Despite similar clinical and pathological features, large numbers of breast cancer patients experience different outcomes of the disease. This, together with the fact that the incidence of breast cancer is growing worldwide, emphasizes an urgent need for identification of new biomarkers for early cancer detection and stratification of patients.n = 20) and nonmalignant breast tissue (n = 5). Biomarker properties of a subset of discovered differentially methylated regions (DMRs) were validated using methylation-sensitive high-resolution melting (MS-HRM) in a case–control study on a panel of breast carcinomas (n = 275) and non-malignant controls (n = 74).We used ultrahigh-resolution microarrays to compare genomewide methylation patterns of breast carcinomas  displayed low-level methylation in nonmalignant breast tissue, independently of locus-specific methylation patterns in cases.Specific loci can undergo either heterogeneous or full methylation during carcinogenesis, and loci hypermethylated in cancer frequently show low-level methylation in nonmalignant tissue. Denmark has the highest standardized incidence of breast cancer in the world, with over 4,500 new cases annually . The breMethylation is a well-established epigenetic process of gene expression regulation. In general terms, methylation of promoter sequences of protein-coding genes results in transcriptional downregulation of the gene, and hypomethylation of previously methylated promoter regions permits transcription . Two advDNA methylation changes have been shown to take part in the very first steps of neoplastic transformation, which makes methylation biomarkers very attractive targets for early cancer detection . MoreoveIn general, clinically useful biomarkers must be applicable in one of the clinical disease management areas: diagnostics, prognostication and treatment monitoring. More than three decades of epigenetic research have provided strong research evidence that methylation-based biomarkers can be applied in all the above areas of clinical use. Nevertheless, current use of methylation biomarkers in clinical cancer management is very limited. The difficulties in clinical implementation of the methylation biomarkers can be attributed mainly to low number of studies focused on extensive clinical validation of the novel (and known) biomarkers.A study aiming to develop a methylation biomarker for clinical use should, apart from utilizing state-of-the-art methylation detection methodologies, consist of five steps:1. Discovery, whereby, in most of the cases genomewide screening is applied in a search for the candidate biomarkers;2. Initial clinical validation, whereby each candidate biomarker must be shown to provide an ability to distinguish nonmalignant healthy tissue from malignant cancer tissue;3. Retrospective validation, whereby archival material is used to determine if there is a significant correlation between specific methylation changes and the disease phenotype. ;4. Prospective validation, whereby the biomarker is used in clinical trials; and5. Long-term monitoring of the biomarker’s clinical use after the above-described biomarker development process to assess its impact.Herein we present and discuss the results of the first two stages of the proposed biomarker development process for breast cancer.Twenty freshly frozen breast carcinomas were obtained from Aarhus University Hospital, and DNA was extracted from those samples using the DNeasy Blood & Tissue Kit  according to the manufacturer’s protocol. Seventy-four nonmalignant breast tissue samples from breast reduction surgeries were collected at the Department of Plastic Surgery, Aarhus University Hospital. The women undergoing breast reduction surgery had had mammograms, and only women without signs of malignancies were enrolled in this study. The breast tissue obtained from breast reductions can potentially differ from healthy breast tissue; however, because of ethical considerations, this type of control material was the only available source for our experiments. DNA taken from breast reduction samples was also extracted using the DNeasy Blood & Tissue Kit. Tumor DNA for screening analyses was obtained from 274 patients diagnosed with sporadic breast cancer. The DNA samples were collected between 1992 and 1994 at Aarhus University Hospital. Complete information about the breast cancer cohort and DNA extraction procedure were previously published ,6. Patie2 signal ratios at each probe. These ratios were averaged within each class of sample to produce a single set of mean ratios per class. The mean ratio sets were processed again with NimbleScan software to generate a relative enrichment score at each probe for each class using a Kolmogorov-Smirnov test in a window (750 bp) around each probe. The enrichment scores for the mean ratios of each class were subtracted to produce a differential score indicating enrichment or depletion of signal in one group relative to the other, and a significance threshold of 2 was applied to the differential scores. Two or more consecutive significant differential scores within 500 bases of each other constituted a differentially methylated region (DMR). Each DMR was mapped to the genome using NimbleScan software. Lists of the annotated regions can be found in Additional file DNA for microarray experiments was extracted from 20 freshly frozen tumor tissue samples. After extraction, the methylated DNA fragments were enriched in each sample using a methylated DNA immunoprecipitation (MeDIP) protocol. A detailed description of the procedure can be found in Additional file 2+, 250 to 500 nM concentrations of each primer and 6 ng of template. PCR amplifications and HRM analyses were performed on the LightCycler 480 platform (Roche) and included 50 cycles at the assay-specific parameters described in Additional file All MS-HRM assays were designed according to previously published guidelines and optimized to allow for highly sensitive methylation detection ,8. In eaTo confirm the MS-HRM results, a subset of MS-HRM PCR products was sequenced using the Sanger method as previously described . In brieThe statistical analyses were performed with the Stata 10.1 software package  and R statistics.PAX2, MYOD1 and PITX2, were also detected in our microarray experiments. For each called DMR, our microarray data analysis workflow (see Methods) provided us with a differential score. This value is in principle a derivative of the methylation difference between cases and controls. We observed that the higher the score, the more pronounced the methylation gain, when we compared both cases and controls. Therefore, 23 DMRs with the highest differential score were selected for the MS-HRM-based microarray validation experiments. MS-HRM assays were targeted to the called DMR or to the closest region that potentially could undergo differential methylation ). The results of the MS-HRM microarray validation corroborated the microarray results for 21 of the selected target sequences . At thihed data ,10. At tTwo of the MS-HRM assays used in the microarray validation experiments did not confirm the microarray results. One possible explanation is that PCR-based MS-HRM assays cover only approximately 100 bp, whereas the region with aberrant methylation called on the microarray can span large genomic regions and the methylation status within those sequence can differ . Design low-level methylation defined as methylation less than 1% standard or any aberrations from the unmethylated profile; and (3) methylation-positive samples. Interestingly, the cancer samples displayed a significant variety of the HRM profiles. The MS-HRM