Reviews of the literature on the health and work environment of ambulance personnel have indicated an increased risk of work-related health problems in this occupation. The aim of this study was to compare health status and exposure to different work environmental factors among ambulance personnel and the core work force in Denmark. In addition, to examine the association between physical and psychosocial work environment factors and different measures of health among ambulance personnel.Data were taken from a nationwide sample of ambulance personnel and fire fighters  and was compared to reference samples of the Danish work force. The questionnaire contained measures of physical and psychosocial work environment as well as measures of musculoskeletal pain, mental health, self-rated health and sleep quality.Ambulance personnel have half the prevalence of poor self-rated health compared to the core work force (5% vs. 10%). Levels of mental health were the same across the two samples whereas a substantially higher proportion of the ambulance personnel reported musculoskeletal pain (42% vs. 29%). The ambulance personnel had higher levels of emotional demands and meaningfulness of and commitment to work, and substantially lower levels of quantitative demands and influence at work. Only one out of ten aspects of physical work environment was consistently associated with higher levels of musculoskeletal pain. Emotional demands was the only psychosocial work factor that was associated with both poorer mental health and worse sleep quality.Ambulance personnel have similar levels of mental health but substantially higher levels of musculoskeletal pain than the work force in general. They are more exposed to emotional demands and these demands are associated with higher levels of poor mental health and poor sleep quality. To improve work environment, attention should be paid to musculoskeletal problems and the presence of positive organizational support mechanisms that can prevent negative effects from the high levels of emotional demands. Systematic reviews of the literature on the health and work environment of ambulance personnel have over the last decade emphasised what appears to be an increased risk of developing work-related health problems among this particular occupational group -3. EarliBecause of the stressful nature of emergency service work, most studies of ambulance personnel have addressed psychological health outcomes -13. PostIn Denmark, five regional counties have the responsibility for hospital and pre-hospital service. Contrary to several other countries, no hospitals in Denmark have their own integrated ambulance departments. Since 1963, there has only been one major ambulance service provider in Denmark. This private provider has covered most of the country apart from areas within four counties where the fire department also runs the ambulance service . Besides ambulance service, the company also performs the majority of other emergency services in Denmark, including fire fighting, animal rescue, patient transportation, roadside assistance, environmental assistance, and various forms of health care education and assistance to private people and public institutions. In 2008, Denmark’s five county governments have started putting ambulance service out to tender every fourth year. The first tender changed little in the overall structure of the Danish ambulance service, with one new private ambulance provider taking over some of the districts that had formerly been run by the original company. Despite this, the biggest ambulance provider still covers 85% of all emergency operations in Denmark. All in all, approximately 4500 persons are employed as ambulance personnel in Denmark. Each county assembles the pre-hospital service individually and puts the service to tender. The set-up and the running of the pre-hospital service differ slightly between the five Danish counties. Some counties have, for example, chosen a model with a specific permanent emergency standby, defining the numbers of ambulances, personnel, ambulance stations, etc. In other counties, there is no minimum permanent emergency standby. The ambulance service provider instead has the responsibility for ensuring specific maximum response times, but has considerable autonomy to set up the emergency standby as it finds it most appropriate to ensure the contractually required response times.Until the middle of the 1990s, Danish ambulance workers received little formal education. However, as the ambulances became increasingly equipped, and especially with the introduction of the defibrillator, a formal emergency worker/ambulance assistant (level 1) vocational education was introduced. Subsequently, an additional 5 week treatment provider (level 2) education was introduced, and in 2004, a formal 11 week paramedic (level 3) education was introduced. Hence, unlike many countries, Danish ambulances are not staffed by nurses. Danish legislation requires that ambulances must be staffed with at least two persons. At least one of these must be an educated treatment provider and the other must have passed an exam as an assistant provider. Most of the ambulance personnel perform more than one job function, e.g., rotating between emergency ambulance driving and transportation of patients over the work week. However, procurement rules require that companies that compete for tender must keep their sectional services separate in the daily operation. This has introduced a more rigid handling of jobs that could affect the work environment. The former board portfolio of jobs gave the local station-manager more flexibility to spare ambulance-workers by assigning them to less demanding tasks such as patient transportation for smaller or longer periods of time.The aim of this paper is threefold: 1) to compare various aspects of health status among Danish ambulance personnel with that of the general work force in order to establish whether prior findings of more musculoskeletal pain and more mental health problems in this occupation can be replicated; 2) to describe the general work environment among ambulance personnel and compare the psychosocial work environment to other occupations; and 3) to examine the associations between the physical and psychosocial work environments and five health outcomes.The data used in this paper stem from the first round of the cohort-study MARS , whose primary aim is to examine the impact of gender identity on risk-taking behaviour, attitudes towards safety, and occupational accidents. Data were collected on standard work environment factors and self-rated general health in order to shed light on some of the lesser known aspects of the work environment exposures in emergency service work. The participants in this study come from the biggest ambulance service provider in Denmark, covering 85% of all emergency calls.In total, 3,888 employees  covering 127 distinct emergency service stations were invited to participate in the study in October 2010. The participants had a choice to fill out the questionnaire either online or using a traditional post-distributed questionnaire. After 5 rounds of invitations and reminders a total of 2,426 completed the questionnaire fully or in part, yielding a response rate of 62.4%. Of these, 54.9%  used the online questionnaire and the remainder filled out the paper-based questionnaire.Examining the characteristics of the non-responders showed that the response rate among women was somewhat lower (56% vs. 63%), and that the older employees were more likely to participate compared to the youngest group. Finally, there were substantial differences across the 66 emergency service stations having more than 15 employees, with response rates ranging from 40.4% to 84.3%. Grouping the stations by geographical region revealed that there were slightly lower response rates in the capital area compared to some of the more rural parts of the country, but the differences were not that great (56% vs. 66%). All in all, the sample is appropriate to capture any differences between emergency departments located in urban and rural parts of the country and the non-responders were not substantially different from those who chose to participate.We asked the participants to indicate all the job functions they were currently carrying out. Additionally, we asked them to specify which of these functions occupied most of their working time. In this analysis, we included those indicating that they worked as emergency ambulance personnel n = 1,691). Figure ,691. FigData from a large national survey  conducted in September 2004 were used to make comparisons of the health status between ambulance personnel and the core work force in Denmark. The national survey contained the same health measures as the current study with only minor differences in response categories and wording. In the current study, participants were asked to indicate the level of pain in neck, shoulders and arms in one single measure, whereas this was asked in two items in the reference sample. The two items in the reference sample were then added up and the mean was taken in order to be able to compare it across samples. Similarly, the two items from the mental health dimension of SF-12 had 6 response categories in the current study and 5 in the reference sample. We collapsed two of the response categories in the current study and recoded the items to a single dichotomous variable indicating low level of mental health. The measures of low back pain and self-rated general health were identical in the two samples. Unfortunately, the reference sample did not include questions on sleep quality, disallowing us from comparing this health measure across samples.No samples were identified that would enable us to compare the physical work environment factors in the current study with the work force in general. Instead, a subsample of all those indicating fire fighting as a job function (n = 397) working within the same emergency service provider was used as an internal comparison group.The developers of the COPSOQ  provided reference material for the psychosocial work environment factors .Five different measures of health were used to assess the health status of the ambulance personnel. Self-rated general health was measured using the single item global measure taken from SF-36 . This va10 items from Dutch Musculoskeletal Questionnaire (DMQ)  were useThe short version of the Copenhagen Psychosocial Questionnaire (COPSOQ) was used to assess the psychosocial work environment among the ambulance personnel . This veFor use in the descriptive analyses and as potential confounder controls in the multivariate analyses, the following variables were included: age, sex, marital status, body mass index, alcohol consumption and level of physical activity. The potential confounders were selected because they could be associated with both the outcome and the work environment exposures, as in the case of age, sex, body mass index and physical activity where this is most likely with physical work environment and musculoskeletal pain. Marital status and alcohol consumption were included as confounders because they have