The immune mechanisms which determine the infection duration induced by pathogens targeting pulmonary macrophages are poorly known. To explore the impact of such pathogens, it is indispensable to integrate the various immune mechanisms and to take into account the variability in pathogen virulence and host susceptibility. In this context, mathematical models complement experimentation and are powerful tools to represent and explore the complex mechanisms involved in the infection and immune dynamics. We developed an original mathematical model in which we detailed the interactions between the macrophages and the pathogen, the orientation of the adaptive response and the cytokine regulations. We applied our model to the Porcine Respiratory and Reproductive Syndrome virus (PRRSv), a major concern for the swine industry. We extracted value ranges for the model parameters from modelling and experimental studies on respiratory pathogens. We identified the most influential parameters through a sensitivity analysis. We defined a parameter set, the reference scenario, resulting in a realistic and representative immune response to PRRSv infection. We then defined scenarios corresponding to graduated levels of strain virulence and host susceptibility around the reference scenario. We observed that high levels of antiviral cytokines and a dominant cellular response were associated with either short, the usual assumption, or long infection durations, depending on the immune mechanisms involved. To identify these mechanisms, we need to combine the levels of antiviral cytokines, including  Respiratory pathogens, which enter the body through the mucosal surfaces of the respiratory tract, are responsible for local inflammation and tissue damages Mycobacterium tuberculosis or the Porcine Reproductive and Respiratory Syndrome virus, replicate in the cells of the respiratory tract, including pulmonary macrophages. They hinder the immune functions of the macrophages and consequently reduce the efficacy of the immune response. With these pathogens, activated macrophages (i) either phagocyte and destroy the pathogen, or are infected and excrete the pathogen; (ii) produce cytokines that promote the migration of immune cells to the infection site; (iii) synthesise cytokines that regulate the adaptive immunity; (iv) express antigen proteins on their cell surface that activate the adaptive response. In turn, the adaptive cell effectors and cytokines regulate the immune functions of macrophages. However, the influence of macrophage–pathogen interactions on the immune response has been poorly studied and needs more insight in vivo and that they have been considered as having little impact compared to the adaptive response.The best strategy to control the severity of respiratory pathogens is to limit the inflammation while maintaining an efficient immune response. Some pathogens, such as influenza viruses, Here, we were interested in identifying the immune mechanisms which determine the infection duration induced by pathogens targeting pulmonary macrophages. The immune response is a highly complex system involving numerous interactions between cells and cytokines. An additional level of complexity is due to the between-host and between-pathogen variability. Pathogens use multiple strategies, that vary among pathogens but also among strains, resulting in various virulence levels. The host response depends on the host genotype or housing conditions, resulting in various susceptibility levels to a given pathogen. Consequently, to explore the impact of pathogens targeting pulmonary macrophages, it is indispensable to integrate the various immune mechanisms and to take into account the variability in pathogen virulence and host susceptibility.Mycobacterium tuberculosisIn this context, mathematical models are powerful tools to represent and explore the complex mechanisms involved in the infection and immune dynamics So we proposed an original model of the immune response to a virus infecting pulmonary macrophages in the lung. We considered with particular attention the macrophage–virus interactions. We highly detailed the mechanisms of the innate response and the cytokine regulations. We included the cellular, the humoral and the regulatory orientation of the adaptive response, as well as their main functions. We represented the interactions between innate and adaptive components. We applied our model to the Porcine Respiratory and Reproductive Syndrome virus (PRRSv). PRRSv is a major concern for the swine industry, as it is responsible for significant economic losses worldwide First, we built our model by synthesising knowledge on the immune mechanisms from published studies on PRRSv. Experimental studies on PRRSv are numerous, but cannot provide all our model parameter values. So we compiled data from the literature by reviewing experimental and modelling studies on pathogens targeting pulmonary macrophages and obtained large value ranges for our model parameters. We explored the influence of these parameters on the viral and macrophage dynamics by a sensitivity analysis. We then identified a parameter set resulting in realistic infection and immune dynamics. Finally, we explored the influence of host susceptibility and viral virulence on the infection outcome and we identified the associated immune mechanisms.In this section, we first present the dynamic model and its calibration, based on literature data. We then describe the sensitivity analysis method used to quantify the influence of model parameters on outputs of interest, among which the viral titer. Finally, we define scenarios which represent the variability in host susceptibility and strain virulence, in order to assess the impact of this variability on the model outputs.We built a deterministic dynamic model of ordinary differential equations to simulate the infection and immune dynamics induced by a pathogen targeting pulmonary macrophages in the lung. The functional diagram of the model appears in The model is characterised by 18 state variables: the free viral particles . For all simulations, the initial conditions were set to represent an initial viral inoculation in a PRRSv-naive host and were chosen as follows: The model was implemented in Scilab 5.3.3  for these analyses.We were interested in identifying the most influential parameters on the infection dynamics thanks to a global sensitivity analysis. Consequently, the first two outputs selected were the viral titer  was used to construct the fractional designs.We selected 30 among the 31 model parameters for the sensitivity analysis. We did not include the proliferation rate of the adaptive effectors  for this analysis.Sensitivity indices were calculated for each parameter on each output in the preliminary analysis  the activation and orientation of the adaptive response. Recent studies hypothesise that these variations of the immune dynamics are due to cascaded reactions initiated by the macrophage–virus interactions Both viral virulence and pig susceptibility seem linked to: (i) the virus capacity to infect the cell and replicate, (ii) the host capacity to synthesise antiviral We used the area under the curve (AUC) to synthesise our model outputs. As the shapes of the immune and viral output curves were similar across the scenarios, characterising each curve by a well-chosen number was appropriate and facilitated the comparisons between scenarios. Choosing the AUC was relevant, as it reflects the entire curve Several linear regressions were performed to extract trends from our results and facilitate the interpretations. In particular, to highlight the links between immuno-regulatory cytokines and the orientation of the adaptive response, we performed linear regressions between (i) the relative AUC of relevant cytokines and (ii) the relative AUC of the adaptive response effectors  was characterised by a 72-day infection duration, an infected macrophage peak at 40% of the total macrophage concentration, a balanced adaptive response orientation and high e.g. 27% for interactions involving In the preliminary sensitivity analysis, with all 30 parameters but no interactions between parameters, the variance explained by the parameters retained for the main sensitivity analysis on each output was 89% for the viral titer, 89% for the cumulative number of phagocyting macrophages and 70% for the percentage of infected macrophages. The results of the main sensitivity analyses with two-parameters interactions are shown in The initial inoculation dose n course –S4.The infection rate The infection rate The 37 scenarios corresponding to graduated levels of host susceptibility and strain virulence were simulated. The results are illustrated in ios SBSA . ConsequThe dynamics of immune components were similarly bell-shaped but differed quantitatively. More severe and longer infections were overall associated with higher levels of immune responses , but theConcerning the innate response, we found a significant and positive correlation  long (scenarios related to macrophage permissiveness) or (ii) short infection durations (scenarios related to cytokine synthesis capacities).In this paper, we presented an integrative dynamic model of the immune response in the lung to a virus targeting pulmonary macrophages: the PRRSv. The complexity level of the model is a good compromise between detailed intra-cellular models which focus on specific immune mechanisms and global models which give general trends The model was built to describe a single infection by a stable pathogen at the within-host scale. We used it to study the impact of PRRSv strains, which exhibit various virulence levels. Our model could be easily adapted to other pathogens targeting pulmonary macrophages, such as influenza viruses. As influenza also infects epithelial cells, these target cells would have to be included in the model. As for other pathogens, the immune dynamic part of our model constitutes a good basis to study the innate response, given the fact that it is strongly simplify in most of the published models.ad hoc method based on large parameter space exploration and sensitivity analysis. We defined a reference scenario, which corresponds to an average dynamics within the observed immune and infection dynamics. To study the impact of host and strain variability, we also defined parameter sets based on published assumptions and resulting in infection durations which were consistent with the literature The variation range of our model parameters were based on literature data. To complement these data and deal with the high variability on the parameter values and output levels, we developed an The sensitivity analysis highlighted five key parameters with a strong influence on the macrophage and virus dynamics: the viral inoculation dose In terms of viral dynamics, the simulated infection durations ranged between 52 and 118 days according to the scenarios. Experimental studies show that the resolution generally occurs in the serum between 28 and 42 days after a PRRSv infection In terms of immune response, the main trends found in the literature are the following: high virulence and susceptibility are associated with (i) a high activation of the immune response The strain virulence and pig susceptibility variability impact the infection duration, but the underlying mechanisms are still incompletely understood. Several hypotheses are formulated to explain PRRSv infection duration. Early immunological findings link prolonged viremia with (i) a weak innate antiviral response, (ii) high levels of immuno-modulatory cytokines  the proportions of Whatever the strain virulence and host susceptibility, the infection duration was linked to some immune variables: (a) the level of pro-inflammatory cytokines was a good indicator of the infection duration; and (b) a dominant regulatory response was associated with a prolonged infection.However, to identify and understand the immune mechanisms responsible for the infection duration, the entire immune response had to be considered. At least (i) the levels of innate antiviral cytokines, (ii) the level of We found that the macrophage permissiveness and the cytokine synthesis capacities both influence the infection duration through various immune mechanisms. Promoting antiviral cytokines or limiting the macrophage permissiveness and viral replication in order to reduce the infection duration has only been suggested Figure S1Preliminary sensitivity analysis: comparison of the phagocytosis and infection activities. This figure results from the 243 simulations performed for the preliminary sensitivity analysis. A: Percentage of phagocyting macrophages among all macrophages over time (maximum 14%). B: Percentage of infected macrophages over time (maximum 100%). C: Phagocytosis activity as a percentage of the phagocytosis and infection flows, i.e. the ratio between the concentration of susceptible macrophages becoming phagocyting macrophages per unit of time and the concentration of susceptible macrophages becoming phagocyting or latent infected macrophages per unit of time (TIFF)Click here for additional data file.Figure S2Parameter space exploration: viral titer. This figure results from the 6561 simulations performed for the sensitivity analysis. A: Viral titer over time (red curve: reference scenario S0). B: Distribution of the viral titer at day 200. Some simulations resulted in infection persistence, others in infection resolution occurring at various dates. The viral titer at day 200 was heterogeneously distributed: 56% of the simulations had a viral titer lower than (TIFF)Click here for additional data file.Figure S3Parameter space exploration: cumulative number of phagocyting macrophages. This figure results from the 6561 simulations performed for the sensitivity analysis. A: Cumulative number of phagocyting macrophages (B: Distribution of C: Distribution of (TIFF)Click here for additional data file.Figure S4Parameter space exploration: percentage of infected macrophages. This figure results from the 6561 simulations performed for the sensitivity analysis. A: Percentage of infected macrophages among all macrophages (B: Distribution of the C: Distribution of the (TIFF)Click here for additional data file.File S1(PDF)Click here for additional data file.


