nm MNP; therefore, the use of smaller nanoparticles appearsto be unreasonable.The absence of nonspecific sorption during signal amplification permits furthergain in sensitivity by using several amplification steps. As an example, if abiotinylated protein with several biotinylation sites is repeatedly passedbetween two runs of streptavidin- conjugated MNP, it is possible tosubstantially increase the number of labels and, hence, the sensitivity.In addition to low nonspecific binding, MNP possess unique properties that canbe used to further develop the proposed techniques. For example, a combinationof magnetic properties with optical detection allows one to realize anoptomagnetic immunoassay [50]. In that assay, the duration of immunochemicalreactions can be significantly reduced due to magnetic stirring by MNP, as wellas by rotation of MNP chains by a magnetic field. It also permits one to enrichthe sample with antigens by magnetic separation and to decrease nonspecificbinding of labels with the surface by removal of the loosely bound particles bya magnetic field of the respective spatial orientation for“magnetic” washing of the labels.We have developed a method for magnetic immunoassay on a glass surface thatallows real-time detection of each stage of the assay and usage as disposablesensor chips of inexpensive cover slips without deposition of any films . Fourschemes of antibody immobilization have been tested and optimized with theirefficacy assessed. As a result, high sorption capacity and proportion of activeantibodies on the surface of the sensor chip have been achieved. Thepossibility of real-time recording of the kinetics of interactions of magneticnanoparticles with biomolecules has been shown. It has been demonstrated thatthe use of magnetic nanoparticles amplifies the signal of spectral correlationinterferometry, which leads to an additional 100-fold improvement of thedetection limit for cardiac troponin. Thus, the developed interferometricbiosensing systems may serve as effective tools for conducting the assays, aswell as for a wide range of applications, including the development andoptimization of immunoassay methods, quality control of immunoreagents, andcontrol of surface sorption properties. The obtained results on the selectionof schemes and optimization of antibody immobilization protocols, as well as onrealtime monitoring of all immunoassay stages and kinetic characterization ofnanoparticles, can be directly transferred to other biosensing platforms,including those based on various labels – magnetic, fluorescent,enzymatic, and others. The proposed biosensing technique is an economicallysound alternative for immunoassays with disposable consumables for diseasediagnostics, detection of pathogens in food, and environmental monitoring.


Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions  in vitro and in vivo. Our membrane elasticity model thus provides insight into the energetics and mechanisms of uncoated vesicle formation.Lipid membranes enclose the cytosol of biological cells and compartmentalize their interior. Vesicles are used to transport membrane proteins between cellular compartments. The ESCRT protein machinery induces the creation of such vesicles away from the cytosol. The resulting vesicles are uncoated by protein. Upon vesicle scission and release into the endosome, the ESCRT proteins are recycled into the cytosol. We develop a membrane-elasticity model that captures this budding process. The model reproduces the vesicle morphologies observed in fluorescence microscopy images, and identifies the energetic driving force of vesiculation. We also characterize possible mechanisms of ESCRT-induced membrane budding. The size of the resulting vesicles is determined by membrane material parameters, explaining the narrow yet different bud size distributions  Lipid membranes enclose the cytosol of biological cells and compartmentalize their interior. The structure and contents of cellular membranes are actively controlled to sustain the vital functions of the cell. Transport vesicles are used to traffic membrane-bound proteins between cellular compartments. The best-characterized pathways of vesicle formation include those facilitated by BAR domain proteins in vitroUntil recently, the membrane remodeling functions of ESCRT were attributed only to ESCRT-III proteins, which assemble on liposomes in vitro experiments showed, however, that ESCRT-I and -II together are responsible for vesicle budding in vitro is thus bare lipid membrane.Recent Fluorescence microscopy These two experimental observations of (i) the formation of ESCRT microdomains on the membrane and (ii) the formation of coatless buds  jointly form the basis for a phenomenological model of ESCRT-induced budding. Observation (i) provides us with the starting point for the budding process and motivates a mechanism that can provide the large energy necessary for membrane deformation. Observation (ii) determines the end point of the budding pathway. In our model, we assume that the ESCRT-I-II supercomplexes have an enhanced affinity for binding to saddle-shaped membrane regions, and that a line tension acts on the outer boundary of an ESCRT-sequestered membrane domain. We cast our model in the framework of membrane elasticity