been found to be associated with mental health and could also have an impact on how the respondents rate their psychosocial work environment. In order for the analyses to be consistent, the complete set of confounders was used for all five outcomes.To compare the health status and work environment of the ambulance personnel with the fire fighters and the reference samples, simple Student’s T-tests of independence and 95% Confidence Intervals were calculated. In order to examine the association between the physical and psychosocial work environment and the five different measures of health status, multiple logistic regressions were carried out. The variables were entered in two steps into two models. First, all physical and psychosocial work environment factors were entered into the regression model one at a time adjusting only for the set of confounders (Model 1). Only physical work environment factors were included as independent variables when musculoskeletal pain was the outcome. For mental health and sleep quality, only psychosocial work environment factors were included as independent variables. For the analysis of self-rated general health, both physical and psychosocial work environment factors were included as independent variables. The second model included all the other physical or psychosocial variables at the same time, still adjusting for the set of confounders. For each of the models, Hosmer Lemeshow Goodness of Fit and Nagelkerke R-Square are reported. In order to discuss the possibility of Type I errors influencing the results, we ran analyses with Benjamini-Krieger-Yekutieli corrected p-values using the smileplot procedure in STATA. All analyses were performed using STATA12 .Table In Table Table Table Table Finally, Table This study compares the health status and exposure to psychosocial and physical work environment factors of ambulance personnel with those of the general work force. In addition, analyses of the association between health and work environment factors were carried out to examine which aspects of the general work environment are important for the health status of workers in this occupation.Prior studies have found high prevalences of both musculoskeletal and mental health problems among ambulance personnel . A JapanDespite the known high levels of exposure to physical work demands required in ambulance work, only a few quality studies on this topic have been conducted on the issue of musculoskeletal disorders. This study shows that ambulance personnel are exposed to a wide variety of factors in the physical work environment including lifting in awkward postures, lifting loads that are hard to hold, performing short, maximal force-exertions and having too few facilities to lean on during work. Of the 10 factors, three were associated with higher levels of musculoskeletal pain after adjusting mutually for each other and for all the other confounder variables. Performing short maximal-force exertions was the only factor associated with both types of musculoskeletal pain. Although none of the estimates had p-values below the Benjamini-Krieger-Yekutieli corrected critical value, we wonder whether this may be too harsh a criterion on which to judge these estimates. Because of the dynamic character of the work, none of the exposures can be seen completely in isolation and for this reason, adjusting for all the other physical work environment factors at the same time may result in over adjustment.A Swedish study identifying physical risk factors as forward bending and twisting postures corresponded to our findings, while they in contrast also found handling of heavy tasks of significant importance . In multMost of the research on musculoskeletal risk factors focuses on heavy lifting, bending, etc. This study points to the need for more detailed knowledge of exposures, such as short maximal force –exertions and brief awkward postures. However, our findings on the ergonomic factors are solely based on questionnaire-derived exposure data, which have been shown to be vulnerable to misclassification due to musculoskeletal complaints, and interpretations must take this into account . ExposurDespite the finding that participants experienced more upper extremity pain and low back pain than the core work force, this cross-sectional study cannot elucidate causal relationships. In addition, only one of the specific items in the physical work environment was associated with both pain measures. This lack of consistency and the fact that the p-values did not reach below the critical values corrected for false discovery rates should stop us from drawing conclusions that are too unequivocal on this. The results could indicate the importance of different physical exposures in the occurrence of pain, but they might also reflect recall bias or Type I errors. Later prospective data will inform us of this in more detail.The current study points to two aspects of the psychosocial work environment where ambulance personnel experience more demanding work conditions than the general work force. Influence at work is substantially lower than in the rest of the work force, while the level of emotional demands is substantially higher. It is hardly surprising that emotional demands are higher and that the influence at work is smaller given the nature of most of the work tasks and the need for urgency related to them. However, the nature of the work serves as a barrier to improving the psychosocial work environment for this particular occupation.On the other hand, the levels of meaning of work and commitment to the workplace reported by the ambulance personnel show that the perception of the work as very important and as having an intrinsic meaning may serve as a buffer against the potential harms of an excessive level of emotional demands. This was the case in a Scottish study where those scoring high on subscales of commitment and control had significantly fewer burn-out symptoms . Other sThe study by Aasa et al. resembles the present one in the purpose of surveying psychosocial factors . They haA Dutch follow-up study found that ambulance personnel scored significantly higher on subscales measuring psychological emotional demands , which iMany studies report significant associations between job stressors and mental health problems, but without controlling for explanatory variables such as personality factors, coping and social support, reliable causal inference cannot be made. The main findings in the present study are the significant effect of emotional demands on mental health and sleep quality. Our study should contribute to the evidence base in the field of psychosocial work environment for workers employed in emergency rescue service, given our large representative study population, relevant comparison group, and control for confounders.The main weakness of the current study is the cross-sectional design. One crucial problem for the analyses is the possibility that health-selection is at work: from our qualitative studies of the company, we know that it has been a practice to transfer personnel with lower work ability to job functions other than ambulance driving. This could have the effect of causing the results of this study to underestimate the true association between the work environment exposures and health. The study, however, is designed as a prospective cohort study and future data will make it more appropriate for us to examine possible causal mechanisms linking different work-environment factors and health status among ambulance personnel more precisely.Nearly all studies of this occupation are of European or Anglo-Saxon origin, and it should be presumed that national differences in organisation, education, urban/rural exposure and many other factors reduce the potential for making generalisations .The differences in psychosocial work-environment factors were small compared to the workforce in general. The lack of contrast was also revealed as rather small associations in the multivariate analysis Table . It lookOur study shows that musculoskeletal problems should be a focus area in improving the work environment for ambulance personnel. Procedures and guidelines for safe lifting are important and are often specified, but adherence to them can be difficult under real life conditions. Therefore, attention should also be paid to the availability and development of new facilities for lifting heavy and difficult loads that would minimize manual lifting in awkward postures and especially the need to perform short maximum force exertions.The nature of emergency work makes it difficult to change the exposure to some of the psychosocial work environment factors, such as unpredictability and high emotional demands. To counteract the potential harmful influence of these factors, focus should be on ensuring the presence of positive organizational support mechanisms, such as influence, social support, organizational justice, and trust, which might serve as protective buffers against mental health problems.All authors declare that they have no competing interests.CH, KN and MK jointly conceived the idea for the paper. CH and JA performed the statistical analyses. All authors interpreted the data. CH, KR, KN and MK jointly drafted the manuscript. JA critically revised the paper. CH will act as guarantor for the paper. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/534/prepub


Macaca fascicularis) usage of the pointing cue. In our baseline condition, with the experimenter fully visible, the monkeys chose the correct container in 61% of the trials. The performance increased significantly, however, when the experimenter was hidden behind a curtain and only the arm of the experimenter, a doll's arm, or a stick was visible. Furthermore, the monkeys performed significantly better when the tip of the pointing finger or device was close to the target compared to the more distant condition. Intriguingly, after these experiments the monkeys' performance was also significantly improved in the baseline condition (70%). Apparently, the monkeys were first distracted by the presence of the experimenter, but then learned to use the cue. These findings highlight the importance of the test conditions, and question some of the assumptions about species-specific differences in the object-choice task.Methodological variations in experimental conditions can strongly influence animals' performances in cognitive tests. Specifically, the procedure of the so-called object-choice task has been controversially discussed; here, a human experimenter indicates the location of hidden food by pointing or gazing at one of two or more containers. Whereas dogs usually succeed, results for nonhuman primates are ambiguous. In the standard version of the task the majority of subjects do not respond appropriately to human pointing. However, modifying the task setup, such as placing the containers further apart, seems to improve subjects' performances, suggesting that cue salience may be an important variable. Here we investigated whether the visibility of the experimenter inhibits long-tailed macaques' ( Methodological variations can strongly influence animals' performances