Urinary tract infection (UTI) is one of the most common bacterial infections in humans. Gram-negative organisms being the most common causative agent, the rising prevalence of resistance to a number of antibiotics and more importantly the production of extended spectrum beta-lactamase (ESBL) by these organisms is a growing concern worldwide. As the scenario is no better in community isolates, the choice of empirical antimicrobials for such infections becomes a great challenge for the clinicians.In this retrospective observational study we aimed at knowing the prevalence of ESBL production by organisms causing UTI in the community and to study the antibiogram of such isolates. Urine samples from patients with suspected UTI in the community were cultured for uropathogen by routine microbiological methods and susceptibility testing was done on Microscan Autoscan 4 (Siemens).Escherichia coli being the most commonly isolated pathogen. Enterobacter spp. showed highest prevalence (80%) of ESBL production from the community samples. Among the ESBL producing strains from the community, the sensitivity to ciprofloxacin, levofloxacin and nitrofurantoin was 18%, 21% and 44% respectively while in the non-ESBL producers the sensitivity rates were 52%, 51% and 73% respectively.Out of 527 isolates of Enterobactereaceae, 314 (59.58%) were ESBL producers from the community samples compared with 315 (67.30%) from hospital samples, with Organisms producing the ESBL phenotype present with an added possibility of being resistant to other broad-spectrum antimicrobial agents which are commonly prescribed in the community to empirically treat such infections. This makes the choice of empirical antibiotic much more challenging in the community, drawing errors in judgment. A possibility of frequent overcorrection lies on the other side of the coin. This study also shows the possible need for empirical institution of class I carbapenems as one of the treatment options and outpatient parenteral antimicrobial therapy.


Pathology of the long head biceps tendon (LHBT) is associated with rotator cuff tears but whether the LHBT texture changes following supraspinatus tendon full thickness tear (SSFT) can be detected at the extra-articular segment remains unknown. This cross-sectional study aimed to explore the morphological differences of the LHBT in shoulders with and without deficient rotator cuffs by using quantitative ultrasound.We selected 145 cases with SSFT and 145 age-and- gender-matched controls. The width, thickness, flattening ratio, cross-sectional area, and echogenicity ratio of the LHBT were measured and a general linear model was used to clarify the relationship between rotator cuff pathology and LHBT morphology. The receiver operating characteristic curves of each parameter were constructed for SSFT discrimination and the maximal Youden indexes were used to define the best cut-off points.2 of the cross-sectional area.We found increased thickness and cross-sectional area but decreased flattening ratio in shoulders with SSFT, and no between-group differences in the width and echogenicity ratio. The LHBT appearance was modified by biceps peritendinous effusion and medial subluxation, but not by the size of SSFT. The flattening ratio was the best discriminator for SSFT with an area under curve of 0.81 . The cut-off values to differentiate between the non-tear and tear groups were 2.00 mm of the thickness, 1.73 of the flattening ratio and 10.53 mmQuantitative ultrasound facilitated the detection of the LHBT morphological changes following SSFT and demonstrated its potential utility in discriminating rotator cuff deficiency. The long head of the biceps tendon (LHBT) is a common origin of anterior shoulder pain and an association between the LHBT pathology and rotator cuff tears has been reported by several arthroscopic case series Among all the biceps pathology concomitant with torn rotator cuffs, tendinopathy is the most common  [2]. CliWe reviewed data from patients referred to our musculoskeletal ultrasound laboratory for the examination of shoulder joints between January 2011 and June 2012. The institutional review board of National Taiwan University Hospital approved the retrospective review of these medical records and waived the requirement for informed consent from the participants because shoulder ultrasound investigation was a routine examination and analyzing the image sets had no risk to violate patient rights. Patient characteristics, age, gender, side of examined shoulder, and summarized clinical and physical manifestations were documented in the report with a check list and image set for recording the presence or absence of certain sonographic abnormalities. The structures were investigated by using a high-frequency 10–14 MHz linear transducer , following the order of the biceps tendon, subscapularis tendon, supraspinatus tendon, infraspinatus tendon, acromioclavicular joint, and posterior shoulder recess. All of the ultrasonographic examinations were conducted by a group of board-certified physiatrists with at least 3 years of experience in performing musculoskeletal ultrasound, following a standard protocol regarding patients' postures and transducer placement One experienced physician, independent from the examinations, selected suitable cases and processed the images for analysis. Patients aged >20 years and with a complete shoulder ultrasonographic investigation were included. Patients with systemic rheumatologic diseases, malignancy, major trauma, or history of past surgeries of the examined shoulders, were excluded. All the information was verified by reviewing the electronic medical records in our hospital. The SSFT cases were then retrieved from the musculoskeletal ultrasound registry. Shoulders with a torn biceps tendon or a supraspinatus tendon partial-thickness tear were excluded. After the case group was finalized, an age and gender-matched control group with intact supraspinatus tendons was selected at a 1∶1 (case∶control) ratio.The diagnostic criteria for tendinopathy included swelling of the affected tendon and abnormal tendon echotexture with a heterogeneous hypoechoic appearance. Calcification was diagnosed based on the presence of hyper-echogenic plaques with or without an acoustic shadow. Partial and total displacements of the LHBT out of the bicipital groove were recognized as subluxation and dislocation, respectively . EffusioFive parameters, comprising width, thickness, flattening ratio, cross-sectional area, and echogenicity ratio were implemented to depict the morphology of the LHBT and were measured in the short axis view at the bicipital groove level. The width was the maximal diameter of the LHBT , whereasThe sample size was estimated from the between-group difference in the thickness of the LHBT. By using a clinically important change of 0.5 mm and a standard deviation of 1 mm, 128 participants were required in each group with a significance level of.05 and a power of 80%. The rationale for assuming 0.5 mm as the between group difference resulted from the measurements of normal LHBTs in a previous study reporting standard reference values for musculoskeletal ultrasonography t-tests and chi-square tests, respectively. The relative risk was used to estimate the ratio of the probability of biceps tendon tear in the SSFT group compared with that in the group with intact supraspinatus tendons. The multiple linear regression of the general linear model was used to explore the relationship between various kinds of shoulder pathology and the morphological parameters of the LHBT Continuous variables and categorical data were compared by using Student's In addition, the receiver operating characteristic (ROC) curves were constructed to assess the ability of each morphological parameter to discriminate between shoulders with and without SSFT From a total of 1349 participants referred for an ultrasound examination of the shoulder, SSFTs were identified in 160 shoulders, 15 of which had a concomitant biceps tendon tear. In the overall study population, 17 shoulders were diagnosed with a biceps tendon tear with the relative risk for coexisting with SSFT estimated to be 8.10  compared to that in patients with intact biceps tendons. After excluding shoulders with torn biceps tendons and including 145 age and gender-matched participants with intact rotator cuff tendons, a total of 290 shoulders were included for further morphological analysis of the LHBT.2 of the cross-sectional area with a sensitivity of 0.57 and a specificity of 0.75.The present study quantified the sonographic morphology of the LHBT and explored the differences between shoulders with and without SSFT. The strength of this research included using a clinical registry, the data of which could be generalized to the population in primary care settings. The most novel finding was that the LHBT morphological differences in shoulders with and without SSFT could be identified using ultrasound at the extra-articular segment of the LHBT through a simple process of quantification. We found that the thickness and cross-sectional area of the LHBT significantly increased but the flattening ratio significantly decreased at the bicipital groove level in shoulders with SSFT. The LHBT appearance was modified by biceps peritendinous effusion and medial subluxation, but not by the size of SSFT. Furthermore, the flattening ratio exhibited the best discriminative ability for the presence of SSFT.We found that the relative risk of SSFT in shoulders with torn biceps tendons reached 8.1  compared with that in patients with intact biceps tendons, implying the LHBT tear  a specifEvaluation of the LHBT appearance using quantitative ultrasound has been proposed since 2006 and most parameters are related to pixel intensities and distributions in the region of interest The morphological changes of the LHBT, including a significant increase in the thickness and cross-sectional area with a decrease in the flattening ratio, were observed using ultrasound in shoulders with deficient supraspinatus tendons. The findings supported our hypothesis that a tendinopathic change such as swelling and enlargement could be detected in the extra-articular LHBT following SSFT. Normal LHBT is oval-shaped in the transverse view at the bicipital groove and the increase in the short-axis length is more easily identified Our analysis indicated that the morphology of the LHBT in large-sized SSFT did not differ from that in small-to-medium sized tears. Several antecedent studies indicated that the tear size played a less important role than the tear location in terms of shoulder pain and function impairment In the present study, the control group had a higher prevalence of clinically diagnosed frozen shoulder. Although the diagnosis was not confirmed by the golden standard Several demographic factors and concomitant sonographic abnormalities were shown to modify the LHBT morphology. Age was positively related to the cross-sectional area, which might be related to a tendency of tendon degeneration in the elderly Our result had two potential clinical implications. First, although it seems easy to identify SSFT in well-trained operators, the rule cannot be applied on less-experienced investigators. For anterosuperior rotator cuff tendon tear, the new learners are vulnerable to miss the diagnosis due to inappropriate shoulder positioning The present research had several limitations. First, we employed a cross-sectional study design, which could merely clarify the association between the LHBT pathological changes and SSFT. We were unable to know how the biceps tendon would change its morphology when the SSFT was left untreated or repaired. A longitudinal follow-up study should be implemented to solve the issue. Second, most participants registered in our musculoskeletal ultrasound registry were symptomatic and our analysis failed to answer whether the association was different in a population without shoulder complaints. We believed that the comparison with the asymptomatic group was of clinical importance, since the grades of LHBT pathology might vary according to their symptom severity. Nevertheless, considering generalization of our results in the hospital-based settings, the use of symptomatic participants without SSFT as controls was still acceptable if the current research aimed at a painful shoulder population referred for ultrasound examinations. Third, our data set did not allow us to explore the association between the location of SSFT and the changes of LHBT morphology, which would be analyzed in our future study using a more standardized measurement of the tear site.SSFT was associated with the morphology changes of the extra-articular LHBT examined using ultrasound, comprising an increase in the thickness and cross-sectional area and a reduction in the flattening ratio. Age, gender, biceps peritendinous effusion, and medial subluxation were shown to modify the LHBT appearance, but the difference between shoulders with small-to-medium and large-sized tears were not significant. Among the morphological parameters of the LHBT, the flattening ratio was the best discriminator of SSFT and may be utilized in future large-scale studies for detection of supraspinatus tendon deficiency of different grades.File S1Baseline and ultrasound characteristics in participants with and without supraspinatus tendon full thickness tears. All the information was anonymized.(XLS)Click here for additional data file.