technology allowed us to evaluate heterogeneous methylation in each sample screened. Heterogeneous methylation is referred to as the presence in the sample of multiple epialleles, each with a different pattern of methylated and unmethylated CpG sites for a given region. Heterogeneously methylated samples display a characteristic HRM profile for the complex mixture of heteroduplexes formed between strands that only differ at a few CpG sites. The evaluation of heterogeneous methylation was not previously performed on a large scale owing to technological limitations of the methods for methylation screening.All DMRs positively validated in the microarray validation experiments were subjected to initial clinical validation screening. In principle, this part of the biomarker development workflow aims to show  the potential of the discovered DMR to distinguish between cancer (cases) and healthy controls. We have screened 275 samples of breast cancer with the same MS-HRM assays used in the microarray validation process as well as 74 DNA samples from breast reduction surgeries . The overall results of the initial clinical validation screening for the 19 DMRs are presented in Tables The significance of heterogeneous methylation is still debated, but this phenomenon may potentially be critical for the clinical application of methylation biomarkers (see Discussion for more details). Therefore, we have chosen to subdivide our results into five groups . In addition, three loci  showed low frequencies of the methylation levels, similar to those observed in cancer tissue. These loci will not be further considered for biomarker development, as their specificity most likely will be very low. Figure HMX2) showed methylation levels between 1% and 10% in the control tissue samples. The methylation levels in controls at all other DMRs were always below 1% when we analyzed the data against the 1% methylation level standard. The high frequency of low levels of methylation in the control tissue hampers the specificity of the biomarkers. However, the quantitative aspect of the MS-HRM technology allows establishment of a cutoff point for low-level methylation.The specificity of each of DMR was evaluated on the basis of MS-HRM screening of the control tissue. The results are presented in Table SIX6 and BC008699) is illustrated in Figure Despite the fact that 17 of the DMRs from our panel showed low levels of methylation in the control tissue, those levels seem to be insignificant compared to the levels of the methylation observed at the same locus in the cancer samples. All tested DMRs showed drastic gains of methylation during carcinogenesis. A general switch in methylation pattern from unmethylated in controls to methylated in cancer samples for two of the screened loci . No heterogeneous methylation was seen for the HOXB13 assay, but 74.5% of samples contained both methylated and unmethylated alleles at this DMR. Only one of the DMRs screened (SLC38A4) showed balanced frequencies of heterogeneous and full methylation of 39% and 34%, respectively.The frequencies of methylation in cancer tissue samples are listed in Table HOXB13 assay has previously been shown to undergo hypermethylation in colon cancer [HOXB13 gene has been shown in cancer [HOXB13 gene and the linkage analyses of the neighboring 17q21–22 region found this region to be involved in the development of different cancers [Functional and clinical effects of hypermethylation of the DMRs from our panel are outside the scope of this paper and will be addressed in future projects. However, it is interesting that the CGI we have targeted with our n cancer . Aberrann cancer . However cancers ,16. Our Overall, our methylation screening results clearly show that a locus can undergo two types of methylation during carcinogenesis: either heterogeneous or full methylation . The type of aberrant methylation seems to be locus-specific, and the mechanism of this process is unknown. To the best of our knowledge, our present study is the first to show this phenomenon, and the physiological and clinical consequences of this process have to be further researched. At the same time, the high locus specificity of the observed methylation changes indicates that our results are not a technological artefact of the methodology used in this study, but instead reflect a biological phenomenon.There is a strong base of research evidence to support the utility of methylation biomarkers in the entire process of clinical disease management, from screening for predisposition through detection of the condition to personalized treatment of the disease.As mentioned in the Introduction, the process of biomarker development for clinical applications can be described in five steps. The data presented herein address the challenges of the first two steps of methylation biomarker development for clinical use: discovery and initial clinical validation. In the biomarker discovery step, microarray technologies and next-generation sequencing) are indispensable tools affording researchers, in a single experiment, the ability to uncover a landscape of methylation changes throughout a cell genome. Currently, however, the complexity of those technologies does not allow for straightforward interpretation of the genomewide screening results. The validation step is especially important when complicated statistical modeling is used to process microarray data. In the present study, we used a very simple statistical model for microarray data processing (see Methods). Despite the simple approach involving very little data processing, validation experiments were necessary. They confirmed low (but present) false discovery results from the genomewide screening. This exemplifies the importance of validation of any genomewide methylation-based study before any conclusions are drawn. At the same time, our results illustrate that simple statistical models can be very effective in the discovery of disease-dependent methylation aberrations.We have implemented an initial clinical validation of the biomarkers as a second step in the biomarker development workflow. This step, in which we initially aimed to answer only the question whether a potential biomarker can distinguish healthy tissue from cancer tissue, allowed us to address two important, recently emerging questions in the methylation biomarker field: (1) the question of the prevalence of low-level methylation of potential biomarkers in healthy tissue and the consequences of this phenomenon for biomarker specificity and (2) the question of the significance of the evaluation of heterogeneous methylation of potential biomarkers.The significance of interindividual differences in low-level methylation of specific loci is frequently discussed in the field of methylation biomarkers ,18. TherAs mentioned above, a second very interesting finding of our initial clinical biomarker validation experiments in a relatively large-scale study is that the frequency of heterogeneous methylation can be very high, with some loci undergoing only heterogeneous methylation during carcinogenesis. Heterogeneous methylation was previously shown to occur at some loci, but this phenomenon has never been studied extensively in a large sample. One reason could be the technological limitations of some of the methodologies used for DNA methylation assessment ,20,21. TOverall, the initial clinical validation step in our biomarker development procedure has allowed us to answer two critical questions for the potential applicability of the biomarker in clinical practice; hence, in our opinion, this step is essential for fast streamlining of the biomarker