theory. A bending elastic model of lipid bilayers was previously used to study the energetics of a possible mechanism of ESCRT-III induced fission of nascent vesicles The analytical solutions of our model allow us to map out different membrane morphologies over a range of possible physical parameters. We identify a regime of membrane bending parameters and line tensions for which the minimum energy configurations are coatless membrane buds with ESCRTs localized in the bud neck. These minimum energy configurations closely resemble the fluorescence images observed in experiment in vitro experiments In the framework of membrane elasticity theory, the energy of membrane deformations consists of the mean curvature termin vitro (in vivo ( vitro m  and in vin vivo , and appHow does the ESCRT machinery deform a flat membrane patch into a bud in vitro experiments The second relevant observation is that late endosomes contain raft-like domains that are rich in cholesterol and sphingomyelin We consider a single lipid-segregated domain in vivo and in vitro. Moreover, the line tension on the boundaries of the lipid-segregated membrane domains As we show below, a minimal model of ESCRT proteins inducing or enhancing lipid segregation can explain central experimental results, in particular the narrow yet different bud-size distributions Our model is defined by the energy functionalIn the following, we first develop an approximate analytic treatment that accurately captures the energetics. We then report the results of numerical calculations that allow us to test the analytical solutions and to provide detailed information about the bud shapes. Finally, we analyze possible budding pathways.To make our model analytically tractable, we applied the spherical cap approximation, which is known to capture the energetics of unassisted membrane budding To test the spherical cap approximation and to calculate bud shapes, we minimized the energy functional ximation .The spherical cap model predicts discontinuous transitions between the different morphological phases, with a kink in the minimum energy at the transition lines. In the complete model, however, the kink is rounded at the transition line bud neck .To additionally quantify the bud morphologies, we determined the tangent angle  see  along thin vitroIt is instructive to have a closer look at the membrane buds shown in the bottom right corner of Within the framework of the spherical cap approximation, we also analyzed possible budding pathways. We focused in particular on activation-less pathways along which the buds grow without having to cross significant energy barriers see . This anIf The detailed physical mechanisms used by the ESCRT machinery to control membrane budding are currently unknown. This lack of knowledge has motivated us to consider a simple, phenomenological model for ESCRT-induced membrane budding. Our model is based on well-established physical principles to ensure robustness. Following earlier studies of membrane tubulation The energetic analysis of our model suggests a three-stage mechanism of vesicle budding induced by the ESCRT proteins. In the first stage, the ESCRT-I-II binding induces lipid phase segregation, consistent with the TIRF experiments in vitro buds with radius in vivo buds with radius The third stage of the budding process described above is dynamically feasible within the overall time scale of the process. The diffusion coefficient of membrane proteins is of order in vitroin vivoOne immediate consequence of the three-stage mechanism is that the complete buds have a radius in vivo buds have the same bending energies and shapes as the buds observed in vitro. This conclusion is a direct consequence of the scale invariance of the membrane bending energy in vivo buds with radius in vitro buds with Despite the 40-times smaller radius We note that the bending rigidity of early endosomes The three-stage budding process discussed above occurs if the Gaussian bending modulus contrast If the affinity of ESCRTs to regions with negative Gaussian curvature is high, In contrast to the three-stage mechanism, in the pathway starting with mini-buds the ESCRTs never enter the bud lumen. It is thus tempting to picture this alternative budding route as follows: ESCRT-0 and -I bind to the membrane with Overall, we favor the three-stage mechanism as a possible route for ESCRT-induced budding. It concisely explains the uniform bud sizes seen in experiment, and makes quantitative and testable predictions for their dependence on physical parameters. In particular, according to A key element of the model is the Gaussian bending energy We consider only axisymmetric vesicle buds as the lowest energy shape by symmetry. Their surface is described in terms of two parameters, the angle The boundaries of the ESCRT-occupied membrane patch are located at It is convenient to use dimensionless variables in numerical calculations. Here we introduceTo make our model analytically tractable, we approximate the nascent membrane bud by a spherical cap of radius Case I: The whole lipid-segregated domain Case II: Only the ESCRT-free membrane patch forms a bud and therefore the membrane surface parametrization is given by In both cases I and II, the determinant of the Point The transition from the initial, flat membrane state at point In the case Another budding scenario is possible if