Biological processes are usually distributed over various intracellular compartments. Proteins from diverse cellular compartments are often involved in similar signaling networks. However, the difference in the reaction rates between similar proteins among different compartments is usually quite high. We suggest that the estimation of frequency of intracompartmental as well as intercompartmental protein-protein interactions is an appropriate approach to predict the efficiency of a pathway.Using data from the databases STRING, ANDSystem, IntAct and UniProt, a PPI frequency matrix of intra/inter-compartmental interactions efficiencies was constructed. This matrix included 15 human-specific cellular compartments. An approach for estimating pathway efficiency using the matrix of intra/inter-compartmental PPI frequency, based on analysis of reactions efficiencies distribution was suggested. An investigation of KEGG pathway efficiencies was conducted using the developed method. The clusterization and the ranking of KEGG pathways based on their efficiency were performed. "Amino acid metabolism" and "Genetic information processing" revealed the highest efficiencies among other functional classes of KEGG pathways. "Nervous system" and "Signaling molecules interaction" contained the most inefficient pathways. Statistically significant differences were found between efficiencies of KEGG and randomly-generated pathways. Based on these observations, the validity of this approach was discussed.The estimation of efficiency of signaling networks is a complicated task because of the need for the data on the kinetic reactions. However, the proposed method does not require such data and can be used for preliminary analysis of different protein networks. Estimation of efficiency of signaling networks is one of the most relevant problems in the study of biological systems. Analysis of effectiveness of biological networks is needed to meet the challenges of medicine and biotechnology ,2. In paOne of the difficulties in modeling a signaling pathway is that biological processes in cells are allocated to different intracellular compartments . Thus, tPreviously we developed the CELLmicrocosmos PathwayIntegration (CmPI) to support and visualize the subcellular localization prediction of protein-related data such as protein-interaction network . Here, wThe method for estimating the efficiency of the pathway is based on consideration of PPI frequencies between different intracellular compartments. We assume that if PPI in general occurs more frequently between proteins from particular compartments, the interactions which contain proteins located in these compartments would be more effective within the pathways. Thus, the optimality of a pathway reaction distribution over the intracellular localization may reflect the efficiency of the pathway, with the most optimal distribution being the one where the frequency of observed interactions between proteins localized in intracellular compartments involved in the pathway has a maximum value.To analyze the effectiveness of intra/inter-compartmental interactions, 15 major locations of eukaryotic cells were selected: Cytoplasm, Nucleus, Secreted, Membrane, Chromosome, Endoplasmic reticulum, Golgi apparatus, Endosome, Lysosome, Mitochondrion, Cell junction, Lipid-anchor, Cell projection, Peroxisome and cytoplasmic vesicle. The localizations were selected by following rules. We considered only the highest hierarchy level of localizations presented in UniProt , data onOn the basis of these data we find efficiency of a reaction and a molecular-genetic network by following approach:i and j, Let q of the network Q, The efficiency There were 282 KEGG human pathways analyzed, including totally 50.000 reactions  of KEGG pathways with the one of random pathways, Eff distributions of 282 KEGG pathways and more than 10000 random pathways were calculated. Random pathways were generated by permutation of KEGG pathways in following way: for each KEGG pathway we generated 1000 of random pathways by replacing the proteins in each reaction by randomly chosen ones from the list of all KEGG proteins. If one protein is involved in several reactions of the pathway - we replace it in all these reactions by the same random protein. It was found that the Eff distribution of KEGG pathways Figure  has a stAlso, it was important to check whether there is a correlation between the length of the pathways and their efficiency. The value of Pearson correlation coefficient was equal to R =- 0.1 . The value of R was low, so we cannot make any concrete conclusions. However, it is negative, suggesting a weak reciprocal relationship with the length of the pathways.To identify similar KEGG pathways, hierarchical clustering was performed on the basis of the correlation distance between pairs of pathways Figure . The corSeven major clusters can be identified in the dendogram presented in Figure KEGG functional classes of pathways were ranked by the mean efficiency of included pathways. The highest efficiency is observed within pathways from "Amino acid metabolism", "Genetic information processing" and "Carbohydrate metabolism" classes."Metabolism" class consists of various subclasses, therefore it is located in the middle of the list. The less effective pathways include "Lipid metabolism", "Nervous system" and "Signaling molecules interaction" classes. Despite the fact that the average efficiency varies slightly between some classes Figure , the disEvaluation of the efficiency of the signaling networks currently remains an important issue. The method for preliminary analysis of networks lacking the data on kinetic parameters was suggested to avoid one of the main obstacles on the way to practical application of existing methods for modeling the dynamic of the molecular genetic systems. The matrix of intra/inter-compartmental interactions efficiencies was constructed for 15 specific human cellular localizations based on PPI data and data on protein distribution over cellular compartments. The analysis of the matrix revealed that the frequency of PPI of proteins from the same compartment is higher in comparison to frequency of PPI of proteins from different compartments. A new method for evaluating pathway efficiency was proposed; all KEGG human pathways were estimated by mean efficiency and clustered based on correlation distances between the distributions of pathway reaction efficiencies. The distribution of pathway functional classes over clusters shows that some classes are mainly presented in one cluster.The proposed method can be used for the preliminary analysis of the effectiveness of various signaling networks, including networks, for which there is not enough data for modeling them with more accurate methods.PPI data was extracted from STRING , IntAct The authors declare that they have no competing interests.OVP, OVS and VAI conceived the method. INL and RH carried out expert assessment and interpretation of results. OVP and OVS implemented the method and BS tested the method. EDP performed the statistical analyses. OVP and VAI drafted the manuscript. VAI supervised the whole studies. All authors read, corrected and approved the final manuscriptClick here for fileClick here for file


The present study illustrates the potential advantages of an eye-tracking method for exploring the association between visual scanning of faces and inferences of mental states. Participants watched short videos involving social interactions and had to explain what they had seen. The number of cognition verbs  in their answers were counted. Given the possible use of peripheral vision that could confound eye-tracking measures, we added a condition using a gaze-contingent viewing window: the entire visual display is blurred, expect for an area that moves with the participant’s gaze. Eleven typical adults and eleven high functioning adults with Autism Spectrum Disorders (ASD) were recruited. The condition employing the viewing window yielded strong correlations between the average duration of fixations, the ratio of cognition verbs and standard measures of social disabilities. The rise of affective computing during the last two decades has offered a wide range of possibilities for designing new tools that foster the study of social and emotional impairments. Affective computing is a stream of research that strives to empower computers with abilities to detect, process and respond to social and emotional signals emitted by human users Picard, . These aSocial misunderstanding in ASD has been linked to atypical visual scanning patterns , ranging from 24 to 40 years with a mean age of 31.82 [SD = 10.01]. Their mean Verbal Intelligence Quotient assessed with the Wechsler Adult Intelligence Scale, 3rd edition  diagnosed with autism by psychiatrists using the DSM-IV R diagnostic criteria. The Autism Diagnostic Interview-Revised . In the gaze-contingent viewing window condition, the entire graphic display was blurred (using Gaussian smoothing), except for a window centered on the focal point of the participant that moved in real time with her/his gaze Figures . Two vidcρ = 0.91. To analyze eye-tracking data, rectangular Areas Of Interest (AOI) were defined around the faces of the protagonists. We used a software prototype developed for a previous study . To calculate this ratio, the number of cognition verbs in each participant’s answer was divided by the total number of verbs employed. The concordance correlation coefficient Lin,  between t-tests that compared the two viewing conditions. We then computed Pearson’s correlation coefficients between the ratio of cognition verbs and the gaze variables.The data analyses were carried out with Statistica software.t(10) = 2.33, p = 0.042]. The average duration of fixations on faces was also higher in the gaze-contingent viewing window condition compared with the normal vision condition . The ratio of cognition verbs did not differ significantly between the two viewing conditions. We did not find any correlations between any of these three variables. Indeed, the ratio of cognition verbs did not correlate with the total fixation time on faces , nor did it with the average fixation duration .In experiment 1, the data of the normative group showed differences in visual patterns between the two viewing conditions. The total fixation time on faces was higher in the gaze-contingent viewing window condition than in the normal