in cognitive experiments. Monkeys, for example, can discriminate quantities much better when the reward and the choice stimuli are separate entities than when they have to choose between different food amounts only One frequently used experiment in comparative cognition is the so-called object-choice task in which subjects are tested for the comprehension of communicative cues. In this experiment a human experimenter usually points at one of two (or more) cups or containers to indicate the location of a hidden reward see , and domThe results from nonhuman primates raised the question of the suitability of the object-choice task to explore and compare animals' socio-cognitive abilities. In its standard version used with nonhuman primates, two cups are placed in-between the subject and the experimenter in relatively close distance to each other. In this setup most nonhuman primates fail to choose the cup pointed at (with the exception of highly enculturated apes Macaca mulatta) tested in the field rather than under standard lab conditions used human pointing cues to locate a food reward In addition to this distance effect, other factors may also influence primates' usage of human communicative cues in an object-choice paradigm. Rhesus macaques , a monkey species performing similar to apes in a range of physico- and socio-cognitive experiments In this study we aimed to achieve a better understanding of the procedural constraints and cognitive mechanisms influencing the usage of human pointing cues in long-tailed macaques .We tested 10 long-tailed macaques - 4 males and 6 females aged 2 to 8 years - at the German Primate Center in Göttingen  living in a social group of 28 animals. The monkeys had access to indoor (25 sqm) and outdoor enclosures (141 sqm). They were individually tested in a small cage  connected to a testing compartment  inside their familiar indoor area. The subjects were free to leave the cage and enter the testing compartment at any time during testing. Tests were conducted once or twice per day and participation was voluntary, that is, dependent on the monkeys' willingness to enter the test compartment. The subjects had already participated in a battery of tasks assessing their socio- and physico-cognitive capacities Two blue opaque cups (Ø 5 cm×5 cm height) were used to hide the reward (raisins or peanuts). The cups were fixed on a sliding board (distance 30 cm) with hinges so that the monkeys could lift them from the front revealing whether a food reward was hidden underneath or not. The sliding board was attached to a fixed table  so that the sliding board could be moved horizontally. The table was attached to a plastic panel, which had two oval openings at the outer sites  to allow the subjects to put one hand through and reach for a cup see .To hide the experimenter we put up a large black curtain (2 m×1.50 m) in front of the testing apparatus. We cut a small hole into the middle of the curtain to allow inserting an arm or stick to point at a cup. Furthermore, we attached a baton to the end of the sliding board, which passed through the curtain and allowed to move the table back and forth from behind the curtain.To point at the cups we either used the arm of the experimenter, a grey coloured stick (Ø 4 cm×80 cm length) or a left and right arm of a regular fashion mannequin (Ø 30 cm×80 cm length), referred to as doll in the remaining text. From behind the curtain it was possible to lower down an occluder of black cardboard (70 cm×100 cm), hanging from the ceiling, to prevent the subjects from seeing the experimenter during the baiting procedure. The experimenter coded live which cup the monkey chose first. The choice was always unambiguous as they only reached for one cup at a time. In addition, all sessions were videotaped (Sony DCR-HC90E) and a second observer coded 20 percent  of all videos resulting in a complete concordance with the initial coding.cross pointing, Before the experiment started the tested subject was separated from the group in its familiar testing cage and received some food rewards to control for its general motivation to participate in the experiment. The experimenter then lowered down the occluder and baited one of two opaque cups out of view of the subject. In the test conditions the experimenter then stepped back behind a large curtain for the remaining session, remotely lifted the occluder from behind the curtain and put her arm, a stick, or the arm of the doll through a hole in the middle of the curtain to point at the baited cup. She always used the arm contralateral to the baited cup, i.e. the left arm to point at the right cup and vice versa .human), the arm of a doll (doll), or a stick (stick) through the curtain to point at the baited cup.After baiting, the experimenter stepped behind the curtain and lifted the occluder. She then either put her own arm  with proximal and six 9-trial sessions with distal cueing. Each session contained 3 trials of each test condition  resulting in 18 trials per condition for the distal and for the proximal variant each. The order of conditions was pseudo-randomised and balanced within and across individuals.After the completion of all experimental conditions, all subjects were re-tested, using the same procedure as in the baseline condition, with the experimenter present. Each subject received two 9-trial sessions with proximal and distal cueing each.Data were analysed using Statistica 9.1  and SigmaPlot 11.0 . As the data were not normally distributed  and the sample size was rather small, we used non-parametric statistics to analyse the data. We used exact, two-tailed Wilcoxon tests to determine whether the performance of the monkeys was above chance level (i.e. 50% correct) in the different conditions. We conducted Friedman ANOVAs to test whether the monkeys' performance differed between the four different conditions , in the proximal and distal cueing conditions. We conducted Post hoc tests (Holm-Sidak method) in case of a significant result. As one monkey participated in the proximal conditions only, we excluded her from this analysis, thus comparing the results of 9 monkeys only. To test whether the monkeys' performance had increased in the re-test condition, we compared these results to the baseline condition using Wilcoxon's exact test.control condition  and thus did not use any cues like smell or sight to find the reward. In the baseline condition, the monkeys performed at chance level with distal cueing  and only slightly better than chance with proximal cueing   and better than chance in the distal stick condition , but in none of the other distal conditions  but not with distal cueing . Post-hoc tests in the proximal conditions revealed a significant difference between the baseline and doll condition (p<0.001) and the baseline and human condition (p = 0.033), but no significant difference between the baseline and the stick condition (p = 0.095). There were no significant differences between the different cue-providing elements within these test conditions .In the baseline and test conditions, Friedman ANOVAs revealed a significant difference between the conditions with proximal  . AlthougOur results show that the performance of the monkeys increased significantly when the experimenter stood behind the curtain and only the pointing cue was visible to the subjects, suggesting that the presence of the experimenter indeed distracted the monkeys in the normal object-choice pointing test. This may be due to two aspects: first, the visual absence of the experimenter may have reduced potential effects of fear (as they may have perceived the experimenter as a competitor), and second, the pointing cue may have been more conspicuous when the rest of the person stood behind the curtain.We believe that our findings are in line with the distraction hypothesis proposed by Mulcahy and Call Our results further suggest that social inhibition may in part explain the failure of primates to use human cues. When the experimenter was hidden behind the curtain, the performance improved considerably, supporting the hypothesis that the monkeys might have been afraid of the experimenter's appearance. Unfortunately, we could not systematically analyse fear responses in our monkeys as these rather subtle reactions were not visible from the videos, but future experiments could try to have a closer look at this potential confounding factor. Even though the experimenter's entire body was hidden in our experiments, the results of previous studies suggest that monkeys are most responsive to the orientation of the head and the eyes; for instance, nonhuman primates preferentially steal food from an experimenter not facing the animals This finding also contributes to the discussion on the superior performance of dogs and other domesticated or trained species in using human communicative cues. Some researchers suggested that through domestication dogs did not acquire special socio-cognitive skills but that learning was facilitated, especially through the reduction of fear and an increased tolerance to the sight of humans Furthermore, we are positive that the procedure, and not learning through a relatively high number of trials during the course of the experiment accounted for the increased performance. We did not see an increase in performance within the first baseline sessions , but the monkeys performed immediately better in the following test conditions . Similarly, also in the studies of Mulcahy and colleagues and gazed at the correct location to enhance the communicative context of the experiment. More specifically, it was argued that establishing eye contact is a necessary prerequisite to effectively use a pointing cue (see In some previous object-choice studies, the experimenter pointed  cue see . The com cue see . In our Similarly, if the monkeys had understood the communicative intent, they should have performed equally in the distal and the proximal conditions, as the distance between the cup and gesture should not matter that much, but this was not the case (see Without additional gazing, proximal pointing is the cue most successfully used by nonhuman primates (e.g. Taken together, when distractors are removed, long-tailed macaques learn relatively quickly to use human pointing cues to locate hidden food, and they can transfer this knowledge to more complex situations, i.e. when the human is visible again. Reducing the ambiguity not only of the cues but of the entire experimental situation apparently facilitates the usage of human communicative cues in nonhuman primates.In sum, our results suggest that the presence of the experimenter can distract nonhuman primates in the object-choice task. Furthermore, in our study simple local enhancement was sufficient to associate the cue with the baited cup. There was no difference in performance concerning social vs. non-social cues, suggesting that the social aspect may not play a major role in the acquisition of this association. These findings highlight the importance of the test conditions, and question the assumption that species-specific differences in the object-choice task are only due to cognitive dissimilarities.