It is known that neural responses become less dependent on the stimulus size and location along the visual pathway. This study aimed to use this property to find evidence of neural feedback in visually evoked potentials (VEP). High-density VEPs evoked by a contrast reversing checkerboard were collected from 15 normal observers using a 128-channel EEG system. Surface Laplacian method was used to calculate skull-scalp currents corresponding to the measured scalp potentials. This allowed us to identify several distinct foci of skull-scalp currents and to analyse their individual time-courses. Response nonlinearity as a function of the stimulus size increased markedly from the occipital to temporal loci. Similarly, the nonlinearity of reactivations (late evoked response peaks) over the occipital, lateral-occipital, and frontal scalp regions increased with the peak latency. Response laterality  was analysed in lateral-occipital and temporal loci. Early lateral-occipital responses were strongly contralateral but the response laterality decreased and then disappeared for later peaks. Responses in temporal loci did not differ significantly between contralateral and ipsilateral stimulation. Overall, the results suggest that feedback from higher-tier visual areas, e.g., those in temporal cortices, may significantly contribute to reactivations in early visual areas. The event-related potentials technique (ERP) has been actively used in brain research for the last half-century. The technique relies on interpreting peaks and troughs in the time course of electric potentials measured on the scalp (EEG). Because the ERP features usually reflect integrated activity of multiple cortical sites interpretation of their neurophysiological bases is problematic making the ERP technique very empirical One potential reason for the response saturation is that receptive field sizes increase along the visual processing stream. Classical models of the visual system’s organization have characterized its function primarily in terms of feedforward processing On the other hand, when receptive fields are comparable or larger in size than the stimulus, the additional stimulus area amounts to a stronger input to the same population of such neurons . This makes the sigmoid input–output nonlinearity typical for neurons  was obtained from all human subjects.The stimulus was a 100% contrast checkerboard grating, 16° in diameter displayed on a medium gray background equated in luminance to the checkerboard stimulus. Seven stimulus configurations were used, they differed only in the area of the visual field stimulated: a full-disk grating, a left half-disk grating, a right half-disk grating, and quarter-disk gratings occupying one of the 4 quadrants of the visual field each. A small fixation cross was shown at the center of the screen at all times. The stimuli are illustrated by the insets in After the onset of the checkerboard stimulus it reversed contrast every second for 10 seconds followed by a 3 second rest interval. Only the fixation mark was displayed on the gray background during the rest interval. The seven stimulus configurations were presented in random order, 200 stimulation epochs (1000 msec each) were displayed for each stimulus configuration; the entire experiment lasted for approximately 30 minutes.The stimuli were generated using an in-house visual psychophysics library (PEACH). They were viewed on a linearized 21″ ViewSonic G225f monitor. The monitor resolution was set to 1600×1200 pixels; for the used viewing distance of 70 cm, a pixel subtended 1 minute of arc. The monitor refresh rate was 75 Hz.EEG was recorded using HydroCell GSN 128-channel nets, amplifiers, and the accompanying NetStation software by EGI Inc. Electrode locations on the head and head landmarks (nasion and tragi points) were measured for each subject using a Polhemus FASTRACK digitizer and in-house software (3Digit). EEG epoch markers were recorded by the NetStation software synchronously with the EEG data acquisition using a DIN signal generated by PEACH via the DATAPixx peripheral (VPixx Technologies). The markers were generated at the beginning of the first CRT raster sweep after the stimulus onset or contrast reversal, which were taken as the beginning of an ERP epoch in our study. The stimulus rendering was completed 13.3 msec later at the end of a video frame. EEG data acquisition was externally triggered by the DATAPixx peripheral synchronously with the monitor refresh signal (75 Hz), the trigger rate was set to 7 samples per videoframe resulting in 525 Hz sampling frequency. EEG and event markers data were saved on a hard drive and processed off-line.Data processing and analysis were performed using an in-house MATLAB software suite (Harmony). EEG potentials were average referenced over all channels. AC line noise frequencies (60 Hz and 120 Hz) were notch-filtered out. Constant and linear terms were subtracted from raw epochs to remove DC components and occasional amplifier reset artifacts. 