vision condition , the total fixation time on faces  and the average fixation duration on faces . Contrasting with the normative group in experiment 1, the total fixation time on faces correlated with the ratio of cognition verbs in the gaze-contingent viewing window condition . The average fixation duration on faces correlated with the ratio of cognition verbs in the normal vision condition  and in the gaze-contingent viewing window condition . In the latter condition, the ratio of cognition verbs correlated negatively with the CARS scores  and the average duration of fixations on faces correlated negatively with CARS scores  and with the ADI-R sub-scores in the Reciprocal Social Interaction domain .In experiment 2, no significant differences were found between the two viewing conditions on any of the measures, that is, the ratio of cognition verbs [Studies using eye-tracking techniques to analyze gaze exploration in ASD have been mostly involved in the description of facial regions of interests and their link with social impairments. The goal of the present study was to pinpoint more precise components of social impairments, by exploring the potential relationship between gaze exploration of faces and inference of mental states in others. Indeed, inferring mental states is often facilitated by deciphering facial expressions. Cognition verbs were used here as indices of mental states processing. Results of experiment 2 show a positive correlation between gaze variables and cognition verbs in participants with ASD. In other words, the more they were attentive to the dynamics of the facial expressions, the more they would use cognition verbs as if the latter were directly derived from the former. This suggests that their mentalistic insight depends predominantly on a face reading strategy whereby mental states are mapped onto behavioral changes perceived on the face. Interestingly, the severity of autism, as assessed by the CARS and the ADI-R sub-scores in the Reciprocal Social Interaction domain, appeared to be related to poor gaze exploration of faces and poor production of mental state terms.In experiment 1, the normative group of typical participants modulated their visual behavior to adapt to the gaze-contingent viewing window by increasing their fixation durations on faces. This is consistent with our previous findings (Grynszpan et al., Recent studies have attempted to tackle discrepancies found in eye-tracking experiments, by devising sophisticated analyses based on the distance between the gaze position of ASD participants and typical gaze patterns (Nakano et al., Most eye-tracking studies used the total fixation time on a given AOI as the main outcome measure (Boraston and Blakemore, It should be noted that even with the limited sample of participants with ASD used in experiment 2, the method we propose revealed strong significant associations between mental state attribution abilities, social competence and visual scanning of faces. The participants in our sample were all adults on the higher range of IQ scores. So the present findings cannot be generalized to the entire spectrum. The gaze-contingent viewing window method could reveal alternative strategies when applied to different sub-groups across the spectrum, shedding light on their distinctive cognitive functioning.The gaze-contingent system that we presented here seems useful to examine visual exploration of social scenes. However, the role of gaze in social communication extends beyond purely perceptual functions. It can also assume an active role in face-to-face interactions as for instance in joint attention situations, where it can be used to orient a partner’s attention towards an object of interest Emery, . The devThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Besnoitia besnoiti (Apicomplexa: Sarcocystidae) is a cyst-forming coccidian parasite that may cause severe lesions  in cattle as intermediate hosts. Wild ruminants are also susceptible. As a unique example among cystogenic coccidia, the main transmission route of B. besnoiti appears to be mechanical by blood-sucking dipterans (tabanid and stable flies), although it is also possible iatrogenically (with hypodermic needles) and most likely with close contact between animals. Bovine besnoitiosis has been endemic to South-Western Europe for more than a century, but a significant geographical expansion was observed during the last decade to other parts of the formerly endemic countries and to countries neighboring France.It usually gains proper attention, when blood-sucking arthropod species in Europe show northward spreading, in part due to climate change and global warming. Accordingly, it is well known, that the geographical regions where pathogens transmitted by these as biological vectors are endemic, also may extend towards the north. However, it is mostly less emphasized, that for vector-borne pathogens present in Western Europe the risk of eastward emergence is also possible, if they have competent vectors in the central and/or eastern part of our continent. B. besnoiti infection), seropositivity among local animals, which originally belonged to the herd, is low. Based on ELISA results venereal transmission  appears to be either rare or unlikely. The seroprevalence of besnoitiosis decreased significantly among calves born to the group of imported mother cows. The risk of infection seems to be high, when calves stay with their mother during suckling (for 6-7 months), and if animals are kept in the same stable  during the main fly season. Confirmation of the ELISA results is done with immunoblot and IFAT. All seropositive cattle are now kept at a distance of several kilometres from other groups of animals, prior to culling. Molecular and sero-epidemiological evaluation of the situation continues with the aim of preventing the spread of the disease and regaining the epidemiological status of Hungary as exempt of bovine besnoitiosis.During the autumn of 2013 bovine besnoitiosis was diagnosed in a beef cattle herd in Hungary , following the import of Aubrac heifers and bulls from France in the previous two years. The preliminary serological herd screening with ELISA shows that even after the 2nd year of the presence of imported animals (with high prevalence of


Prolactin (PRL) is one of the most versatile hormones known. It is considered an adaptive hormone due to the key roles it plays in the modulation of the stress response and during pregnancy and lactation. Within the brain, PRL acts as a neuropeptide to promote physiological responses related to reproduction, stress adaptation, neurogenesis, and neuroprotection. The action of PRL on the nervous system contributes to the wide array of changes that occur in the female brain during pregnancy and result in the attenuation of the hypothalamic–pituitary–adrenal axis. Together, all these changes promote behavioral and physiological adaptations of the new mother to enable reproductive success. Brain adaptations driven by PRL are also important for the regulation of maternal emotionality and well-being. PRL also affects the male brain during the stress response, but its effects have been less studied. PRL regulates neurogenesis both in the subventricular zone and in the hippocampus. Therefore, alterations in the PRL system due to stress or exposure to substances that reduce neurogenesis or other conditions, could contribute to maladaptive responses and pathological behavioral outcomes. Here, we review the PRL system and the role it plays in the modulation of stress response and emotion regulation. We discuss the effects of PRL on neurogenesis and neuroprotection, the putative neuronal mechanisms underlying these effects, and their contribution to the onset of psychopathological states such as depression. Prolactin (PRL) is a pleiotropic pituitary hormone with more than 300 known physiological effects. This protein hormone has a regulatory control on reproduction, immunomodulation, angiogenesis, energy metabolism, osmotic balance, and development. In addition to its peripheral functions, PRL also plays many important roles as a neuropeptide. PRL crosses the blood–brain barrier, and local hypothalamic production contributes to reach several brain regions producing strong modulatory effects. In particular, PRL contributes to the regulation of the stress responses through the inhibition of the hypothalamic–pituitary–adrenal (HPA) axis. Moreover, PRL modulates anxiety and depressive-like behaviors. PRL also regulates neurogenesis, the generation of new neurons, in both the subventricular zone (SVZ) and the hippocampus. PRL levels and environmental conditions may induce changes in neurogenesis, potentially leading to an impact on emotional behavior. Indeed, reduction of PRL levels during early pregnancy affects olfactory bulb neurogenesis and increases postpartum anxiety in the female rat. In addition, PRL may exert neuroprotective effects in the hippocampus of adult animals exposed to chronic stress or subjected to hippocampal infusions of kainic acid. However, PRL reduces neurogenesis, when administered, during the early developmental stages and promotes depressive-like behavior in adulthood. Some of these effects are mediated by the activation of different neuronal signaling systems and ion channels. Here, we will summarize some aspects of PRL actions within the brain and their contribution to the onset of psychopathological states like depression.In situ hybridization showed PRL expression in the medial preoptic area (MPOA), periventricular preoptic nucleus, the bed nucleus of the stria terminalis, the PVN, and the lateral septum in the domestic turkey and in both the fetal and adult sheep (Prolactin is produced mainly by the lactotroph cells in the anterior pituitary. PRL is under the inhibitory control by dopamine released from the tuberoinfundibular dopaminergic (TIDA) neurons . Despitelt sheep , 6. Recelt sheep .Peripheral PRL is considered the major effector within the brain. It has been hypothesized that PRL may cross the blood–brain barrier through a receptor-mediated mechanism occurring in the cells of the choroid plexus . HoweverProlactin receptors belong to the class 1 cytokine receptor superfamily . Both lovia an antisense probe strongly increases the stress-induced ACTH secretion in virgin and lactating rats, suggesting that PRL plays an inhibitory role on HPA axis reactivity  in the PVN, which promotes the secretion of adrenocorticotrophin (ACTH) from the pituitary. In turn, ACTH triggers the release of glucocorticoids from the adrenal glands. PRL is also secreted from the pituitary in response to a number of stressors. Initial studies suggested that PRL may counteract glucocorticoid actions on the immune system during the stress response . Howeveractivity , 17. It activity . Additioactivity .Prolactin has been recently associated with resilience in a model of chronic mild stress (CMS). Adult rats, previously subjected to CMS, were selected as responders and non-responders, according to their reaction to stress. The resilient animals  presented higher plasma levels of PRL, and higher PRL-R mRNA in the choroid plexus than their vulnerable  counterpart . AccordiThe effects of PRL on anxiety and depressive behavior have been studied, but they differ depending on the species used and the physiological state.Intracerebroventricular or intravenous administrations of PRL exert dose-dependent anxiolytic effects in both male and virgin female rats . AnxietyStudies on inbred lines of rodents have shown an association between elevated levels of PRL and increased anxiety. Increased basal and stress-induced levels of PRL were reported in male rats bred for high anxiety behavior (HAB) as compared to low anxiety behavior (LAB) rats . The RomArtificially inducing hyperprolactinemia in adult male rats by placing pituitary grafts of a donor rat in the kidney of the receptor animal was shown to exert antidepressive-like effects in the forced swimming