Although many methods have been proposed to reconstruct gene regulatory network, most of them, when applied in the sample-based data, can not reveal the gene regulatory relations underlying the phenotypic change . In this paper, we adopt phenotype as a variable when constructing the gene regulatory network, while former researches either neglected it or only used it to select the differentially expressed genes as the inputs to construct the gene regulatory network. To be specific, we integrate phenotype information with gene expression data to identify the gene dependency pairs by using the method of conditional mutual information. A gene dependency pair  means that the influence of gene A on the phenotype depends on gene B. All identified gene dependency pairs constitute a directed network underlying the phenotype, namely gene dependency network. By this way, we have constructed gene dependency network of breast cancer from gene expression data along with two different phenotype states (metastasis and non-metastasis). Moreover, we have found the network scale free, indicating that its hub genes with high out-degrees may play critical roles in the network. After functional investigation, these hub genes are found to be biologically significant and specially related to breast cancer, which suggests that our gene dependency network is meaningful. The validity has also been justified by literature investigation. From the network, we have selected 43 discriminative hubs as signature to build the classification model for distinguishing the distant metastasis risks of breast cancer patients, and the result outperforms those classification models with published signatures. In conclusion, we have proposed a promising way to construct the gene regulatory network by using sample-based data, which has been shown to be effective and accurate in uncovering the hidden mechanism of the biological process and identifying the gene signature for phenotypic change. In order to understand the regulatory mechanism in biological process, it is important to identify gene regulatory network (GRN) by using gene expression data. A lot of methods have been proposed to solve this problem, such as Bayes network These days, many approaches based on mutual information (MI) from information theory Gene dependency is common in biological process. For example, the activities of many transcription factors regulating their targets depend on other modulators In this work, we propose a novel method to construct the gene dependency network underlying the alteration of the phenotype, by integrating the phenotype information and the gene expression data. We first apply conditional mutual information (CMI) to identify the gene dependency pairs underlying the phenotypic change . We then use permutation test to select the significant pairs. Finally we construct the gene dependency network by combining all pairs with significant gene dependency relationships.Breast cancer has been widely researched and many multi-gene markers In this work, we first construct the gene dependency network of breast cancer by integrating the gene expression data and the corresponding metastasis clinic information. Then we functionally analyze the hub nodes in the network to investigate their relations with breast cancer and cancer metastasis. Finally, we select the discriminative hub genes to form the signature to distinguish the distant metastasis risks of breast cancer patients in six breast cancer data sets.Gene expression data sets of breast cancer and the corresponding clinic information were downloaded from NCBI  with accession number GSE2034 In order to construct the gene dependency network, we first discretized the clinic information and the gene expression levels. If the distant metastasis occurred within 5 years, we set the phenotype status as 1; if the distant metastasis occurred after 5 years, we set the phenotype status as 0; the other data were abandoned. The gene expression value was set to 0 if it was lower than the median of the gene expression levels of all samples; otherwise, it was set as 1.The human protein interaction data was downloaded from HIPPIE (Human Integrated Protein-Protein Interaction rEference) Our work aims at identifying the gene dependency pairs such that the relationship between one gene (denoted as A) and the phenotype  depends on another gene (denoted as B). In this case, we say that gene A depends on gene B in the context of phenotype P. In this work, we used CMI, calculated by the way similar to We used permutation method to evaluate the significance of every gene-gene dependency relation. For each candidate gene pair , we first calculated its real CMI value as described above. Then we randomly permuted the expression level of gene A and the phenotype state value across the samples 1000 times and calculated out 1000 CMI values as the null hypothesis distribution. Then the order (descending) of the real CMI value in the null hypothesis distribution divided by 1000 was taken as the significance p-value of . Finally we used a threshold (0.05 in this work) to decide whether the pair was significant or not.The significant gene dependency pairs were preserved to construct the gene dependency network, in which, nodes are genes, and the directed edge (B→A) represents the dependency relationship of gene A on gene B, with CMI value being the corresponding edge weight.The flow chart of network analysis is shown in In the gene dependency network, the out-degree of a gene indicates the number of genes related to phenotypic alteration that it influences. Therefore, the genes with higher out-degrees should be more critical to the phenotypic change. In this work, we selected the top 20% genes with the largest out-degrees (not less than 4) as hub genes of the network for further analysis.In order to find out the biological functions of hub genes, we used DAVID To select the genes significantly related to the distant metastasis risks of the patients, we adopted the strategy similar with ScoR ix (or jx) represents the expression level of the gene i (or gene j) which has positive (negative) Cox coefficient with the metastasis risk.In order to evaluate the distant metastasis risk of a patient, we calculated the Risk Score for each patient based on following formula by using the determined signature genes. This method is similar to the strategy of Gene expression Grade Index (GGI) http://www.mathworks.com/matlabcentral/fileexchange/22317-logrank).Following the general way, we divided the patients in a data set into two groups with the same size. That is, we assigned 50% of the patients with lower risk scores into the low risk group and vice versa. After that, we used log rank test to test if the risks of the two groups were significantly different . Kaplan Meier curves and the log rank test were performed by a tool  as follows:Where n is the number of the data sets, and The gene dependency network was visualized by Cytoscape 2.8.2 and the topology analysis was performed by the Network Analyzer plug-in for Cytoscape We used GSE2034 to infer the gene dependency pairs underlying the breast cancer distant metastasis. As a result, we got 17,511 significant gene dependency pairs  involvinAmong the 1,281 hub genes, 380 (about 30% of hub genes) genes are in the breast cancer related gene set , includiBy enrichment analysis, 64 KEGG pathways are shown to be significant with the hub genes (with FDR less than 0.05) . Among tIn order to validate the inferred gene dependency relations, we checked whether the relations between famous cancer genes have been reported before.As we know, TP53 is one of the most famous cancer genes and its mutation has critical influence on cancer prognosis MYC is deregulated or overexpressed in most cancer cells and MYC inhibition is a therapy for cancer In the dependency network, another famous breast cancer gene BRCA1 has a significant dependency relation with gene JAK1 (Entrez ID: 3716) , while BRCA1 has been reported to up-regulate JAK1 to govern cellular proliferation, differentiation, apoptosis and transformation; and all these processes are involved in breast tumorigenesis In conclusion, the literature has confirmed the validity of some gene dependency relations. The validation of the gene dependency relations together with the significant biological meanings of the hub genes may indicate that the gene dependency network can indeed reveal hidden mechanism of cancer metastasis.To understand the metastasis mechanism, it is essential to identify gene signature that can distinguish the metastasis risks of cancer patients. Many gene signatures have been identified to solve this problem, however, few of them are satisfactory because of the poor generalization In the gene dependency network, a gene pair indicates