5% to 25% of raw epochs were rejected due to muscle activity artifacts determined by potential thresholding. Because subjects were encouraged to blink during the rest intervals the rejected epochs often included the first epoch after each rest interval. The remaining epochs (approximately 180 per condition) were averaged for each subject. Noisy electrodes were identified based on the proportion of the rejected epochs, and, if detected, were replaced by thin-spline interpolating data from the remaining electrodes . The experimental noise variance on each electrode and its covariance among electrodes (the noise covariance matrix) were estimated by projecting out the averaged epoch from each raw epoch and then averaging the remaining signal covariance over the epochs.Across-subjects data averaging was performed using scalp interpolation. Electrode locations on each scalp were measured with respect to the subject’s head landmarks . Conversely, the surface Laplacian provides a unique solution for the skull-scalp currents best matching the observed EEG distribution on the scalp in a parameter-free fashion except for an overall scaling factor To improve spatial resolution of our analysis we used a surface Laplacian method . This technique aims to reduce strong blurring of EEG potentials resulting from abrupt electric conductivity changes at the boundaries of the skull and the surrounding tissues Importantly, no anatomical MRI images were necessary for the transformation to the skull-scalp currents. The only used parameter was the scaling factor casting the surface Laplacian of the scalp potential into the units of electric current density on the skull-scalp interface. This factor depends only on the head radius, scalp thickness, and scalp conductivity Twenty observers  24–40 years of age, with normal or corrected visual acuity participated in the study. Three of the observers were the authors. The remaining observers were naive to the purpose of the study and did not take part in EEG experiments before. Observers were trained for a short time to get acquainted with the stimuli and the task. They were instructed to fixate at the fixation cross at the center of the screen at all times and to minimize head and eye muscle activity during the visual stimulation intervals (10 seconds long). Otherwise, no task was performed. Five subjects showed excessive eye-muscle activity (blinks and eye movements) and their data were excluded from the study.In the first part of this section responses to the quadrant and half-disk stimuli located in the left and right visual half-fields were pooled so that response nonlinearity as a function of the stimulus area was analyzed irrespectively of the stimulus location. Conversely, in the second part responses to the quadrant and half-disk stimuli were pooled so that response laterality  was analyzed irrespectively of the stimulus area.Scalp VEPs 100 msec after the stimulus onset averaged across observers are shown in The maximal absolute values over the VEP epoch at each location are shown in Skull-scalp current patterns  the magnitude  of the first clearly identifiable peak was plotted vs. the stimulus area in To quantify (ii) we repeated the above analysis for other peaks, wherever they could be clearly identified for at least two stimulus sizes. These peaks were indicated by gray, blue, and green arrows in The fitted The left and right LO and TP ROIs were well separated on the scalp and hence skull-scalp currents evoked in response to contralateral and ipsilateral stimulation could be compared for these loci. First, responses to the individual quadrant and half-disk stimuli in the respective  visual half-fields were summed. Then, the results were averaged between the left and right ROIs. The resulting amplitudes for the first three LO peaks indicated in To quantify the observed differences between contralateral and ipsilateral responses we defined the laterality index, In this study the effect of the stimulus size and location on VEP magnitude was measured at different scalp ROIs. Early VEP peaks showed little response saturation at occipital locations, but the saturation increased via lateral scalp regions toward temporal and frontal regions. This trend can be seen in Amplitudes of the first clearly identifiable peaks at the chosen ROIs were analysed to measure the response nonlinearity (saturation) across various scalp regions. The nonlinearity increased significantly from weak saturation for the regions above early visual areas  to strong saturation over temporal poles . The TP The reactivation (late) peaks indicated by gray, blue, and green arrows in There are several possible explanations for the increase in the response saturation with the peak latency. VEP response can saturate with time as a result of neuronal adaptation through the course of the VEP epoch. We find this unlikely for two reasons. First, the stimulation was repeated every second for 30 minutes, and one would expect all adaptation to occur in the first few minutes of the experiment or even prior to the experiment during the training session. Second, given the small receptive field sizes  the adaptation magnitude would be expected to be the same for all three stimulus sizes and therefore would not produce the observed saturation effect.Alternatively, surround suppression could produce the observed response saturation as the stimulus area increased. However, neurophysiological