test .In humans, some patients with hyperprolactinemia exhibit depressive symptoms. However, no differences were observed in the prevalence of depression in hyperprolactinemic patients compared to controls , 26. RemSeveral animal studies report a causal link between reduced maternal care and depressive-like state or alterations of emotional behavior in the offspring. Stress exposure during pregnancy alters the neural mechanisms that prepare the female to her maternal role and contributes to the development of psychiatric diseases such as depression or anxiety , 31. FemDysregulation of PRL-R responsivity could be associated with postpartum disorders . Indeed,Chronic stress exposure and depressive states are known to affect neurogenesis. Neurogenesis is the process that produces new neurons throughout life. New neurons are thought to transiently increase neuronal communication. Two neurogenic niches have been recognized in the adult brain: the hippocampus and the SVZ. The hippocampus exerts a negative control on the HPA axis activity and it is involved in emotional modulation. Antidepressive treatments increase hippocampal neurogenesis and show a correlation with mood improvement . OlfactoProlactin is a regulator of neurogenesis. PRL receptors are expressed in the SVZ and the hippocampus –48. Initin vitro  and in vivo (direct injection into the dentate gyrus of adult mice) that PRL administration increases the neurosphere number, suggesting a direct effect of this neuropeptide on the hippocampus . ERK5 is expressed in the neurogenic niches of the brain . Moreove regions . In the  regions  and prom regions . Activat regions . HoweverProlactin alters neural circuits to help the individual to cope with stress. Reduced activation of neural inputs, activation of ionic channels, or the modulation of several signaling pathways are some of the putative mechanisms of action underlying the effects of PRL on brain circuits. It is unknown how PRL regulates the HPA axis function during the stress response. PRL could affect hypothalamic and/or hippocampal activity to regulate emotionality. The contribution of PRL to the onset of postpartum depression is still unknown. Low PRL levels in nursing women have been associated with postpartum depression. Inhibition of PRL reduces neurogenesis in the olfactory bulb, but the mechanisms that result in a postpartum depressive state are unknown. Besides the pathways affected by PRL to regulate anxiety are not yet known, and whether the PRL effects on hippocampal neurogenesis contribute to modulate anxiety is still uncertain. Administration of PRL in the early postnatal stages reduces neurogenesis in the hippocampus and the olfactory bulb and promotes a depressive-like state in adulthood. This suggests that PRL affects a critical window during brain development and that PRL actions are dependent on the age during which it is administrated. Further studies are needed to understand the mechanisms of action of PRL in the brain, and how this hormone modulates emotionality and anxiety at different developmental stages.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest.


Trastuzumab therapy given in combination with one of several chemotherapy regimens is currently considered the standard of care for the treatment of early-stage, human epidermal growth factor receptor-2 (HER2) -positive breast cancer. The treatment with trastuzumab is due to a significant impact on the survival part of the standard adjuvant treatment of patients with HER2-positive breast cancer. Patients treated with postoperative breast or chest wall irradiation receive trastuzumab concomitant with radiotherapy. In a small proportion of patients trastuzumab causes cardiotoxicity. Preclinical findings indicate a radiosensibilizing effect of trastuzumab in breast cancer cells, but it is not yet clear whether it radiosensibilizes cells of healthy tissues too.Special attention is required when left breast or left thoracic wall is irradiated in patient receiving trastuzumab, because long-term effects of the concurrent treatment with trastuzumab and radiotherapy are not yet known. In an era where more patients are surviving a diagnosis of breast cancer, better understanding and earlier detection of therapy-induced cardiac toxicity will be of paramount importance. The exact mechanism of action that leads to the clinical efficacy of trastuzumab is not yet entirely clear, although its antitumor effect in vitro has already been shown before 20 years. Trastuzumab was approved by the US Food and Drug Administration in September 1998 for the treatment of metastatic breast cancer. Nowadays it is widely used in the metastatic and adjuvant systemic treatment for breast cancer.Trastuzumab  is 15–25% of breast cancers.The meta analysis, published in Cohrane Database in April 2012, which included eight studies involving 11.991 patients, found that the combined hazard ratios (HR) for the overall survival and disease-free survival significantly favoured the trastuzumab-containing regiments  used in the treatment for early and locally advanced breast cancer.9Clinical guidelines based on these findings, therefore, recommended the introduction of trastuzumab before postoperative radiotherapy and after the treatment with anthracyclines.11The treatment with trastuzumab is well tolerated by most patients. In a small proportion of patients treatment may be necessarily temporary or permanently discontinued due to the resulting damage to the heart.Anthracyclines, which are received by most patients with early breast cancer during the adjuvant treatment with CT, may cause cardiotoxicity type I.14et al. report on the reversibility of trastuzumab-induced reduction in LVEF after discontinuation of therapy.14Trastuzumab could cause cardiotoxicity type II.So far there is very little known about the long-term significance of asymptomatic LVEF reductions. Cardiologists warn that it is necessary to monitor these patients annually even after the treatment with trastuzumab.20Irradiation of the heart may be associated with multiple effects on the heart; the result could be acute or chronic pericarditis, pericardial effusion, constrictive pericarditis, coronary vascular disease, restrictive cardiomyopathy, valvular heart disease or malfunction of the conductive system of the heart. The development of these defects depends mainly on the dose received by the heart and the proportion of the heart, which is exposed to radiation.22versus not, also reported that there was, at least with some of the older radiotherapy regimens, a significant excess mortality from heart disease . It was slight during the first five years, but continued after year 15.Cardiotoxicity of adjuvant radiotherapy in patients with breast cancer is the subject of many studies.Meta-analysis, which allows the identification and abstraction of critical information from different randomized, controlled trialsOlder radiotherapy techniques  radiotherapy) and irradiation devices , which were used in the past for the treatment of patients with a breast cancer did not allow for as good protection of the heart as it is possible with newer radiotherapy techniques  conformal radiotherapy) and modern irradiation devices (linear accelerator) . In the Despite progress in radiotherapy for the patients with breast cancer, there is still some risk of a cardiac damage due to irradiation. The reason for the damage is the anatomical position of the heart, which lies just below the breast or chest wall, which is irradiated with the therapeutic dose.in vitro and in vivo studies have shown that the cascade of events through the HER2 receptor is involved in tumour radiosensibility28Preclinical Currently the most important question remains whether the concomitant therapy with trastuzumab and radiotherapy increases cardiotoxicity of the treatment. In the literature, there are limited data about the safety of concomitant therapy with radiotherapy and trastuzumab. The observation period in the studies was short, the longest reported median observation period after the completion of concomitant treatment with radiotherapy and trastuzumab was 3.7 years.29vs. 7%).In the study which included a retrospective series of 218 patients with advanced breast cancer at MD Anderson, a significantly higher rate of cardiovascular events was identified in patients who have completed left breast irradiation, as it was found in patients after irradiation of the right breast (26% vs. only radiotherapy).In the monocentric prospective study from the Institute Curie in Paris acceptable skin toxicity and cardiac toxicity was reported after a median observation period of 13 months after the completion of the adjuvant treatment with trastuzumab and concomitant radiotherapy.NSABP-B31 study did not allow radiotherapy of parasternal lymph nodes. It included 1503 patients and did not show any differences in the incidence of cardiovascular events, regardless of whether the patients were irradiated to the left or right breast/thoracic wall.31vs. non-irradiated patients), and there were no significant differences in comparison with the irradiated side.Sub-analysis of NCCTG N9831 study has included 1286 patients, of whom in the adjuvant treatment 908 patients received concomitant trastuzumab and radiotherapy and 378 patients received only trastuzumab.The Canadian study included 59 patients, of whom 44 were treated with concomitant trastuzumab and radiotherapy.In the HERA studyThe optimal method, duration and frequency of cardiac monitoring for patients receiving trastuzumab combined with radiotherapy have not yet been established. The role of careful history, physical examination, electrocardiograph and chest radiograph is crucial.40Cardiac troponins I and T are early, highly specific and sensitive markers of myocardial injury.BNP is a member of natriuretic hormones family. Their effect is vasodilatative, natriuretic, diuretic and hypotensive. They inhibit renin-angiotensin system and enhance neurohormonal activation in heart failure patients.35Standard transthoracic two-dimensional echocardiography is already a golden standard in the evaluation of carditoxicity in cancer patients.49in vivo technetium-99m labelled red blood cells and observes their intracardiac accumulation during different stages of the heart cycle with gamma camera in a standard left anterior oblique view.40Another useful diagnostic tool in the evaluation of cardiotoxicity is radionuclide ventriculography. This radionuclear technique uses Data from the studies published so far indicate that the concomitant therapy with radiation and trastuzumab is likely safety; irradiation should not have a significant additional effect on cardiotoxicity detected after the treatment with trastuzumab. However, so far only data obtained solely some years after the treatment are published. Currently there is no evidence that such therapy is safe even after a long observation period. In the previously mentioned studies biomarker NT-proBNP wasn’t measured, which could possibly earlier than the measurement of LVEF show a significant difference in cardiac toxicity between the two compared groups of patients .Because we know that trastuzumab in terms of cardiotoxicity in most patients causes only asymptomatic decrease in LVEF51As yet there are no guidelines for the follow-up of the patients after the treatment with trastuzumab. Perhaps particularly young patients would need a regular follow-up by the cardiologist after the treatment.Since the new target drugs from the group of HER2 inhibitors  have been used alone or in combination with trastuzumab, the question of co-toxicity of trastuzumab and radiation is even more important.Because the prognosis of patients with HER2-positive early breast cancer, which is considered a more aggressive type of breast cancer, with new types of treatment is improving and their expected lifespan is extending, the determination of the toxicity of treatment has an increasing importance.