that the relation between one gene and the distant metastasis risk depends on another gene. Thus, the distant metastasis-specific gene dependency network may be more likely to identify the hidden driver genes that influence the metastasis of cancer patients by modulating other passenger genes, which is very different from other methods that select the genes directly associated with the outcome. Therefore, the hub genes in the constructed network are more likely to distinguish metastasis risks of cancer patients than those selected through other methods.Among 442 genes significantly correlated with the metastasis risks  , 43 geneTo investigate the robustness of the signature, we did similar discrimination test on other six independent data sets as well. The results are shown in From 442 genes significantly correlated with the metastasis risks , we chosConsidering most published signatures on independent test sets have been argued to be very poor, even not significantly better than random signatures with the same sizes In the first test, we randomly selected 43 genes from the whole gene set  to form a random signature, and the patients from each of the six independent data sets were divided into good and bad prognosis groups according to the metastasis risks computed via the random signature, based on which Dscore was calculated. The above procedure was repeated 1000 times to obtain 1000 Dscore values. As a result, we found the Dscore followed a chi-squared distribution , which iIn the second test, we randomly selected 43 genes from 442 genes that were significantly associated with the metastasis risk  and perfIt has been argued that most published signatures are not significantly better than the random signatures Although many methods have been proposed to construct gene regulatory networks, few of them investigates the gene dependency relationships specific to phenotypic change that are common in biological process. In this work, we have proposed a novel methodology to infer the gene dependency pairs underlying the specific phenotypic change. Concretely, we applied CMI to identify the gene dependency pairs, each of which indicates that the correlation between one gene and the phenotype is dependent on another gene. All the significant gene pairs are combined together to construct the gene dependency network that could characterize the gene regulatory mechanism underlying the alteration of the phenotype. When applied to sample based gene expression data of breast cancer (with distant metastasis or non-distant metastasis clinic information), the network has been demonstrated to be able to uncover the biological mechanism in the metastasis process. The hub genes in the network have significant intersection with the breast cancer related genes, and the functional analysis shows that the hub genes in the network are biologically significant. Moreover, many gene dependency relations reported previously in literature can be detected in the network. Furthermore, the hub-based signature can distinguish the cancer prognosis of breast cancer patients with robust performances across various independent data sets, which is significantly superior to most published gene signatures that have been argued to perform badly in the independent tests and even not to be significantly better than the random signatures The good performance of hub-based signature may owe to the following merits of our method. First, we directly included the phenotype information in the inference of the gene dependency relations, thus the gene dependency network can reveal the gene regulatory relation during the alteration of the phenotype. Second, gene dependency network focuses on modulators that can influence the phenotype via other genes, so the network can uncover the hidden biological mechanism that may be ignored by former researches. Therefore, the hub genes modulating the largest number of genes may be the key genes influencing the distant metastasis of breast cancer and have robust discrimination capability across different data sets.As illustrated from the results, our methodology can reveal the hidden mechanism in the metastasis of breast cancer. In fact, it can also be applied in other fields, such as the mechanism in the disease progress, embryo development, and other aspects as long as there are enough gene expression data and the corresponding phenotype information. In this work, our methodology succeeded in the binary phenotype data sets (metastasis and non-metastasis). However, it is also suitable for the data sets with more than two phenotype states.Of course, the methodology proposed in this paper only works on the sample based data with more than one phenotype states. In the case of single phenotype state, for example, only non-metastasis samples, our method fails to work. In addition, the calculation of CMI would be inaccurate if there are not enough samples in the data set. These limitations should be addressed in future study.Figure S1The power law fit of the in-degree.(TIF)Click here for additional data file.Figure S2The power law fit of the out-degree.(TIF)Click here for additional data file.Table S1Data sets used in this work. All the data sets are available at NCBI GEO.(XLSX)Click here for additional data file.Table S2All significant gene dependency pairs. All the gene pairs in the table are with p-values less than 0.05. Genes in the first column are the modulators of the gene pairs.(XLSX)Click here for additional data file.Table S3The selected hubs in the network. The genes with the highest out-degrees are selected as hubs .(XLSX)Click here for additional data file.Table S4Enriched pathways. The significant enriched pathways in the table are with Benjamini FDRs less than 0.05.(XLSX)Click here for additional data file.Table S5All the candidate genes which are significant with the metastasis risk of breast cancer. A gene is selected as a candidate when it is significant in 90% of the 400 runs.(XLSX)Click here for additional data file.


The study aims to introduce a hybrid optimization algorithm for anatomy-based intensity modulated radiotherapy (AB-IMRT). Our proposal is that by integrating an exact optimization algorithm with a heuristic optimization algorithm, the advantages of both the algorithms can be combined, which will lead to an efficient global optimizer solving the problem at a very fast rate. Our hybrid approach combines Gaussian elimination algorithm (exact optimizer) with fast simulated annealing algorithm  for the optimization of beam weights in AB-IMRT. The algorithm has been implemented using MATLAB software. The optimization efficiency of the hybrid algorithm is clarified by (i) analysis of the numerical characteristics of the algorithm and (ii) analysis of the clinical capabilities of the algorithm. The numerical and clinical characteristics of the hybrid algorithm are compared with Gaussian elimination method (GEM) and fast simulated annealing (FSA). The numerical characteristics include convergence, consistency, number of iterations and overall optimization speed, which were analyzed for the respective cases of 8 patients. The clinical capabilities of the hybrid algorithm are demonstrated in cases of (a) prostate and (b) brain. The analyses reveal that (i) the convergence speed of the hybrid algorithm is approximately three times higher than that of FSA algorithm; (ii) the convergence (percentage reduction in the cost function) in hybrid algorithm is about 20% improved as compared to that in GEM algorithm; (iii) the hybrid algorithm is capable of producing relatively better treatment plans in terms of Conformity Index (CI) [~ 2% - 5% improvement] and Homogeneity Index (HI) [~ 4% - 10% improvement] as compared to GEM and FSA algorithms; (iv) the sparing of organs at risk in hybrid algorithm-based plans is better than that in GEM-based plans and comparable to that in FSA-based plans; and (v) the beam weights resulting from the hybrid algorithm are about 20% smoother than those obtained in GEM and FSA algorithms. In summary, the study demonstrates that hybrid algorithms can be effectively used for fast optimization of beam weights in AB-IMRT. Recently, there has been a growing interest in aperture-based inverse planning (ABIP) for IMRT, as ABIP can significantly reduce the number of segments and monitor units.2 This isThe simulated annealing method simulates the slow cooling of a sample to find low-energy states. This technique has been applied to problems in radiotherapy, especially in IMRT.–5 RecentOn the other hand, several very efficient exact optimization algorithms have been developed in recent years.