Instead, our results suggest that the reactivations of early visual areas observed as the later VEP peaks are due to a feedback from higher-tier visual areas such as cortical areas in temporal lobes. Because the TP peak precedes the reactivations peaks by 150 msec (LO), 180 msec (FP), and 470 msec or more , we hypothesize that these peaks have their origin in a feedback from the TP area. This explains the strongly increased response nonlinearity for the later peaks. Three results in particular support this hypothesis: (i) due to its early nonlinear response TP is clearly different from the rest of the ROIs; (ii) the measure of response nonlinearity plotted in the last panel of This hypothesis is further supported by the laterality analysis of the LO and TP responses presented in Numerous experimental findings indicating feedback interactions in the visual system of human and nonhuman primates provide support to the feedback hypothesis. Single-cell recordings in nonhuman primates have shown that inactivation of higher-order areas modulates neuronal responses in lower-order areas


Laparoscopic liver resection has been performed mostly in centers with an extended expertise in both hepatobiliary and laparoscopic surgery and only in highly selected patients. In order to overcome the obstacles of this technique through improved intraoperative visualization we developed a laparoscopic navigation system (LapAssistent) to register pre-operatively reconstructed three-dimensional CT or MRI scans within the intra-operative field. After experimental development of the navigation system, we commenced with the clinical use of navigation-assisted laparoscopic liver surgery in January 2010. In this paper we report the technical aspects of the navigation system and the clinical use in one patient with a large benign adenoma. Preoperative planning data were calculated by Fraunhofer MeVis Bremen, Germany. After calibration of the system including camera, laparoscopic instruments, and the intraoperative ultrasound scanner we registered the surface of the liver. Applying the navigated ultrasound the preoperatively planned resection plane was then overlain with the patient's liver. The laparoscopic navigation system could be used under sterile conditions and it was possible to register and visualize the preoperatively planned resection plane. These first results now have to be validated and certified in a larger patient collective. A nationwide prospective multicenter study (ProNavic I) has been conducted and launched. Laparoscopic liver surgery has been performed throughout the last 20 years. Fabiani et al. first described the minimally invasive fenestration of large solitary liver cysts in 1991 . In 2000The first navigation system for laparoscopic surgery in the abdomen was presented by Ellsmere et al. . The goaIn order to compensate the limited tactility in laparoscopic liver resection and to use guidance by preoperative resection planning data, we developed a laparoscopic navigation system (LapAssistent). It enables the intraoperative presentation of preoperative 3D-image data of the liver, including intrahepatic vessels, tumor location, and estimated residual functional liver volume. The technical aspects of the LapAssistent system are as follows: (1) presentation of all views provided by MeVis planning, (2) no visibility problem in tracking, (3) preoperative calibration, (4) preregistration manually or by landmark selection, (5) documentation of intraoperative tumor location, (6) online update of resection planning, and (7) navigation guidance for laparoscopic tumor ablation.In this paper we describe the LapAssistent system and exemplarily report the navigated laparoscopic liver resection in a patient with benign liver cell adenoma in segment VI. Due to an uncertain raise in alcalic phosphatase and unspecific pain in the right upper abdomen a 28-year-old female patient  underwent further diagnostic exploration. The abdominal ultrasound revealed a lesion of low echogenicity in the right upper abdominal quadrant with relation to the liver. A subsequent MRI scan showed an oval lesion at the right lower lobe of the liver measuring 5 × 9, 5 cm in craniocaudal extension. According to the imaging there were no signs of an infiltration of healthy liver tissue. Also, there were no signs of pathologically enlarged lymph nodes or ascitis. The preliminary diagnosis was a liver cell adenoma.The preoperatively generated MRI-data of the abdominal scan were provided to the cooperation partner MeVis  in Bremen, Germany, for additional calculation and generation of three-dimensional data reconstruction . Besidesvirtual reality navigation ,laparoscopic screen ,real-time ultrasound picture ,menu for changing views,menu with general applications such as the setting of landmarks and starting of wizards.The hardware components of the LapAssistent consist of a touch-screen monitor, a SpaceMouse, a keyboard, and a standard PC . The essential part of the system is the electromagnetic tracking device  with connection plugs, tracking cable, transmitter, interface and a rack. The components of the navigation software consist of a main window with the following applications:The entire system has to be calibrated to the individual setting before the beginning of