validation process.Our results show that loci undergoing hypermethylation during carcinogenesis can acquire either heterogeneous or full methylation. Furthermore, low-level methylation at those loci in nonmalignant tissue can be a common event. Consequently, researchers in studies addressing the clinical applicability of hypermethylated loci as biomarkers should evaluate the clinical relevance of both specific types of methylation, as well as low-level methylation of those loci in nonmalignant tissue.TKW and LLH are listed as inventors on a patent pending application for the aspects of MS-HRM technology. TKW, JO and LLH are also listed on a patent pending application for the aspects of the biomarkers discovered during this project. Roche Diagnostic, Penzberg, Germany, has partly supported this project based on the research agreement with the University of Aarhus, Denmark  but had no influence on the reporting of the results. A provisional patent application has been filed for the aspects of the new biomarkers by the University of Aarhus with TKW, JO and LLH listed as inventors.TKW, JAW and BBT performed the experiments and analyzed the data. TKW and LLH drafted the manuscript. TED supervised obtaining ethical approval and collecting the specimens for the project. JO and LLH supervised the project. TKW, LLH, JO and TED conceived the project. All authors contributed to the editing of the final version of the manuscript and approved final version for publication.Detailed methylated DNA immunoprecipitation (MeDIP) protocol used in the study.Click here for fileList of the differentially methylated regions obtained after processing of microarray data as described in the Methods section.Click here for fileDetails of the methylation-sensitive high-resolution melting assays used in the validation experiments.Click here for file


Shaker/Kv1 and Shal/Kv4. We find that these two channel types, which are functionally distinguished by their inactivation properties, exert different control mechanisms on the transitions between rest and repetitive spiking in neurons. Bifurcation analysis is used to characterize the influence of each channel and the overall behavior of the membrane as a function of the relative presence of the two A-type channels. We found that the influence of each channel type depends on the resting potential of the neuron, suggesting that modulation and/or network states may shift their relative contribution. For example, at a particular membrane potential, Shal/Kv4 channels are more likely to mediate delays to first spike in response to excitatory input. Overall, this work increases our understanding of the interaction between intrinsic properties and synaptic input in creating neuronal response profiles. Further, we provide a link between relative gene expression and bifurcation structures in dynamical systems modeling neuronal membranes.Neurons receive input from thousands of excitatory and inhibitory synapses, but they are not merely passive receivers and transmitters of information. The intrinsic properties of neurons, and more specifically the collection of ion channels expressed, influence a cell's excitability and integration processes. The role of different voltage-dependent transient (A-type) and persistent (delayed rectifier) potassium currents in shaping the activity of neurons in response to synaptic input is not well understood. We developed a biophysical model incorporating realistic synaptic input  to inves


Sensor technology has matured enough to be used in any type of environment. The appearance of new physical sensors has increased the range of environmental parameters for gathering data. Because of the huge amount of unexploited resources in the ocean environment, there is a need of new research in the field of sensors and sensor networks. This special issue is focused on collecting recent advances on underwater sensors and underwater sensor networks in order to measure, monitor, surveillance of and control of underwater environments. On the one hand, from the sensor node perspective, we will see works related with the deployment of physical sensors, development of sensor nodes and transceivers for sensor nodes, sensor measurement analysis and several issues such as layer 1 and 2 protocols for underwater communication and sensor localization and positioning systems. On the other hand, from the sensor network perspective, we will see several architectures and protocols for underwater environments and analysis concerning sensor network measurements. Both sides will provide us a complete view of last scientific advances in this research field. New technologies have brought us new ways to monitor and sense aquatic environments. Marine surveillance, river and sea pollution detection and monitoring, and oceanographic data collection are needed to explore, protect, and commercial exploit the aquatic environment. Many potential applications exist in this environment such as fish and mussels grow observation ,2, deep-Sensor technology has matured enough to be used in any type of environment. As time goes on, the market offers smaller sensor devices, with lower power consumption and higher processing capacity. Moreover, the appearance of new physical sensors has increased the range of aquatic parameters for gathering data. Because of the huge amount of unexploited resources in the ocean environment, there is a need of new research lines in the field of sensors and sensor networks . FurtherThis special issue has tried to collect the recent advances on underwater sensors and underwater sensor networks. It has been mainly focused on measuring, monitoring, surveillance and controlling underwater environments. The topics of interest in the call for papers are shown in This Special Issue has finally included 28 papers, which have been classified as shown in To provide a better view of this classification when studying every item, we also present The structure of the paper is as follows: Section 2 presents the papers published in this Special Issue in the topic Sensor Nodes. The papers included in underwater communications topics are described in Section 3. Section 4 discusses the papers included in the underwater sensor networks topic. Finally Section 5 draws the conclusions of this Special Issue.2.This section shows the papers accepted in the Special Issue focused on underwater sensor node topics.et al. design, build, and test a portable, watertight, and user-friendly an autonomous underwater sound recording device (USR) that monitors the underwater sound and pressure waves generated by anthropogenic activities, such as underwater blasting and pile driving. It uses two hydrophones or other dynamic pressure sensors which allow approximately up to 1 h and 55 min of data collection from both sensors simultaneously. The gain can be independently set for each sensor. They proposed two versions. The first one is a submersible model deployable to a maximum depth of 300 m and the second one is a watertight but not fully submersible model. The submersible version is appropriate for collecting long-duration measurements at depths that would require very long hydrophone cables or extension cables, while the non-submersible version is better for short duration underwater monitoring cases. Their test demonstrates that the device operates as designed and performs as well as larger commercially available data acquisition systems. Moreover, they present a case study with a prototype measuring blast pressures while investigating the effect of underwater rock blasting events while simultaneously exposing juvenile Chinook salmon and rainbow trout held in