–12 TheseIn this work, a simple and efficient optimization algorithm for AB-IMRT, called “hybrid algorithm,” is introduced in response to the drawbacks mentioned above. Our proposal is that by integrating an exact optimization algorithm with a heuristic optimization algorithm, the advantages of both the algorithms can be integrated into the created hybrid algorithm, which will lead to an efficient global optimizer solving the problem at a very fast rate. Our hybrid approach combines Gaussian elimination method (GEM) (exact optimizer) and fast simulated annealing (FSA) algorithm  for the optimization of beam weights in AB-IMRT. The numerical and the clinical characteristics of the hybrid algorithm are compared with those of the GEM and FSA algorithms. In the numerical analysis, the numerical characteristics of the hybrid algorithm, such as convergence, convergence rate, consistency, number of trails and overall optimization speed, were analyzed for 8 patients in comparison with those of the GEM and FSA algorithms. The clinical capabilities of the hybrid algorithm were demonstrated in (i) prostate and (ii) brain cases.We used a simple anatomy-based segmentation method–5 for maThe proposed hybrid approach combines GEM algorithm (an exact approach) and FSA algorithm (a heuristic approach) for the optimization of beam weights in AB-IMRT using a quadratic dose-based cost function. In linear algebra, GEM is a powerful algorithm that can be used to determine the exact solutions of a system of linear equations.–17 Recen® (4.3.1) treatment planning system, and the same system is used for dose calculation as well. In this planning system, the dose is calculated using a fast convolution superposition algorithm./DprescribedHI = [Dmin (dose to 2% of the PTV), Dmax (dose to 98% of the PTV) and Dprescribed are the minimum, maximum and prescribed doses, respectively.where DThe numerical analysis shows Figures –5 that tThe convergence is about 20% improved in hybrid algorithm as compared to that in GEM algorithm and is comparable to that in FSA algorithm in most of the cases. In our study, the convergence was measured by the percentage reduction in the cost function for a particular case. In other words, the more the percentage reduction in the cost function, the better is the convergence of the optimization algorithm. But the percentage reduction in the cost function value does not necessarily mean an equal amount of improvement in the required outcomes. However, the dose distributions obtained in the 8 patient cases used for the numerical analysis indicate that the plan with lower final cost function value appears to have apparently better dose distribution as compared to the plan with higher final cost function value. This feature is common for all the 8 patients cases included in the numerical analysis and 2 patient cases included in the clinical analysis. This observation confirms that in a single-criterion optimization, the reduction in the cost function can be used as an approximate indication of the corresponding improvement in the dose distribution. The numerical analysis also points out that an algorithm having relatively good convergence characteristics will obviously lead to better dose distribution.The hybrid algorithm was used to generate an AB-IMRT plan for a prostate case. In this case, we planned a dose of 57.6 Gy for the gross disease in a single phase comprising 37 fractions. The gross disease was delineated using CT images and was defined as clinical target volume (CTV). The planning target volume (PTV) was drawn with a 3-mm margin to the CTV. The volume of PTV was 160 cc. The geometry of the PTV was very complex as it was overlapping on the nearby rectum and bladder volumes. Moreover, we wanted to restrict the dose to bladder and rectum as much as possible. Because of the geometric and dosimetric complexities, we considered to execute AB-IMRT plan instead of 3DCRT plan for this case.The summary of the treatment goals for this case is given in The case is a typical brain lesion of volume 308 cc (PTV). A total dose of 50.4 Gy in 25 fractions was prescribed for the PTV in this case. The sensitive normal structures such as brainstem, optic nerves and eyes were close to the target volume. Especially, we wanted to restrict the dose to the portion of the brainstem volume which was not overlapping on the target volume (rest of the brainstem), while maintaining good dose coverage to the PTV. Hence it was decided to go for AB-IMRT plan instead of 3DCRT. The summary of the treatment goals for this case is given in First, the comparison of the numerical capabilities of the hybrid algorithm with those of the GEM and FSA algorithms as shown in Figures The dose distribution and DVH comparisons demonstrate that the plan obtained using hybrid algorithm offers better PTV dose conformity and dose homogeneity as compared to the plans obtained using GEM and FSA algorithms. On an average, one can observe about 2% to 5% improvement in dose conformity and 4% to 10% improvement in dose homogeneity in plans optimized using hybrid algorithm as compared to GEM- and FSA-based plans. From Figures The plan quality obtained with hybrid algorithm is improved as compared to that obtained with GEM-based algorithm, which is due to two reasons: First, the GEM component in the hybrid algorithm gets the initial set of solutions, which when fed into the FSA component drives the heuristic process towards the goal in an effective way. Secondly, the simulated annealing component in the hybrid algorithm escapes the solution from the possible local minima during the optimization. The general observation is that the dose conformation to the tumor is very good in both the cases; and furthermore, the dose gradient in the region proximal to the OAR is steep, ensuring a good OAR protection.Moreover, Figures There is an extensivedebate about whether the cost functions really possess local minima, and whether they are sufficiently close to the global minimum so that the finding of global minimum becomes insignificant. However,A hybrid optimization algorithm for anatomy-based IMRT (AB-IMRT) is introduced, which integrates an exact solver (GEM) with a global optimizer (FSA) in order to get better solutions at faster rate. For the cases presented, the implemented hybrid optimizer was able to produce treatment plans comparable to FSA-based plans in terms of target coverage and OAR-sparing with a remarkable improvement in the optimization speed . We believe that such an improvement in optimization speed will lead to highly efficient workflow in AB-IMRT optimization, which in turn can be helpful to produce better treatment plans.


For this reason ophthalmic drug delivery, particularly targeted to posterior segment, is one of the most challenging endeavors facing the ocular pharmacologists. Topical route represents a safer administration, therefore a major challenge to the scientists is to overcome the ocular barriers and reach the tissue target. Systemic route  is also used to reach the eye, though the drug transport across the ocular barriers  is quite difficult.Topical administration is the most common route of ocular drug delivery. Despite its apparent easy accessibility, the eye is well protected from foreign materials and drugs by a number of very efficient mechanisms such as blinking, induced lacrimation, tear turnover, nasolacrimal drainage, which cause rapid removal of substances from the eye surface and by the cornea, which forms the physical-biological barrier Figure . ConsequUnder normal condition the human eye can hold about 25–30 μl of an ophthalmic solution; however after a single blink the volume is reduced to 7–10 μl through nasolacrimal drainage which cause the drug to be systemically absorbed across the nasal mucosa or the gastrointestinal tract. A significant systemic loss from topically applied drugs also occurs from conjunctival absorption into the local circulation Figure . In ordeThe success of nanoparticle systems for ocular drug delivery may depend on optimizing lipophilic-hydrophilic properties of the polymer-drug system, optimizing rates of biodegradation, and safety. Polymers used for the preparation of nanoparticles should be mucoadhesive and biocompatible. The choice of polymer plays an important role in the release kinetics of the drug from a nanoparticle system. Ocular bioavailability from a mucoadhesive dosage form will depend on the polymer’s bioadhesion characteristics, which are affected by its swelling properties, hydration time, molecular weight, and degree of crosslinking. The binding of drug depends on the physicochemical properties of the molecule as well as of the nanoparticle polymer, and also on the manufacturing process for these nanoparticle systems. The polymers used in ophthalmic drug formulations are poly (PACA)  will bring, very soon, to a clinical use of these innovative nanosystems for the pharmacological management of sight-threatening eye diseases.