the surgical procedure . This inFollowing the calibration of the laparoscopic instruments a pre-registration of the patient's liver is performed. The aim of this step is a rapid and crude measurement of the liver. For this, the field generator is positioned next to the right upper abdomen of the patient. Then, four defined spots on the liver surface  are focussed by using the ultrasound device Figures . The detY-position. The sterile navigation system LapAssistent was placed on the right side of the patient (The Patient was kept in  patient . First oTo control the resection outcome we accomplished MRI on day 8 after surgery . The resInterventional navigation systems have been widely used in neurosurgery, otolaryngology, and orthopedic surgery to improve intraoperative orientation and to increase accuracy of tumor localization or bone resection. This was possible due to the rapid progress in new imaging modalities in radiology together with the continuous improvement in high performance computing technology –18. We h


When regional lymphatic flow is insufficient to maintain tissue fluid homeostasis, interstitial fluid accumulates and swelling ensues. Furthermore, in addition to this readily observed role in the maintenance of tissue fluid homeostasis, functioning lymphatics are crucial to the traffic of immunocompetent cells from the tissue periphery to the lymph node, where antigenic processing can occur.6 that include fear, affective disorders, and loss of self-esteem and body image.7 Lymphedema is a chronic debilitating disease for which there continues to be a great deal of clinical confusion and treatment delay.8Lymphedema is the all-too-frequent clinical consequence of impaired lymphatic function. The condition does not typically threaten survival, yet the advent of lymphedema can significantly undermine productivity and quality of life for affected individuals. The sequelae of lymphedema include loss of function, restriction of movement, risk of infection, and profound alterations in psychosocial adjustment9 In recent years, however, the identification of the molecular components of the lymphatic developmental apparatus has made it feasible to contemplate the therapeutic administration of lymphatic-specific growth factors (therapeutic lymphangiogenesis) to alleviate the tissue consequences of lymphatic vascular insufficiency.15The current available treatments for lymphedema do not address the causal molecular pathophysiology and thus provide only modest delays in the progression to end-stage sequelae of lymphedema.18 Furthermore, it seems that the ultimate ability of the lymphatic vasculature to regenerate after injury might be governed by an exquisite balance between prolymphangiogenic and antilymphangiogenic cytokines.19Despite initial perceptions of benefit, the enthusiasm for growth factor therapy has not been universal. The precise mechanism for vascular endothelial growth factor receptor 3–mediated amelioration of secondary lymphangiogenesis is unclear. There are currently contrasting observations that exogenous growth factor administration to sites of lymphatic injury augments early lymphatic endothelial cell proliferation but without the development of functionally competent vasculature.Journal of the American Heart Association, Shimizu et al20 address this persistent quandary in experimental therapeutics with a modification of the prolymphangiogenic approach. Specifically, they administered adipose-derived regenerative cells to a murine tail model of acquired lymphedema to assess whether these cells might serve to augment meaningful lymphatic neovascularization. The findings of this investigation include the observation of a direct lymphangiogenic response to implantation of adipose-derived regenerative cells into the zone of lymphovascular deficiency. In addition, the researchers observe that these cells in culture produce vascular endothelial growth factor C, which is capable of augmenting lymphangiogenesis. Finally, they report that the in vivo administration of the cellular material resulted in augmented recruitment of bone marrow–derived M2 macrophages that could serve the role of lymphatic endothelial progenitor cells. These findings are certainly congruent with other recent observations.21In the current issue of the 22 The authors used a frequently studied small rodent model of acquired lymphedema19 that offers a high-throughput means to readily observe histological, microvascular, and molecular events. However, the ability to translate therapeutic observations in this model to human pathology might be limited. Cytokine-stimulated therapeutic lymphangiogenesis, whether via direct ligand administration or, as used here, indirect cell-mediated augmentation, simply might be insufficient to foster the development of the competent lymphatic architecture that is needed to restore lymph flow. Nevertheless, continued investigation and the creative approaches described here certainly are warranted. Patients with lymphedema undoubtedly will one day be the beneficiaries of these focused investigations.The functional significance of the authors' findings ultimately must be demonstrated through further investigation. First, it must be acknowledged that the animal models for lymphedema are imperfect.