cages to the same sound and pressure waves. Device withstood operation in harsh environments, thus it is a valuable tool for collecting field measurements.In , Martineet al. present in [Ardid esent in  the deveet al. [The paper authored by Baronti et al. , presentet al. in [The Ocean Bottom Seismometer (OBS) presented by Manuel t al. in  has beenet al. present a case study of a submersible UV-Vis absorbance spectrophotometer to determine stream organic carbon dynamics within a headwater catchment. The experiment was placed near Campbell River . Field measurements were made before and after forest harvest. The system has high temporal resolution compared to traditional grab sampling and laboratory measurements, which allows resolving variations at small time scales and the observation of concentration changes over longer time periods, and thus demonstrating DOC concentration dynamics over the pre and post harvest periods. One of its main advantages is the ability to remotely monitor the system through a wireless connection. The system is able to react when environmental or power disturbances cause system interruption and data loss.The effects on ecological productivity and biogeochemical cycling can be evaluated by determining how land-use changes affect dissolved organic carbon (DOC) concentrations and bioavailability within aquatic ecosystems. In , Jollymo3.Won and Park show the design and Implementation of an Omni-Directional Underwater Acoustic Micro-Modem Based on a Low-Power Micro-Controller Unit in . The woret al., describes the physical layer of a new low-cost acoustic modem called ITACA. Its architecture combines a typical microcontroller-based core with energy-efficient mechanisms. One of the main features of the modem is the implementation of an ultra-low power asynchronous wake-up system for underwater acoustic transmission that is based on a low-cost off-the-shelf RFID peripheral integrated circuit. This allows a reduced power dissipation in stand-by mode and very low power values during reception and transmission. The modem uses coherent-FSK modulation. It also incorporates clear channel assessment (CCA) detection using both carrier frequency detection and the RSSI level to support CSMA-based medium access control (MAC) layer protocols and an efficient development of upper communication layers by supporting additional RTC  asynchronous wake-up and clear channel assessment (CCA). This asynchronous wake-up design has been tested for non-detections and false positives with excellent results.The paper in , authorePark, and Shin propose in  the SeleKim and Park propose in  a novel In , Llor anet al. show in [Zhang  show in  how to ra priori, because underwater medium is assumed to be composed of multiple isogradient sound speed profile (SSP) layers where in each layer the sound speed is linearly related to the depth. They used a ray-tracing approach for high-frequency signal transmission to model the propagation and a set of polynomial root finding equations to distinguish among different possible transmission paths between the nodes, and determine the fastest one. Each sensor node is able to measure its depth and can exchange this information with other nodes. Then, they relate the pair-wise time of flight (ToF) values with nodes positions in order to propose a novel ranging algorithm for an underwater medium. The proposed ranging algorithm considers reflections from the seabed and sea surface and selects the fastest transmission path based on the measured ToF and the nodes' depth measurements. The algorithm estimates iteratively the range of a target by minimizing the difference between the measured ToF and the constructed ToF estimated from the known map. The authors evaluate the performance of the proposed algorithm by running several simulations and comparing successfully the results with other existing algorithms.In , Ramezanet al. propose the use of EM waves in the 2.4 GHz ISM frequency band in underwater environments when more bandwidth is needed, despite the high attenuation of the water at these frequencies. In [One of the main drawbacks in underwater communications is the low available data rate due to the use of low frequencies. Lloret cies. In , they pret al. investigate the tradeoff between the energy efficiency and the network connectivity through the selection of the transmission range in [Gao range in . They pr4.etc. but it can be sufficient for certain data gathering applications such as water monitoring and climate change monitoring.In , Gómez aet al. present in [etc.).In recent years, the localization problem in Underwater Wireless Sensor Networks has resulted in many innovative solutions and ideas. Han esent in  a compreet al. propose a novel 3D centralized localization scheme for mobile underwater wireless sensor network, named Reverse Localization Scheme (RLS) in [Moradi (RLS) in . RLS is In , Lee andet al. discuss and compare the reliability challenges, characteristics, advantages, and disadvantages and enhancement approaches for these network architectures. The reliability evaluation has been performed in terms of network connectivity, the continuity of power supply for the network, and the physical network security. Authors developed an analytical model to compare the reliability of different architectures for monitoring underwater pipelines. Moreover, they also develop and evaluate a hierarchical sensor network framework for underwater pipeline monitoring. In this architecture, the node batteries are only used if there is a fault in the wires providing power to the nodes, so nodes would have better chance to have power supply in integrated wired/wireless sensor network than in wireless sensor networks. The integrated wired/wireless sensor network can provide good sensing and communication reliability. Moreover, they have less physical security concerns than underwater wired sensor networks because physical attacks on the network will not stop the connectivity of the network in some degree.There are different sensor network architecture designs that can be used for monitoring underwater pipeline infrastructures. They are underwater wired sensor networks, underwater acoustic wireless sensor networks, Radio Frequency (RF) wireless sensor networks, integrated wired/acoustic wireless sensor networks, and integrated wired/RF wireless sensor networks. In, , Mohamedet al. propose in [Macias opose in  a multimet al. propose an architecture and protocol design for marine vehicles including Autonomous Underwater Vehicles (AUVs), Autonomous Surface Vehicles (ASVs) and Autonomous Underwater Gliders (AUGs), used as a mobile sensor network, for taking samples of a regional ocean area, ocean phenomena observing and ocean feature tracking. Authors analyze the marine vehicle dynamic control, multi vehicles mission assignment and path planning methods, ocean feature tracking and observing techniques. They integrate the control and plan of the sensor platforms with data assimilation to obtain more sample data. Combined with the observation plan in the South China Sea, authors provide numerical simulations of ocean models and data assimilation, and autonomous trajectory planning and optimization, which demonstrate the effectiveness of the system.In , Zhang eet al. discuss a new architecture and routing protocol for underwater sensor networks called Energy-efficient aDaptive hiErarchical and robusT Architecture (EDETA) and EDETA-e protocol respectively in [Climent ively in . AuthorsTaking into account that in underwater sensor networks, the probability of node failure is high because sensor nodes are deployed in harsher environments than