Vibrio harveyi (VH) which is infected with the VHS1 (Vibrio harveyi Siphoviridae-like 1) bacteriophage. The lysogenic Vibrio harveyi undergoes an unequivalent division of the extra-chromosomal VHS1 phage genome and its VH host chromosome and produces a true lysogen (TL) and pseudolysogen (PL). The PL is tolerant to super-infection of VHS1, as is of the true lysogen (TL), but the PL does not contain the VHS1 phage genome while the TL does. However, the PL can become susceptible to VHS1 phage infection if the physiological state of the PL is changed. It is postulated that this is due to a phage receptor molecule which can be inducible to an on-and-off regulation influence by an alternating condition of the bacterial host cell. This characteristic of the PL leads to speculate that this phenomenon can also occur in high organisms with low immunity such as shrimp. This article proposes a hypothesis that the viral receptor molecule on the target cell can play a crucial role in which the invertebrate aquaculture animals can become tolerant to viral infection. A possible mechanism may be that the target cell disrupts the viral receptor molecule to prevent super infection. This concept can explain a mechanism for the prevention of viral infection in invertebrate animals which do not have acquired immunity in response to pathogens. It can guide us to develop a mechanism of immunity to viral infection in low-evolved-immune animals. Also, it can be an additional mechanism that exists in high immune organism, as in human for the prevention of viral infectionA pseudolysogen (PL) is derived from the lysogenic  Vibrio harveyi which is infected with its specific temperate phage called VHS1 (Vibrio harveyi Siphoviridae-like1) [Lysogenic infection of the phage-bacterium relationship is considered similar to persistent infection. According to the lambda phage, its life cycle of lytic and lysogenic infection is dependent mainly on the role of the cI and Cro proteins. The predomination of cI protein maintains the phage in lysogenic stage while the Cro protein dominates the lytic infection ,2. It ise-like1) . It is eIt is well accepted that lysogen can tolerate  a super-infection to the same phage. The explanation for this phenomenon has never been clearly explained. Besides, there are at least three different definitions of PL. The earliest report of PL concerns those infected cells that produce a defective phage genome as proposed by Cambell . The othImmunity to a second phage super-infection in lysogenic bacteria is similar to the interference phenomenon of the eukaryote; the target cell which is infected with the first virus would not be infected with the second viral infection. This can happen either among the same virus or the same subgroup  ,10. It aOn the other hand, the interference phenomenon is not always true since dual viral infection in the same cell has been reported ,23. ThisHence, we present the hypothesis based on the inducible viral receptor concept that the host cell disrupts the expression of the receptor molecule after the first virus enters the cell. This mechanism can prevent not only the same virus entry  but also any other viruses  that use the same viral receptor for entry into the target cell ,27. If tMonodon baculovirus [In the mean time, there were independent observations in the shrimp aquaculture industry concerning the persistent infection of various viruses such as ulovirus , Yellow ulovirus ,31, Whitulovirus ,33 and Tulovirus . These vulovirus ,31. Howeulovirus . Thus, tulovirus . Additioulovirus ,34. Instulovirus ,34,35. Iulovirus . The mecTo explain the persistent viral infection in shrimp by the inducible receptor hypothesis; it can be explained that after viral infection into the cells of the target organ, the neighboring cells which are not infected process a mechanism to disrupt the viral receptor molecule so the virus cannot attach to them. This can prevent the virus to spread to the entire organ of shrimp to cause death. It should be mentioned that these persistent viral infected shrimp do not show any gross sign of pathogenesis of persistent viral infection and the virus can be detected by only the sensitive techniques such as the polymerase chain reaction (PCR) ,37. ThisIn addition to using the viral inducible hypothesis to explain the appearance of PL in the VHS1-infected VH and the persistent viral infection in shrimp, the concept can be applied to interpret the report of Burivong et al  which coBased on the inducible viral receptor concept, the result of Burivong et al ,39 couldIn higher animals and humans who possess acquired immunity, there are some examples of viral infection which can be explained by the viral inducible receptor hypothesis. In case of persistent chronic hepatitis B carriers, the virus particles are produced continuously without any evidence of disease but cause pathological transmission to other humans ,47. HoweThis paper presents an additional theory of an inducible viral receptor concept that the cell, in general, can adapt itself to prevent the viral infection. This process should be named as tolerant, instead of resistant, mechanism since the cell loses its prevention mechanism if exposed to inappropriate conditions such as low resources and toxic substance. This inducible viral receptor hypothesis can be used to explain the incidences by (1) the VH-PL phenomenon, (2) the toleration to viral infection in shrimp (3) the alternative interpretation of the reports of the Densovirus persistent C6/C36 cell and (4) the persistent infection of the Hepatitis B viral particle in the carriers. Mainly, it explains that each individual cell has an ability to learn to prevent the secondary viral infection by itself after the primary viral infection. This mechanism can explain the phenomenon of the viral toleration in either the low or high immune organism. In high immune organisms, the mechanism might be the additional pathway to help the memory lymphocytes to respond to the latter viral infection more promptly and effectively. In case of the primitive immune animals which lack lymphocyte to create the memory lymphocyte to respond to prevent the secondary viral infection, the phenomenon of the inducible viral receptor can explain the incidence of viral protection by the cell toleration as witness from the incidences in VH and shrimp. Moreover, it can explain why some researchers found that vaccination is possible in shrimp which possess lower immunity and do not have lymphocyte to create adaptive immune response and memory cells. However, this hypothesis still cannot explain it all especially the dual or multiple infection of the viruses in the same cell. I do wish more researchers would investigate the possibility of this concept.Vibrio harveyi; VHS1: Vibrio harveyi siphoviridae-like 1 phage; TL: true lysogen.PL: pseudolysogen; VH: The author declares that he has no competing interestsTP prepares and writes up the whole manuscript. He also read and approved the final manuscript.


Calcium imaging has become a standard technique for the measurement of the activity of a population of cultured neurons. Typically these recordings are slow compared to the cell dynamics and display a low signal-to-noise ratio, but they allow for the simultaneous recording of hundreds of neurons.We are interested in reconstructing an approximation of the structural connectivity of a culture of neurons. This would allow for characterization of the bulk properties of these networks, such as the dependence of connection probability of two nodes on the distance between them, the degree distribution or the clustering coefficient, which are currently inaccessible with single-cell or even typical multi-electrode techniques. In order to benchmark our connectivity inference methods, we first study simulations of fluorescence signals and examine established methods of inferring the topology. It turns out that we can improve on these methods if we turn to measures from information theory, which do not rely on a linearity assumption.Because we are interested in directed networks, our measure of choice is Transfer Entropy ,2. It tuWe demonstrate post-processing improvements of the reconstruction using the Data Processing Inequality that are only possible in the case of information theoretical measures. These methods, already applied with success in the reconstruction of gene regulatory networks , help toWe then apply our algorithm to real data from large cultures of hippocampal neurons in vitro stained with Fluo-4 AM dye. We probe and quantify the distance-dependent probability of connection and other topological properties of the reconstructed network, finding deviations from a random topology.Finally we point out and quantify which experimental parameters would be most relevant for an improved reconstruction using our method.


This review focuses on the application of functional magnetic resonance imaging (fMRI) to the investigation of blast-related traumatic brain injury (bTBI). Relatively little is known about the exact mechanisms of neurophysiological injury and pathological and functional sequelae of bTBI. Furthermore, in mild bTBI, standard anatomical imaging techniques (MRI and computed tomography) generally fail to show focal lesions and most of the symptoms present as subjective clinical functional deficits. Therefore, an objective test of brain functionality has great potential to aid in patient diagnosis and provide a sensitive measurement to monitor disease progression and treatment. The goal of this review is to highlight the relevant body of blast-related TBI literature and present suggestions and considerations in the development of fMRI studies for the investigation of bTBI. The review begins with a summary of recent bTBI publications followed by discussions of various elements of blast-related injury. Brief reviews of some fMRI techniques that focus on mental processes commonly disrupted by bTBI, including working memory, selective attention, and emotional processing, are presented in addition to a short review of resting state fMRI. Potential strengths and weaknesses of these approaches as regards bTBI are discussed. Finally, this review presents considerations that must be made when designing fMRI studies for bTBI populations, given the heterogeneous nature of bTBI and its high rate of comorbidity with other physical and psychological injuries. This review focuses on the application of functional magnetic resonance imaging (fMRI) to investigate blast-related traumatic brain injury (bTBI). Relatively little is known about the exact mechanisms of neurophysiological injury and pathological and functional sequelae of bTBI. Furthermore, in mild bTBI, standard anatomical imaging techniques [MRI and computed tomography (CT)] generally fail to show focal lesions and most of the symptoms present as subjective clinical functional deficits. Therefore, an objective test of brain functionality has great potential to aid in patient diagnosis and provide a sensitive measurement to monitor disease progression and treatment. The heterogeneity of injury in the bTBI population and resulting lack of a specific anatomical target present a challenge when attempting to develop an fMRI protocol sensitive to TBI in blast victims. The goal of this review is to present the relevant body of blast-related TBI literature and, combined with a brief review of some currently used fMRI techniques, present