This article describes an esthetic rehabilitation of a case of severe fluorosis associated with tooth mobility and gingival recession. Direct composite technique was applied to improve the color, shape and alignment of the teeth using direct composite veneering and replacement of the missing tooth by fiber-reinforced composite bridge. One year follow up have displayed acceptable outcomes and esthetic appearance. Dental fluorosis is caused by an excessive fluoride intake during tooth formation. Fluoride-containing dental products and drinking water are two main potential sources for this developmental tooth disorder. Fluoride-related alterations in enamel lead to surface hypermineralization and subsurface hypomineralization which are characterized by white opaque appearance with secondary brown stain-2.The successful treatment of fluorosed teeth is a subject of interest in the literature. An appropriate treatment plan may be selected depending on the severity of the fluorosis-2. In thFrequently, the management of fluorosis involves resin composite restorations. In this situation, some concerns raise about the effect of etching and bonding agents on the fluorotic enamel and dentin. This arA 21-year-old woman was referred to the department of operative dentistry in the faculty of dentistry for the esthetic rehabilitation. The patient was not satisfied with her smile appearance because of the discolored tooth and excessive length of maxillary incisors. She also needed replacement of the extracted teeth .She was healthy, having no systemic diseases and she did not have any contraindication for dental procedures.The patient’s dental history showed an aggressive periodontitis in her adolescence. Radiographic evaluation and clinical examination revealed bone loss especially in the area of mandibular incisors and first molars which led to mobility of the other incisors . Oral hyThe patient presented generalized areas of fluorosis representing as opaque patches, subsurface brown staining and small pits in enamel representing severe fluorosis. The diagnosis was made based on the Dean's fluorosis index. The clinical and diagnostic cast examinations revealed the rotation of tooth #7, the cross bite in tooth#11, excessive length of clinical crowns of maxillary central teeth and flaring of the anterior teeth proceeding to upper midline diastema . At the The color was recorded using the Vita Classical shade guide, and the shade A2 and A3 was considered as the initial color.The tooth preparation involved a minimal chamfer in the facial surfaces and additional reduction for correcting the length and orientation of teeth was limited to these areas. Cotton rolls, salivary ejectors, and retraction cords were used for field isolation. The enamel surface was acid etched using 35% phosphoric acid gel  for 15 second, rinsed for 10 seconds and dried. A self-etch, two-component adhesive system  was applied on the prepared enamel and dentin surface and light-cured for 10 second with an intensity of 1100 mW/cm2 . Direct composite veneers were contoured regarding the diagnostic wax up. A stratified layering technique was used to fill the tooth with microhybrid resin composite  shade A2, 3 and Pearl Neutral. The diastema was closed and the alignment and length of teeth were corrected where it was necessary. The composite was light-cured through the matrix for 10 seconds on each surface. After removal of the matrix, each surface was polymerized from incisal/occlusal, facial, and lingual aspects for additional 20 seconds. The fiber-reinforced composite bridge was made in order to replace tooth #25 and splint the mobile mandibular incisors simultaneously. An appropriate length of polyethylene fiber  was cut and saturated with bonding agent .The interproximal and lingual surfaces were prepared and treated as described before. Flowable composite  was used to make interproximal composite connectors and cover the splinting fiber .Finally, the occlusal adjustment of the restorations was performed and the canine guidance discluded all the posterior teeth in the eccentric mandibular movements.The contouring and finishing was accomplished with finishing burs . The polishing was performed using polishing disk, polishing points and cups  and diamond polishing paste .The smile of the patient revealed good esthetics after the performed treatments . AdequatThe aim of the treatment in this case was to restore the patient esthetics and self-esteem. Different treatment plans have been proposed for the treatment of discoloration in the fluorosed teeth depending on the severity of the fluorosis. Since sAlthough using direct composite provides excellent esthetics; the fracture resistance, wear resistance and color stability of composite resin is lower than indirect porcelain restorations. FurtherIt is recommended to grind the fluorosed enamel surface to remove the hypermineralized layer. Etching with phosphoric acid for 15 seconds achieved the best results in the normal enamel. While