ground-based networks, H. Min at al. propose in  a checkpet al. propose a new underwater routing scheme, namely Autonomous Underwater Vehicles aided Underwater Routing Protocol (AURP), which uses both heterogeneous acoustic communication channels and controlled mobility of multiple autonomous underwater vehicles (AUVs) to maximize the data delivery ratio and minimize the energy consumption of underwater sensor nodes. The network architecture consists of U-sensor nodes, gateway nodes (GWs), AUVs, and surface unit/mothership. AURP minimizes the total number of data transmissions using the following procedure. Underwater sensors send the sensed data, using a midrange underwater acoustic channel, to the GWs directly or in a multihop fashion. By exploiting controlled mobility of AUVs and the fact that AUVs can move closer to other underwater acoustic sensor nodes, a short-range high data rate underwater channel for large amount of data transmissions let GWs forward the aggregated data to an AUV. AUVs act as relay nodes, which collect sensed data from GWs and then forward them using a short-range high rate acoustic channel to the sink, which will in turn forward the data to the surface unit. AUVs have another acoustic interface for a long distance and low data rate communications to receive control signal and to send urgent data from/to the mothership via a surface unit. This procedure lets the system improve the network performance in terms of data delivery ratio and energy consumption. Authors simulated AURP performance using NS2 and they compared it with another Directed Diffusion based routing protocol. Their simulations used a realistic underwater acoustic communication channel model to evaluate the performance of the proposed scheme. Their results indicate AURP has higher delivery ratio and low energy consumption than the other. Moreover, they have observed that it could be used for applications that require mass data transmissions, such as multimedia applications.In , Yoon etet al. propose both a security suite, which is designed with the goal of reducing the communication overhead introduced by security in terms of number and size of messages between autonomous mobile underwater sensors, and a cooperative algorithm in which the mobile underwater sensors  respond to simple local rules based on the available information. The cooperative algorithm relies on the fact that the available information from the other sensors, though not necessarily complete, is trustworthy. It considers that when there is a loss of communication among the vehicles, the coverage performance is degraded but not lost. The proposed security suite is aimed at communication integrity and confidentiality, which jointly with the robustness of the cooperative algorithm, which is intrinsically reactive against DoS due to the emergent behaviour approach, makes it ideal to perform a mission and maintain the communication link with the network in the underwater scenario. Authors also explain the implementation details of the integration between both and provide statistics of the system performance collected from a real project. Finally, they report some results from an Underwater Acoustic Network project to characterize its application level performance including the cooperative strategy and the security suite proposed in the paper. They demonstrate that the security solution is efficient in terms of number of messages and message size, reducing the time and the energy of the transmission.In  Caiti etet al. propose in [Wu opose in  a serieset al. examine the hydrological and biogeochemical variables obtained from the Guadalquivir estuary during three years through a real-time remote monitoring network. This network was developed with the aim of providing accurate data in order to study the influence of hydrodynamical and hydrological features within the estuary on the functioning of the pelagic ecosystem and know the trophic status of the aquatic environment. This information could be used for research or educational applications and by policy-makers or agencies in charge of the management of the coastal area. Obtained measurements showed that several sources of physical forcing  were responsible of the spatio-temporal patterns of the dissolved oxygen, salinity and turbidity in the estuary. The river discharge was identified as the main forcing agent of the hydrology inside the estuary. Using network values, they related episodes of elevated turbidity, together with episodes of low salinity and dissolved oxygen to the increase in water supply from a dam located upstream. The real-time monitoring network can be applied to generate early warnings for navigation, turbidity increases, hypoxia events, and toxic algal blooms, and for earlier decision making processes to prevent or mitigate undesirable events.In , Navarroet al. propose an Artificial Neural Network (ANN)-based architecture for completion and prediction of data retrieved by underwater sensors in [Balandrón nsors in . The sys5.Almost all the papers have highlighted the need of new technology for underwater communications because the range of applications in underwater environment is growing fast.Most researchers have point out the challenges and drawbacks when developing new technologies in this research field.The issues that should be taken into account in sensing systems are different depending on the size of the area to be monitored. It can be used one or few independent sensors to monitor a localized zone or a sensor network for large zone monitoring.The fact of including video cameras and the increase of the number of sensors placed inside the water has emphasized the need of higher bandwidth to transmit more data in the wireless channels.This Special Issue has received more papers focused about underwater architectures and protocols than the rest of the topics proposed in the call for papers.Although underwater sensor nodes and underwater sensor networks have been a hot topic since several years ago, it seems that this area will continue to be a hot topic for many more years to come due to the need for more technology in this field.After having organized this special issue, we can draw several conclusions from the published papers:


Giardia lamblia has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of Giardia parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host.Compared with many protists, G. lamblia life cycle we compared the transcriptome of G. lamblia trophozoites and cysts using single-color oligonucleotide microarrays. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. The comparison of the transcriptome of cysts generated in culture or extracted from feces revealed little overlap, raising the possibility of significant biological differences between the two types of cysts.To investigate the magnitude of the transcriptional changes occurring during the G. lamblia cyst and trophozoite transcriptome showed that transcripts of most genes are present at a lower level in cysts. This global view of the cyst and trophozoite transcriptome complements studies focused on the expression of selected genes during trophozoite multiplication, encystation and excystation.The comparison of the  Giardia lamblia  is a diplomonad parasite which causes over 20,000 reported cases of giardiasis a year in the United States .Comparison of Cy3 fluorescence emitted by microarrays hybridized with assemblage A and B trophozoite cDNA. Fluorescence values are means of two replicate microarray spots and are ranked in order of decreasing intensity, as in Figure 1. All datasets are biologically independent; the 3-digit microarray number is shown in the legend. Fluorescence and rank are plotted on a log scale. Isolate WB (red lines) is assemblage A, isolate GS (green line) assemblage B.Click here for fileGene ID of 215 cyst and trophozoite genes which generated the highest mean Cy3 fluorescence. Microsoft Excel fileClick here for file