Phaseolus vulgaris leucoagglutinin (Pha-L) allow brain-wide mapping of connections through analysis of large series of histological section images. We present a workflow for efficient collection and analysis of tract-tracing datasets with a focus on newly developed modules for image processing and assignment of anatomical location to tracing data. New functionality includes automatic detection of neuronal labeling in large image series, alignment of images to a volumetric brain atlas, and analytical tools for measuring the position and extent of labeling. To evaluate the workflow, we used high-resolution microscopic images from axonal tracing experiments in which different parts of the rat primary somatosensory cortex had been injected with BDA or Pha-L. Parameters from a set of representative images were used to automate detection of labeling in image series covering the entire brain, resulting in binary maps of the distribution of labeling. For high to medium labeling densities, automatic detection was found to provide reliable results when compared to manual analysis, whereas weak labeling required manual curation for optimal detection. To identify brain regions corresponding to labeled areas, section images were aligned to the Waxholm Space (WHS) atlas of the Sprague Dawley rat brain (v2) by custom-angle slicing of the MRI template to match individual sections. Based on the alignment, WHS coordinates were obtained for labeled elements and transformed to stereotaxic coordinates. The new workflow modules increase the efficiency and reliability of labeling detection in large series of images from histological sections, and enable anchoring to anatomical atlases for further spatial analysis and comparison with other data.Axonal tracing techniques are powerful tools for exploring the structural organization of neuronal connections. Tracers such as biotinylated dextran amine (BDA) and  Axonal tract-tracing methods have been widely used over several decades for mapping the wiring patterns of the brain. A large number of tracers are available to fulfill various experimental requirements  labeling in series of section images, as well as a module performing measurements relevant for describing the extent and location of plexuses and terminal fields of labeling and injection sites. To support spatial analysis and integration with other data we further extended the workflow toward assignment of atlas coordinates to the labeling. Thus, we apply a registration method that allows us to define the spatial location of each section image relative to a volumetric atlas . The new workflow modules extend the functionality toward image processing, analysis, and alignment to atlases (steps 5–7). Key metadata are collected at each workflow step and stored along with the original (“raw”) and processed image data. Steps 5–7 involve the use of software tools and analysis procedures intended for integration in a common application but currently managed as separate stand-alone tools.The original workflow for brain-wide mapping of axonal connectivity described in (1) The first step of the core workflow (“Experiment”) includes a series of procedures leading to the injection of an axonal tracer into a target region of the brain of the experimental animal, followed by the survival of the animal, allowing axonal transport to take place before perfusion and extraction of the brain from the skull.(2) The second step (“Microtomy”) covers the preparation of the fixed brain tissue, the sectioning of the brain, and the storage (and possibly mounting) of sections before further treatment.(3) The third step is the histochemical processing of the sections, often done on free floating sections, before mounting on glass slides. Through this processing step, the injected tracer and the elements  containing the tracer are labeled.(4) The fourth step completes the core workflow at the level of high-resolution image acquisition of the sections using robotic microscopes or slide scanners. For brain-wide mapping, extensive series of sections covering the entire brain are collected and submitted to image acquisition.(5) The fifth step provides automated processing of large series of high-resolution images to detect neuronal cell bodies or axons labeled with the injected tracers. Image processing parameters are determined based on a set of representative images, followed by processing of the full image series producing binary maps of the detected labeling for each section.(6) The sixth step defines the spatial location of each section relative to a volumetric brain atlas. First, the angle of orientation (slicing angle) of the series of sections is replicated and applied to the 3D atlas to generate customized atlas plates with a corresponding angle and spacing. These plates are then superimposed on the section images by affine transformations, and used to extract atlas coordinates for labeled elements identified in the co-registered section images.(7) The seventh step involves tools for analysis of the extent and location of labeling in full image series, including automated measurements of labeled area and centroid position for injection sites, larger plexuses, or terminal fields.1 provided by Pha-L), was injected in the cerebral cortex of anesthetized rats. After 7 days, animals were transcardially perfused with 4% paraformaldehyde, and brains were removed for histological processing. Coronal sections with a thickness of 50 μm were cut on a freezing microtome, and every second or fourth section was processed to visualize BDA . Depending on the type of labeling and the presence and quality of counterstaining in the images, different image processing methods are used to extract labeled elements from the sections using a combination of custom-made macros and modified plugins for ImageJ . Series of section images from a single brain that share identical staining and imaging history are processed in an identical way. In the first phase of the image processing procedure, representative images are selected from the series, and multiple image characteristics are analyzed to determine the optimal set of processing steps and parameters for the whole image series. In the second phase, the entire image series is processed automatically based on the previously determined setup without a need for further human interaction. Image processing parameters can be adjusted and reused for similar image series.Microscopic images resulting from steps 1–4 of the original workflow are processed using a new workflow module (step 5) to automatically detect labeling in each section . For RGB images where the color and intensity of the labeling is substantially different between labeling and background, the image channel providing the best signal-to-noise ratio  is used for generating a grayscale image. In cases where these parameters are close to the section background or the counterstaining, a modified version of the Colour Deconvolution plugin . Customized parameter sets are acquired for different types of staining combinations using a parameter acquisition plugin. We acquired and added new Colour Deconvolution parameters for BDA, BDA combined with Neutral Red, Pha-L, Pha-L combined with thionine, and BDA combined with cytochrome oxidase. These standard parameters can be used as they are or further adjusted to fit individual image series.Native RGB images are stepwise converted to binary labeling maps via intermediate grayscale images . The binary images resulting from the thresholding operations are optionally further enhanced by noise filtering using a standard median filter with a typical radius of a few pixels. Depending on the needs of further analysis, larger clusters of a specified size (area) range of interest are automatically selected, and all smaller background elements are removed. Clusters are then automatically measured for labeled area and centroid position (2F). The results are collected in a comprehensive table for all images in the series. Binary labeling maps are saved in compressed Graphics Interchange Format (GIF) allowing significant reduction in image size without loss of image quality.In the next step, multiple automatic thresholding methods are tested to reach optimal signal to noise ratio when generating binary labeling maps from the grayscale images . We then compared the binary maps generated by the automatic labeling method with the distribution of labeling as observed in the original section images, and with results obtained in a previously published study using the same series of images but a rigorous manual mapping approach . (2) Modest amounts of labeling feature relatively fewer fibers, separated but not readily counted. (3) Low amounts of labeling comprise a countable number of single fibers. The comparison of the binary maps with the results of the manual analysis, taking into consideration the three categories , allowed us to evaluate the automatic labeling detection method with regard to the amount of undetected labeling , the detection limit for labeling, the level of noise, and the consistency of the signal-to-noise ratio throughout large series of images.The aim of the image processing, referred to as automatic labeling detection, was to detect labeled axons or cell bodies , and discard other entities including non-labeled tissue elements and diffuse non-specific background (noise). We tested the image processing workflow module on several series of images with varying densities of BDA and approach . LabelinTable 1 provides an overview of findings in a case containing BDA labeling3 . Results from the extensive manual analysis performed by r = 2 pixels), two of the seven previously detected subregions remained detectable. It should be noted that the detailed manual analysis by Pha-L) and independent of counterstaining method , was also seen in the other cases . Low amounts of labeling could be detected at about the same levels as reported for the case shown in Table 1. Thus, some instances of very low amounts of labeling reported by Fully reliable detection  for high and modest amounts of labeling, for both tracers . The main functionality of this tool is to generate arbitrarily positioned, sized and oriented rectangular slices from NIfTI-1 volumes (Neuroimaging Informatics Technology Initiative4). Re-slicing calculations are realized in platform-independent Java code running on a Web server. Custom slices are produced interactively using a Flash-based web interface allowing the user to set the clip region while the selected microscopic section image is co-visualized with the atlas volume. For this project, we used the open access WHS atlas of the Sprague Dawley rat brain which consists of a structural MRI and diffusion tensor imaging (DTI) template . Briefly, (1) an approximate anteroposterior position is determined; (2) the height and width, as well as the in-plane rotation of the MRI slice is adjusted to fit the section image; (3) MRI re-slicing angle and anteroposterior slice position are adjusted to match the section image; and (4) optionally the slice is moved in-plane to allow fine alignment