Pneumatisation of styloid process is a very rare finding and has never been reported previously. We present a unique case of a pneumatised styloid process with a cholesteatoma arising within the cavity. We describe the clinical features, associated radiological findings, and management of this lesion. Styloid process develops from the dorsal part of the second branchial arch (Reichert cartilage) at around fourth week of intrauterine life , 2. Its A 43-year-old female presented to our otolaryngology department with an 11-month history of recurrent, right-sided otalgia and foul-smelling otorrhoea. The condition had been misdiagnosed as recurrent otitis externa. Patient denied any history of hearing impairment, vertigo, tinnitus, facial nerve palsy, or a history of head trauma. Clinical examination revealed squamous debris and purulent material in a grossly eroded floor of bony meatus with the lower half of the tympanic membrane hanging in the breeze. The facial nerve function was normal and there were no other neuro-otologic signs. Examination of contralateral ear and rest of the ENT examination were unremarkable.CT scan of the temporal bone demonstrated an extensively pneumatised styloid process containing a soft tissue mass Figures . FurtherThe patient subsequently underwent a right combined approach tympanoplasty with extended posterior tympanotomy approach continuing the dissection inferiorly to the hypotympanum and down to the styloid apparatus. The lesion in the pneumatised styloid process contained a fibrous cyst wall full of cholesteatoma. This was completely excised and the cavity was obliterated with fat. Cartilage was used to reconstruct the defect in the floor of the bony external auditory canal. There were no postoperative complications and recovery was uneventful. Patient remains disease free until the last followup (14 months after surgery). The exact gestational stage at which pneumatisation of temporal bone starts is controversial; however, it is thought to start sometime between 24 and 38 weeks in utero and then continue into adult life [Anatomically the pneumatisation of temporal bone can be divided into five regions, mastoid, middle ear, perilabyrinthine, petrous apex, and accessory region. The detailed description of temporal bone pneumatisation is beyond the scope of this paper and is well described by Virapongse et al., 1985 . It is tPneumatisation of temporal bones is thought to be necessary for lightening the weight of the skull, providing a reserve volume of air (air reservoir action), insulation, reception, resonance, and acoustic dissipation of sound. Any alteration of the reservoir function of temporal bone and eustachian tube dysfunction leads to generation of negative pressure within the middle ear culminating into retraction of tympanic membrane and cholesteatoma formation , 4. Cholesteatoma most frequently involves the middle ear and mastoid but may develop anywhere within the pneumatised portions of the temporal bone including the petrous apex . The litBy the nature of the disease cholesteatoma causes destruction of the surrounding bone and local erosions which at times may be extensive . It is nDiagnosis of cholesteatoma is made clinically although radiology is helpful in difficult cases as described here. CT and MRI with their various sequences  complement the clinical examination  as in ouSurgical resection is the management of choice for both acquired and congenital cholesteatoma. The two primary goals of surgery are to remove the disease whilst managing complications and to reconstruct the middle ear. Prognosis of both congenital and acquired cholesteatoma is generally good.In summary, we present the first reported case of pneumatisation within the styloid process. Unusually, the patient in this case also developed cholesteatoma within the cavity. Pre-treatment scanning of the temporal bone and stylomastoid apparatus should always be performed before planning the surgery so as to minimize chances of leaving behind the residual disease at unusual places of pneumatisation of temporal bone including the skull base.