The intracranial localization of large artery disease is recognized as the main cause of ischemic stroke in the world, considering all countries, although its global burden is widely underestimated. Indeed it has been reported more frequently in Asians and African-American people, but the finding of intracranial stenosis as a cause of ischemic stroke is relatively common also in Caucasians. The prognosis of patients with stroke due to intracranial steno-occlusion is strictly dependent on the time of recanalization. Moreover, the course of the vessel involvement is highly dynamic in both directions, improvement or worsening, although several data are derived from the atherosclerotic subtype, compared to other causes.We report the clinical, neurosonological and neuroradiological findings of a young woman, who came to our Stroke Unit because of the abrupt onset of aphasia during her work. An urgent neurosonological examination showed a left M1 MCA stenosis, congruent with the presenting symptoms; magnetic resonance imaging confirmed this finding and identified an acute ischemic lesion on the left MCA territory. The past history of the patient was significant only for a hyperinsulinemic condition, treated with metformine, and a mild overweight. At this time a selective cerebral angiography was not performed because of the patient refusal and she was discharged on antiplatelet and lipid-lowering therapy, having failed to identify autoimmune or inflammatory diseases. Within 1 month, she went back to our attention because of the recurrence of aphasia, lasting about ten minutes. Neuroimaging findings were unchanged, but the patient accepted to undergo a selective cerebral angiography, which showed a mild left distal M1 MCA stenosis.During the follow-up the patient did not experienced any recurrence, but a routine neurosonological examination found an unexpected evolution of the known MCA stenosis, i.e. left M1 MCA occlusion. Neuroradiological imaging did not identify new lesions of the brain parenchyma and a repeated selective cerebral angiography confirmed the left M1 MCA occlusion.Regardless of the role of metabolic and/or inflammatory factors on the aetiology of the intracranial stenosis in this case, the course of the vessel disease was unexpected and previously unreported in the literature at our knowledge. It has been known since several years that ischemic stroke related to large intracranial arteries steno-occlusion has a poor prognosis, if the vessel recanalization does not occur within 1 hour ,3. ConveA 26 years-old woman came to our attention because of the abrupt onset of aphasia during her work (she was a lower school teacher). This symptom lasted several days with a progressive improvement during hospital staying and a final complete recovery. The patient arrived in the Emergency Department four hours after the onset of symptoms and an unenhanced brain CT did not show abnormalities. Urgent blood tests was also normal. The neurological evaluation reported a mild aphasia with prevalent anomic and paraphasic alterations, without motor deficits; the National Institute of Health Stroke Scale (NIHSS) score was 1.®). It confirmed the presence of a left distal M1 MCA stenosis; moreover this finding was congruent with signs and symptoms of the patient. The ultrasound grading of the MCA stenosis was performed according to the Baumgartner's velocity criteria [At this point an urgent neurosonological study was performed both in the extracranial and intracranial vessels, showing a normal appearance of the extracranial carotid and vertebral axis and a suspected left distal M1 MCA stenosis, by using unenhanced Transcranial Colour-Coded Sonography (TCCS). Therefore, because of the suboptimal temporal bone window, contrast-enhanced TCCS was performed by intravenous injection of sulphur hexafluoride  showed a left caudate head and temporal cortical ischemic damage and the Magnetic Resonance Angiography (MRA) confirmed the left MCA stenosis Figure , row A.The past medical history of the patient was significant only for a previous diagnosis of hyperinsulinemia, treated with metformine; overwheight is present (BMI 26). The diagnostic pathway included blood testing and instrumental evaluation to rule out a primary or secondary central nervous system vasculitis. An accurate anamnestic survey was performed about the previous occurrence of any event that could point to immunological and/or rheumatologic or infectious processes, with negative results. Blood samples were taken for screening of autoimmune diseases and common acquired or congenital thrombophylic factors or coagulation abnormalities, both with negative findings. The titration of autoantibodies did not show any abnormality, also including antiphospholipid and anti-beta2 glycoprotein 1 antibodies. Serological screening for borreliosis, herpesviruses (comprising Varicella-Zoster virus - VZV) and influenza viruses was not suggestive for a first or recurrent infection. Also the lymphocyte typing and the immunofixation electrophoresis of immunoglobulins were normal.Antiplatelet and HMGCoA reductase inhibitor therapy was early started and a selective Digital Subtraction Angiography (DSA) was proposed, but the patient initially refused it.After 20 days the patient was again evaluated because of the recurrence of a ten minutes-lasting episode of aphasia. Both neurosonological and neuroradiological examinations were repeated and their findings were unchanged; at this point the patient agreed to undergo selective DSA of the cerebral vessels. Therefore DSA was performed after 10 days. Meanwhile a dual antiplatelet therapy with acetyl salicylic acid and clopidogrel was suggested. The DSA findings  2.5 mL was injected by the right antecubital vein. A left M1-M2 MCA occlusion was diagnosed, with a large early branch, arising from the M1 MCA segment just before the stop (Figure In the following ten months no other event has been reported by the patient and she was phone called in order to undergo a contrast-enhanced TCCS as a follow-up test. TCCS was performed in second harmonic imaging with pulse inversion and sulphur hexafluoride (SonovueIn the following 16 months neurosonological control examinations overlapped the last findings and no neurological event was reported. Also neuropsychological testing was normal and the patient was treated with a Serotonine Selective Reuptake Inhibitor, because of an associated post-stroke depression.This case, during its course, raises some relevant discussion points to be clarified during the clinical reasoning.First, the infarction localization at the caudate nucleus is seldom documented in the literature in case of MCA stenosis ,6. This Second, the difference in the grading of the MCA stenosis between TCCS and DSA was somewhat surprising initially, and we have not a definite explanation about this single case from the literature data. It is possible that a high flow condition was present in order to maintain a sufficient perfusion downstream the stenosis and to sustain the collateral circulation. A similar situation has been seldom demonstrated in the post-acute phase of striato-capsular infarction, although it was considered a transient phenomenon . The timThe third but maybe more relevant point is the course of the vessel lesion, dramatically worsening, associated to a clinical