of selected regions of interest in the section image to corresponding regions in the volumetric atlas . Once optimal alignment parameters are acquired for the original section image, the position, size, and orientation of this section is saved, and concurrently processed versions of the same section, e.g., binary labeling maps, can automatically be viewed in alignment with the custom-angle atlas slice (MRI or delineations). To facilitate alignment of large series of section images, propagation of alignment parameters is provided between manually anchored sections. First, 5–10 sections spread throughout the series are aligned manually. The position of intermediate sections is then interpolated, and a provisional alignment is applied to each image. Manual adjustments are applied to complete alignment of the full series.We developed a new tool for registration of section images (2D) to volumetric (3D) atlases . Centroid coordinates are then obtained manually for each region through the active cursor functionality in the AligNII tool. In the current implementation, NIfTI coordinates and Sprague Dawley rat WHS v1.01 coordinates  to the stereotaxic atlas of Figure 5G). We used bregma as the common reference point between the two atlases to transform WHS coordinates to stereotaxic space. The transformation takes into account the rotation between the two coordinate systems (–4.085°) and an overall size difference between the WHS template and the After alignment of the section images to a selected atlas, the final step in the workflow is to acquire anatomical coordinates for regions of interest such as labeled clusters. We here used the 2D centroid of labeled objects  to represent the location of each region of interest on a section. Centroids are calculated and plotted in ImageJ for clusters of a specified size (area) range measured in the binary labeling maps  license. The image processing modules are shared on the ImageJ Documentation WikiWe have extended a workflow for brain-wide mapping of axonal connectivity with automatic detection of labeling in series of microscopic section images, semi-automatic alignment of these images to a volumetric atlas, and assignment of anatomical location to the detected labeling. Our results include an evaluation of the possibilities and limitations of the image processing methods applied for detecting axonal labeling, and a demonstration of how the alignment and coordinate transformation process facilitates data integration across 2D and 3D atlases.We used publicly available standard histological material originally produced to map axonal connections across the rat brain  to test Compared to the manual analysis , which sPha-L. The same approach can also be adjusted for detection of other combinations of axonal tracers and counterstaining. Fluorescent tracers represent another tracer category potentially well suited for automatic detection and quantification provided that background fluorescence is sufficiently low.We have set up the image processing procedure to detect distinct light-microscopic labeling with BDA or nth) of the sections is stained for cytoarchitecture in order not to obscure labeling or interfere with quantification. Our registration tool handles such images displaying limited structural contrast without the need for complete series or even consistent angle of sectioning. The interactive registration of individual sections is complemented by propagation of alignment parameters, enabling fast registration of large series of images. The precision of the alignment and the subsequent coordinate transformation process is constrained by affine registration of the WHS template to individual section images and to the stereotaxic atlas. This can be improved by local alignment of selected regions of interest on the section images, and by using multiple in-brain landmarks or non-linear methods to define the transformation field.Assignment of anatomical location to labeling is a key step in the analysis of whole brain axonal connectivity data. In the present study, we have taken the individual section images as a starting point for registration to the volumetric atlas. A range of other registration approaches and methods have been reported, including reconstruction of an image series to form a volume, followed by volume to volume registration . AutomatAt the new workflow endpoint, histological image series and associated maps of auto-detected labeling are assigned anatomical reference, and basic quantitative measures of the position and amount of labeling are provided. Subsequent analytical options may include a wide range of approaches, depending on the research questions asked. For studies of topographical organization, it will be relevant to combine labeling data from several different experiments to detect changes in spatial distribution as a function of the size and position of the tracer injection site . InvestiEAP, TBL, and JGB designed the workflow, analyzed results from automatic labeling detection and wrote the manuscript. EAP developed the image processing modules and calculated the transformation matrix between atlas spaces. GC provided input to the manuscript and implemented the AligNII tool for alignment of sections to atlas space.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Topical medicinal patches have been popular for the treatment of minor injuries like sprains and avulsions. Other inflammatory conditions like chronic musculo-tendinous pain and or fasciitis are also taken care of by local ointments or rubs. In the oriental communities, medicinal herbs frequently form the major components of the patches. In spite of the lack of scientific evidence of efficacy, the popularity of such traditional application persists for centuries. In this era of evidence-based clinical treatment, there is an urgent need to look into this traditional practice. The purpose should include a scientific verification of the efficacy of the practice, and once proven, further explorations would be indicated to bring the practice to a higher level. A system of comprehensive exploration was proposed and practiced in the past years to fulfill the aspiration. The research consisted of four areas:(1) Identification of the suitable medicinal herbs for the topical study;(2) Study of the biological activities of the selected herbs, concentrating on the areas of anti-inflammation, anti-oxidation, angiogenesis and cellular proliferation;(3) Study on the transcutaneous transport of the chemicals of the selected herbs to deeper tissues; and(4) Pilot clinical studies on common superficial inflammatory musculo-skeletal conditions to give objective clinical evidences to the topical applications.Five herbs were identified as suitable candidates of study. They were put into relevant laboratory platforms and were proven to be anti-oxidant, anti-inflammatory and pro-angiogenic. Three of the herbs were prepared as topical patches with an enhancer and used to treat three common ailments in pilot clinical trials, viz., plantar fasciitis, undisplaced metatarsal fracture and tendonitis of the wrist (de-Quervain’s disease) and the elbow (Tennis elbow). The clinical results of the pilot studies were very positive. It is therefore concluded that further explorations are justified to create medicinal herb patches of even greater efficacy. Centuries before the emergence of chemical drugs and pharmaceuticals, our ancestors have been making use of special plants for the control of disease symptoms and clinical problems. They could have followed the examples of animals which, by instinct, are capable of creating their primitive way to combat their odd infections and parasitic infestations . SuccessDuring the long era of Traditional Medicine, the best known and probably the most widely practiced clinical offer has been herbal pastes for topical use . The verSince 10 years ago, the Institute of Chinese Medicine at The Chinese University of Hong Kong has engaged in a comprehensive study on topical herbal agents used for skeletal injuries. The scope of study included the selection of herbs; investigation on the biological effects of the herbs; the cross-skin barrier effects; and lastly the clinical evidences.Study of old classics revealed that over 100 herbal items have been used as topical agents in the treatment of musculo-skeletal injuries . How areCarthamus tinctorius L. (flower)], Dipsaci Radix [Dipsacus asperoides C.Y.Cheng & T.M.Ai (root)], Rhei Rhizoma [Rheum officinale Baill. (root and rhizome)], Angelica Sinensis Radix [A. sinensis (Oliv.) Diels (root)] and Achyranthis Bidentatae Radix [Achyranthes bidentata Blume (root)]. Carthami is well known for its antioxidant effects. Dispaci has been described as an “bone repairing” agent. A. Sinensis, Achyranthis Bidentatae and Rhei have been studied for their anti-inflammatory and neo-vascularisation effects Undisplaced fracture of the distal metatarsal bone. Swelling and pain commonly persist in spite of fracture healing. Topical agent to consolidate the bone healing and to better control the symptoms of pain and swelling is a logical consideration.(b)Plantar fasciitis in the foot classically lead to long lengthy duration of heel pain which affects walking and standing. Topical treatment over the heel was chosen as another study target.(c)In the upper limb, tendon inflammation like de Quervains disease of the short extensor and abductor of the thumb and wrist extensor origin tendonitis (tennis elbow) are common conditions suitable for clinical study of topical application.Table 1. The topical treatment for all three sites had been very well accepted by patients. Recurrences after cessation of local treatment were observed in some cases, but the intensities were apparently much less than before treatment (Henceforth, three pilot studies were organized to serve as “proof of concept” trials in preparation for larger scale studies in future. The study design followed the standard requirements for similar clinical trials so that proper clinical data could be built up to be compared with other options of treatment. The study protocols followed principles recommended by the Declaration of Helsinki and had been approved by the hospital ethical committee. The methodology of study and results of treatment are presented in the reatment ,b.Transdermal topical drug delivery offers a non-invasive route of drug administration. Ten years of large scale explorations on the clinical science of traditional herbal medicine applied as topical agents for anti-inflammatory and pain control measures, using standard laboratory platforms and small scale pilot clinical studies, have given us confidence that the traditional method of treatments does have objective evidences of efficacy. Established surgeons and physicians should refrain from teasing topical agents as “myths,” “counter-irritants” or “ritual practices.” Topical agents providing anti-oxidant and anti-inflammation effects on the diseased tissues after penetrating the skin barrier is now a proven fact. The vascular promotion ability observed further supports the revascularization