suggestions and considerations in the development of bTBI fMRI studies.Traumatic brain injury following blast exposure is quite prevalent in soldiers returning from the conflicts in Iraq and Afghanistan. Although there is a range of estimates regarding the occurrence of TBI and bTBI in the recent military operations  or hemorrhage. In addition to these direct effects, the injury may lead to secondary injury, such as the creation of regions of edema or ischemia in the brain  loss of consciousness for more than 30 minutes, 2) after 30 minutes, a Glasgow Coma Scale score of 13-15, and 3) a period of post-traumatic amnesia not to exceed 24 hours.”Subjects with closed-head TBI are usually broken down into three classifications of injury severity: mild, moderate, and severe. A fourth category of TBI, penetrating TBI, is used when an object penetrates the dura and enters the brain. In military TBI, this is most commonly a bullet or metallic fragment. Classification of closed-head TBI is generally made via a combination of Glasgow Coma Scale score  in an attempt to reduce the relative effects of these injuries on study imaging results. With regard to fMRI, localized injury visible via anatomical imaging will obviously have a significant effect on a subject’s blood oxygen level dependent (BOLD) signal in the injured region and, potentially, regions with which the injured tissue normally communicates. However, the goal of and motivation behind a given study will ultimately dictate how secondary and tertiary blast effects are handled when forming population groups.The variety of potential injury mechanisms associated with TBI creates a challenge for identifying the nature and extent of damage in an individual TBI patient. In cases of moderate to severe TBI, anatomical imaging techniques such as CT and standard MRI can provide details about the localization and extent of major injuries such as bone fracture, hematomas, or hemorrhage. Other anatomical sequela such as a shift in the midline of the brain or the presence of a mass effect can also be visible with CT or MRI Broder, . This stIn cases of mild TBI (blast- and non-blast-related), regions of injury are generally less observable. CT and MRI have been used to identify regions of mild injury to some extent, mostly identifying micro-hemorrhages and contusions  sequences, images of the whole brain can be obtained in 2–3 s. This rapid imaging rate, along with the BOLD signal, allows a measure related to blood oxygenation to be made at a relatively good temporal resolution (∼2 s) relative to blood flow changes, and forms the basis of fMRI.In order to determine which areas of the brain are activated by the presented stimuli or task, the time-dependent fMRI signal from each image voxel  is compared to an estimate or model of the signal expected from an ideal activation. The signal model usually takes the form of a prescription for the task or stimuli presentation timing convolved with a hemodynamic response function (HRF). The HRF is the theoretical BOLD response to a single short stimulus. Convolution of this HRF with the time series of the stimuli thus creates a model of the theoretical response of a region of the brain activated by the stimuli. Those voxels with time-courses found to be statistically significantly similar to the modeled stimuli response are characterized as relevantly activated.The specific questions and hypothesis of an investigation play a large role in the design of the task or stimuli presented to the subject. fMRI tasks broadly fall into two categories: (i) block designs and (ii) event-related designs. Studies aimed at investigating a prolonged mental state associated with a given task typically use block designs. Event-related designs, on the other hand, attempt to investigate brain activation in response to individual short-duration events. Generally, a block paradigm is more efficient , but is constrained by the need for a paradigm that maintains the same mental state for the duration of each block. An event-related paradigm permits additional measurements related to magnitude and shape of the HRF, allowing the estimation of hemodynamic responses associated with individual cognitive processes.Functional magnetic resonance imaging has been used to investigate many categories of neural processes and responses such as working memory, emotional processing, attention, along with auditory and visual processing. Those with particular relevance to bTBI are discussed below.Working memory encompasses the mental processes of encoding, storing, manipulating, and retrieving information over relatively short time frames. These processes are essential for most day-to-day tasks and interactions. Difficulty with working memory is a commonly reported impairment for victims of mild to severe blast and non-blast TBI  has been implicated in assisting with both the manipulation and maintenance of information while the vasolateral pre-frontal cortex (VLPFC) may be more involved with maintenance alone  back of previous stimuli. Typically, the N-back task is run with N = 1, 2, or 3 stimuli back. A “0-back” version can also be used, in which the subject is given an initial target stimulus and simply responds based on whether each subsequent stimulus is the same or different than the single target stimulus. As the task progresses in the 1-, 2-, or 3-back cases, subjects must continually update and maintain information in working memory. The 3-back level is considered very difficult for most people, taxing the working memory networks to a high degree. There are many variations on the N-back task, including the type of stimuli used  and the method of presentation .Of the various tasks that have been used in fMRI studies to investigate working memory, some of the most common are the N-back task and various forms of a delayed recall (DR) task. In the N-back task, the subject is presented with a rapid series of stimuli and asked to compare each stimulus to one of those previously presented. For each stimulus, the subject must respond whether it is the same or different than the stimulus shown some number , and is then presented a probe asking the subject to recall one or multiple objects from the initial stimulus group. Alterations to this task can ask the subject to perform mental manipulation of the stimulus objects during the delay period, invoking executive control as well as memory maintenance and recall.An advantage of the delayed recall task, as with the N-back, is the ability to present varying degrees of working memory challenge . Inclusion of manipulation of the objects held in memory allows a direct within-subject comparison between the encoding-retrieval tasks and the encoding-manipulation-retrieval tasks.Like the N-back task, DR tasks have been shown to activate various brain regions associated with working memory in healthy controls including the frontal cortex, DLPFC, VLPFC, and parietal regions  differences in activation patterns within a subject may help in the localization of mild bTBI and perhaps lead to a better understanding of the subject’s specific functional impairment.Many individuals with bTBI also report problems with attention. In general, maintaining attention is the ability to consistently direct mental effort and vigilance toward a target task or goal. This requires the discrimination of related stimuli from unrelated or distracting stimuli as well as control of mental focus. Like working memory, attention is necessary for many day-to-day tasks, especially in a work environment. Impairment of attention due to bTBI can significantly impact quality of life.The attention neural network consists of several regions of the brain including the DLPFC, ACC, striatum, VLPFC, parietal cortex, and possibly areas of the cerebellum Bush, . AlthougAs with working memory, attention may be affected by TBI through damage to gray matter components of the associated neural network or through damage to white matter tracts that facilitate communication between these components. Axonal injury in the anterior limb of the internal capsule or to the anterior corona radiata may inhibit communication between regions in the frontal cortex and the striata or anterior cingulate.A common functional MRI task used to engage and investigate selective attention is the Stroop task Stroop, . While tFunctional MRI studies of healthy adults have shown activations associated with the Stroop task and the response interference condition it presents in areas including the anterior cingulate cortex, medial and inferior frontal lobe, and inferior parietal lobe  may be compared to performance in response to generally negative words . Williams et al.  provide The relationship between bTBI and the alteration of emotional processing is far from apparent. Conclusive findings have yet to be made tying region-specific injury to emotional consequences  in healthy controls , Bryant et al.  saw incrThe presence of PTSD or MDD has also been shown to correspond to alterations in resting state fMRI data. Studies have reported PTSD-related changes in resting state connectivity between regions of the limbic system and cortical regions or between the PCC and cortical or subcortical regions. A seed-based analysis performed by Yin et al.  showed gResting state studies of MDD have reported a variety of changes in connectivity associated with MDD. While, in general, these studies do not seem to reach a consensus about functional connectivity changes introduced by MDD, several show changes in the DMN. A recent study by Zhu et al.  that invGiven the rates of comorbidity of PTSD and MDD with mTBI and the effects these disorders can have on brain activation patterns in response to a variety of fMRI tasks, measurements of PTSD and MDD presence or severity [such as the PTSD checklist  which is invariably accompanied by several sources of noise. Since an fMRI experiment is typically composed of multiple measures compared to an ideal model, the signal as well as the noise is difficult to estimate. Furthermore, most fMRI results are not a simple magnitude or effect size, but rather are cast in terms of statistical reliability. Care must be taken when interpreting statistical results, as this will obviously affect any neuro-functional conclusions.Given the inherent heterogeneity of bTBI and traumatic brain injury in general, there are several important considerations to be made when interpreting group analysis results. Several papers have addressed the effects of inhomogeneous activation within populations consisting of healthy subjects These effects may be even greater in TBI populations.Thirion et al.  present p-value thresholds ranging from 0.000001 to 0.001. However, even at smaller sub-group levels the majority of voxels reported as activated in the sub-groups were also seen as activated in the full group activation map. With a sub-group size of 20 subjects, 80% of the activated voxels seen in the sub-group activation map were also seen in the full group activation map for p-value thresholds from 0.01 to 0.0001. This behavior led to the conclusion that the sub-group activation maps contained regions of false negative voxels  but not necessarily a large number of false-positive voxels  patient populations and matched numbers of controls. Measures of group data heterogeneity and susceptibility to outliers would also support interpretations of group results.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.