silence. The asymptomatic course can help to explain the difference in grading, discussed above, because of the efficient recruitment of collateral channels, of which the high flow velocity of the left MCA at the site of the stenosis may be a marker. Several previous studies showed that intracranial atherosclerosis is an highly dynamic process, reporting both progression or regression by using neuroradiological  and neurIn the case series of Kern and colleagues  only twoThe evolution of the MCA stenosis in our case was not previously described in the literature, at our knowledge, not only for intracranial atherosclerotic stenosis but also for other causes of intracranial vessel disease. Diagnostic investigations about the causes of the MCA stenosis in our patient were substantially negative and the neuroradiological findings were not pathognomonic for vasculitis, dissection or a moiamoia disease, although this last condition can be very challenging in the initial phase and a longer follow-up could allow to identify the involvement of other intracranial vessels. At the moment, however, a clear definition about the causes of the MCA stenosis has not been reached in our case.It is possible that inflammatory and metabolic factors, potentially related to each other, have played a role in the evolution of the vessel history, but both vascular and clinical course of this case are previously unreported in the neurological literature at our knowledge.Written informed consent was obtained from the patient for publication of this case report and any accompanying images.The authors declare that they have no competing interests.GM revised the draft and performed the first ultrasound evaluation; MZ wrote the draft and performed the follow-up of the patient; GDB revised the neuroradiological examinations and co-revised the draft; MM performed the neuroradiological follow-up and co-revised the draft; NM revised the draft.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/11/154/prepub


The dynamics of individual neurons are crucial for producing functional activity in neuronal networks. An open question is how temporal characteristics can be controlled in bursting activity and in transient neuronal responses to synaptic input. Bifurcation theory provides a framework to discover generic mechanisms addressing this question. We present a family of mechanisms organized around a global codimension-2 bifurcation. The cornerstone bifurcation is located at the intersection of the border between bursting and spiking and the border between bursting and silence. These borders correspond to the blue sky catastrophe bifurcation and the saddle-node bifurcation on an invariant circle (SNIC) curves, respectively. The cornerstone bifurcation satisfies the conditions for both the blue sky catastrophe and SNIC. The burst duration and interburst interval increase as the inverse of the square root of the difference between the corresponding bifurcation parameter and its bifurcation value. For a given set of burst duration and interburst interval, one can find the parameter values supporting these temporal characteristics. The cornerstone bifurcation also determines the responses of silent and spiking neurons. In a silent neuron with parameters close to the SNIC, a pulse of current triggers a single burst. In a spiking neuron with parameters close to the blue sky catastrophe, a pulse of current temporarily silences the neuron. These responses are stereotypical: the durations of the transient intervals–the duration of the burst and the duration of latency to spiking–are governed by the inverse-square-root laws. The mechanisms described here could be used to coordinate neuromuscular control in central pattern generators. As proof of principle, we construct small networks that control metachronal-wave motor pattern exhibited in locomotion. This pattern is determined by the phase relations of bursting neurons in a simple central pattern generator modeled by a chain of oscillators. The dynamics of individual neurons are crucial for the functionality of neuronal networks. Precise timing and reliability of temporal responses are critical for memory, pattern recognition, and especially motor control Drosophila larvae Rhythmic motor functions are executed by precisely coordinated oscillatory patterns of contracting muscles. These functions require flexibility of rhythmic patterns to cope with environmental conditions such as temperature, load, or the demand for speed of locomotion. Accordingly, some rhythmic behaviors scale their pattern, maintaining phase relations across a wide range of periods, for example, according to different speeds of locomotion. Examples of pattern scaling behaviors include the pyloric rhythm in Crustacea, crayfish swimmeret beating, the leech heartbeat, leech swimming, lamprey swimming, and crawling of the Rhythmic behaviors are controlled by devoted oscillatory neuronal networks: central pattern generators (CPGs) Bifurcation theory explains how dynamical systems like neurons precisely change their dynamics in response to the variation of a controlling parameter like neuromodulatory tone or descending drive Our study emphasizes the dynamics of single neurons as an organizing principle underlying pattern formation. We present a global codimension-2 bifurcation and assert that this so-called cornerstone bifurcation can precisely control the regulation of temporal characteristics in periodic and transient neuronal activity. We suggest that this bifurcation generates a family of cellular mechanisms which can answer the aforementioned questions. These mechanisms also explain the operation of conditional oscillators in parameter space near a functional bursting regime.In neurons, control over regimes of activity is ubiquitously executed through neuromodulation. Potassium currents and hyperpolarization-activated currents are common targets for neuromodulation Drosophila larvae.Biophysically accurate neuronal models allow the utilization of well developed techniques from bifurcation theory. In a model, bifurcations can predict the dependence of temporal characteristics of oscillatory regimes near the bifurcation 2+ and 4-aminopyridine (4-AP). Application of Co2+ blocks Ca2+ currents and the synaptic current. Application of 4-AP blocks most of the K+ currents. The remaining currents in this model are the leak current, the non-inactivating potassium current, We developed a generic low-dimensional Hodgkin-Huxley type neuronal model stemming from a model of the leech heart interneuron under certain pharmacological conditions The parameters  spiking . For higactivity . For higs silent . For low silence .We determined the boundaries between bursting and tonic spiking and between bursting and silence by integrating the system over a range of values for interval . The trantly of  . The traence on  . As the ecreased .We associated saddle-node bifurcations of equilibria and periodic orbits with the transitions between different regimes. The transitions between We show that the saddle-node bifurcations along the border between w motion ; Text S2w motion ; Fig. S1urcation –B. The pduration –D. The cn curves . We alsoFor parameter values in regions  and  , tonic s region  . For exale burst  A–C. Thests grew . Even thEq. 1 in  , organizes bursting, spiking, and silent regimes . The bifw motion .At the codimension-2 bifurcation point, the slow motion manifold determined by the equilibria of the fast subsystem is obstructed by a saddle-node equilibrium, and the slow manifold for spiking is obstructed by a saddle-node bifurcation for periodic orbits .Perturbations in the –either