and regeneration of tissues accordingly. On the other hand, users and advocates should not dogmatize the old view that the topical agent heals perfectly on its own. Instead, the topical agent helps to control symptoms, whereas the primary causes of the pain and swelling could be the result of more complicated pathologies that requires the joint efforts of caring teams to achieve the best clinical outcome .The common conditions studied had known predisposing causes of chronic stress and/or repetitive minor injuries. To ensure cure and prevention of recurrence, the primary causes must be dealt with at the same time. Without taking effective care of the causative mechanisms relief of the symptoms could be transient. Topical agent administration therefore needs to go hand in hand with active rehabilitation measures.Further work needs to be done so as to give more comprehensive understanding of the mechanisms of action of the herb extracts. The molecular aspects of the biological effects need to be explored so that more suitable medicinal herbs could still be identified and either small chemical molecules or groups of chemicals from the gross extracts of the herbs could be used to give more potent effects.On the skin surface penetration, particle sizes manipulation has been the many innovations of the nanotechnology experts. The application of nanotechnology would be able to enhance penetration, while at the same time ensure that the ideal quality and quantity of the chemical components with desirable effect be adequately delivered .PL had responsibility for all parts of the manuscript. WS, EK, and CL had worked on the herbs biological and mechanistic and pre-clinical data mentioned in the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A 74-year-old male patient presented to the outpatient department with left-sided epiphora and chronic dacryocystitis, without any history of head trauma or previous nasal or paranasal sinuses surgery. No abnormalities were noted at the time with the use of nasal endoscopy. The computed tomography scan however revealed an osteoma of the medial wall of the left maxillary sinus. An endonasal endoscopic dacryocystorhinostomy (DCR) with osteoma removal by using a drill with temporary silicone stenting of the nasolacrimal duct system was performed. Due to a granuloma formation at the DCR-window site 2 months postoperatively a revision-DCR was performed and the new window remained patent at control 6 months after surgery. Acquired nasolacrimal duct obstruction (NLDO) may be due to various causes including idiopathic stenosis of the nasolacrimal duct, with or without formation of nasolacrimal duct mucoceles, trauma, or surgery to the paranasal sinuses and tumors of the nasal cavity.Osteomas are benign tumors developing in the paranasal sinuses and the nasal cavity. They usually grow in the frontal sinus near the nasofrontal duct and, in decreasing order of frequency, in the ethmoid, sphenoid, and maxillary sinus. They usually remain asymptomatic and they tend to be an incidental finding on radiographic studies. Rarely, they extend out of the sinus limits.To our knowledge, acquired NLDO associated with an osteoma of the medial wall of the maxillary sinus has not been reported in medical literature so far.Various methods such as laser beams, forceps, curettes, neurosurgical microrongeurs, and drills have been used for the formation of the lateral nasal wall osteotomy during endonasal dacryocystorhinostomy (DCR) . By meanA 74-year-old male patient presented to the outpatient department with left-sided epiphora and chronic dacryocystitis, without any record of head trauma or previous surgery of the nose or paranasal sinuses. On nasal endoscopy no abnormalities were noted. A diagnosis of saccal NLDO was made by clinical examination including lacrimal irrigation and fluorescein tests (Jones I and II tests) as well as probing of the canaliculi with a blunt probe up to bony contact with the lacrimal sac fossa performed by an ophthalmologist. The imaging studies (CT scan) revealed a 1 cm osteoma of the ventral half of the medial wall of the left maxillary sinus causing total obstruction at the level of the lacrimal sac Figures , and 4.Under general anesthesia, an endonasal endoscopic dacryocystorhinostomy (DCR) without flaps  using a A lacrimal duct system irrigation with natural saline solution into the inferior canaliculus was performed by an ophthalmologist once a week during the first month and twice a month during the second month. An endoscopic endonasal examination to test the patency of the duct system was performed 15, 21, and 30 days postoperatively. The silicone stent was removed 6 weeks postoperatively. An anatomical and functional obstruction of the DCR-window due to granuloma formation at the DCR-window area was noted 8 weeks after surgery on the combined nasal endoscopic and ophthalmologic examination. An endoscopic revision-DCR procedure with granuloma removal under local anaesthesia was performed. The DCR-window remained functionally and anatomically patent 6 months after surgery , with the maxillary and sphenoid sinuses as well as the nasal cavity being rare sites of occurrence (10%) . The cauSome possible causes of a maxillary sinus osteoma include trauma, previous surgery, inflammation, or developmental abnormalities. Given that there was no history of previous trauma, paranasal surgery, or chronic sinusitis for this particular patient, the cause of osteoma formation is unclear.In their early stages, osteomas of the paranasal sinuses are usually asymptomatic and are incidentally detected on imaging examinations for other conditions. Symptoms vary depending on tumor size and location. The most common symptoms include headache, facial pain, and nasal obstruction. Osteomas causing nasolacrimal duct obstruction with symptoms of epiphora or dacryocystitis are usually associated with the ethmoid sinus. A review of osteomas causing epiphora is summarized on In asymptomatic cases, excision is not always indicated and should be considered when the osteoma is responsible for occurring symptoms, if medical treatment of the symptoms is unsuccessful. In cases of osteoma extension beyond the sinus margins, surgical treatment is mandatory. Surgical approaches include external, endoscopic, and combined endoscopic and external procedures.In cases where a sinus osteoma causes obstruction of the nasolacrimal duct and in the management of the diseases of the lacrimal system in general, close cooperation between the ENT surgeon and the ophthalmologist is of major importance. The combined nasal endoscopic and ophthalmologic examination, before and after the excision of the osteoma and the treatment of the obstruction, will ensure the functional and anatomic success.


Posttraumatic intradiploic leptomeningeal cyst is an exceedingly uncommon complication of skull fracture in childhood with only about twenty-one cases described in literature till now. We report 2 such cases of intradiploic leptomeningeal cyst of occipital bone in two 17- and 21-year-old males presenting with headache with history of occipital bone fracture in childhood and briefly discuss its pathogenesis and differential diagnosis. A 17-year-old male presented with history of intermittent headache in the occipital region for 12 months, not relieved by medications. Patient also noticed a swelling in the occipital region which was progressively increasing over a period of 2 years. On examination, an ill-defined smooth hard lump was felt in the occipital region; however the rest of the physical and neurological examinations were normal. Patient gave a past history of head trauma at the age of 12 years due to fall from staircase. A CT scan of head performed at that time revealed linear undisplaced fracture in the midline involving both the inner and outer tables of the occipital bone Figures . The injPresent CT brain revealed a CSF density collection in the diploic space of the occipital bone with a break in the continuity of the inner table and expansion of the diploic space Figures . MagnetiA 21-year-old male with a past history of trauma 10 years back which was managed conservatively presented in neurology OPD with history of headache for 5 months. No significant finding was noted on clinical examination. As the pain was not getting relieved on standard medications, patient was referred for CT head. CT revealed widening of diploic space of the occipital bone caused by a CSF density lesion which was seen communicating with the cisterna magna through a break in the inner table with scalloping of outer table. No break in outer table was however noted Figures .Posttraumatic intradiploic leptomeningeal cysts are rare variants of growing skull fractures . The first case of PTIDLC was reported by Weinand et al. in 1989 , intraosPosttraumatic intradiploic leptomeningeal cysts are extremely rare complications of calvarial fractures occurring in paediatric patients –6. TheseThe most widely agreed-upon hypothesis proposes the herniation of leptomeninges into the intradiploic space through the traumatic rent in the dura mater and inner table. The ball and valve effect due to child's growing brain and the continuous CSF pulsations act as driving expansile forces facilitating the growth of the intradiploic cyst over the years with resultant thinning of the outer table , 7, 10. Communicating type of hydrocephalus is often seen to occur along with PTIDLC and may be attributed to the intraventricular haemorrhage at the time of initial trauma . A possiCommon neuroimaging includes a plain radiograph of skull showing eggshell expansion of diploic space with intact outer table. A Computed Tomography (CT) scan of head gives information about the extent of bony defect, intactness of the outer table, and three-dimensional reconstruction aids in surgical planning. Magnetic Resonance Imaging (MRI) of brain is the investigational modality of choice and helps in diagnosis of PTIDLCs by excluding dermoid and epidermoid cyst . In PTIDPTIDLCs need to be differentiated from leptomeningeal cysts (growing skull fractures). The later entity develops as a consequence of diastatic fractures causing laceration of dura mater as well as both the inner and outer tables , 13, 14.The other differential diagnosis includes intradiploic arachnoid cyst. These are probably congenital in origin and are formed because of obstruction to the flow of CSF from the arachnoid granulations into the venous system. These cysts usually present late in life with local pain, swelling, seizures, or neurological deficit. While radiologically it is difficult to distinguish between the two, a history of trauma and characteristic location in the occipital region favour the diagnosis of PTIDLC.Surgical management is the mainstay of treatment of PTIDLCs and indications for surgery include large disfiguring swelling and persistent headache , 15, 16.PTIDLC is a rare cause of headache and occurs secondary to calvarial fractures in paediatric age group. Knowledge about the entity is important for the radiologist and clinician as a treatable cause of headache.