continuous and smooth or discontinuous and catastrophic–in response to a smooth change in a physical parameter. Applications of bifurcation theory in physics, engineering, and neuroscience have established a new interdisciplinary field: bifurcation control Bifurcation theory has been applied to describe the general laws of neuronal dynamics that govern characteristics of spiking and bursting activity as a controlling bifurcation parameter approaches a transition between qualitatively different regimes of activity Similarly to spiking activity, temporal laws have been described for bursting activity where the length of the burst duration or interburst interval is subject to control by a bifurcation parameter Examples of control over burst duration and interburst interval have been shown in a model of the electrosensory lateral line lobe pyramidal cell Another key feature that distinguishes ghostbursting from our scenario, is that in the ghostbursting model the SNLC describes the transition from tonic spiking to chaotic bursting, and SNFP describes the transition of chaotic bursting into silence. Doiron et al. show that these transitions to chaos are intermittency type I Drosophila larvae, and the lamprey Drosophila larvae can crawl with body contractions of cycle period from 0.6 s to 1.3 s DrosophilaWe suggest that these cellular mechanisms could contribute to control of various types of motor activities. For example, phase shifts of a metachronal-wave pattern could be determined by the duty cycle of each element in a chain of inhibitory coupled bursting neurons. Metachronal-wave patterns are ubiquitous in animal locomotion such as that of the leech, the crayfish, Drosophila larvae crawl by means of peristaltic contractions of body wall muscles. Posterior-to-anterior waves of contraction propel the animal forward at varying speeds. The segmental phase delay of the propagation of these waves is proportional to the cycle period of oscillations Drosophila (see Methods). By preserving duty cycle and varying cycle period we achieved maintenance of delay in phase between neighboring neurons in the chain. Moreover, the duty cycle of a model of a single cell predicts the phase delay in the network. Our model demonstrated a five-fold scaling of the metachronal-wave pattern, which is comparable to the two-fold scaling of the motor pattern in DrosophilaWe modeled this phenomenon in a chain of coupled bursting neurons. The model corresponded to segments three through seven of the larval Dynamical mechanisms govern rhythmic neuronal activity in the vicinity of bifurcations. These types of mechanisms are often discussed within the context of steady state activity. Stereotyped transient responses and activity could process or represent sensory stimulation These parameters provide two types of control over temporal characteristics for two types of transient activity. First, we demonstrate control of the duration of pulse-triggered bursts in a silent neuron. As the voltage of half-activation of In this article, we showed that the silent model could respond to a hyperpolarizing pulse by a stereotypical burst of spikes. We report that the burst duration of the pulse-triggered bursts scaled as Our model produces a stereotypical latency to spiking after a brief hyperpolarizing pulse in a spiking neuron. By coordinating In Steuber et al., pauses in Purkinje cell firing are implicated in pattern coding Potassium and hyperpolarization-activated currents have been implicated in the dynamics of delay to spiking. In Meng et al., the authors show control over delay to firing by manipulating the ratio of transient potassium conductances Another important feature of the dynamics of our model is that it shows bistability of spiking and silence region , . PerturbIn the example presented here, a codimension-2 bifurcation that satisfies the criteria for the SNIC and the blue sky catastrophe controls burst duration, interburst interval, pulse triggered bursting, and latency to spiking. In conclusion, we suggest that the control over the temporal characteristics of neuronal activity presented here is critical for designing functional artificial neurons in biomedical and neuroengineering fields + current, + current, We developed a Hodgkin-Huxley style neuronal model. It contains three voltage gated currents: a fast NaThe activation of 2+ currents are blocked with divalent ions and most of the K+ currents are blocked with 4-aminopyridine The majority of the parameters of this model were pulled directly from our previous model Drosophila larvae. It follows the generic motif of a chain of coupled oscillators Drosophila, such that the most anterior neuron corresponded to the third segment, and the most posterior neuron corresponded to the seventh segment. Coupling was accomplished through inhibitory synapses. The nearest of the posterior cells provided the strongest synaptic input. The synaptic input of the anterior cell was weaker than any of those from posterior cells. The total synaptic current onto cell We built a model representing the CPG that produces a metachronal-wave pattern of locomotion in We defined For re  and  .http://www.gnu.org/software/gsl/). The continuation of stationary states, periodic orbits, and most bifurcations was performed using CONTENT We performed numerical integration using the 8–9 order Prince-Dormand method from the GNU Scientific Library . We computed burst duration, interburst interval, cycle period, and duty cycle. Burst duration is the time from the first spike in a burst to the last spike in a burst. Interburst interval is the time from the last spike in a burst to the first spike in the next burst. Cycle period is the time from the first spike in a burst to the first spike in the next burst. Duty cycle is burst duration divided by cycle period.−9.In on Eq. 2 , the coeIn Figure S1Interburst interval and burst duration are scaled according to saddle-node bifurcations. Graphs are plotted in the log-log scale. The interburst interval and burst duration are depicted as blue dots. The curves fitted to these data are depicted as red curves. Curve fits for the interburst interval took the form (TIF)Click here for additional data file.Figure S2The dependence of equilibria and periodic orbits on the parameter . For each orbit, we plot the maximum, minimum, and average voltage. The green curves represent the evolution of a stable orbit as (TIF)Click here for additional data file.Figure S3Structure of the manifolds of slow motion. (A) The slow motion manifolds for parameter values of both the SNIC and the blue sky catastrophe calculated at  of  see . The ave(TIF)Click here for additional data file.Text S1Inverse-Square-Root Curve Fits Confirm SNIC and Blue Sky Catastrophe Bifurcation Curves.(PDF)Click here for additional data file.Text S2Computing the Manifolds of Slow Motion.(PDF)Click here for additional data file.


The paediatric flat foot is a common concern and has long been regarded as a problem, and feared to be potentially disabling. Definition of what exactly constitutes a flat foot remains surprisingly debatable, given its common presentation. Estimates of flat foot prevalence in children have been influenced by the varied the methods of assessment used to assess the foot posture, and the subsequent criteria employed to delineate feet as flat versus not flat. The aim of this literature review was therefore to evaluate the techniques of foot posture assessment in children and the reported reliability and validity of these measures.A systematic search of electronic databases including Medline (1966-present), CINAHL, SportDiscus, Embase, Scopus and Pedro occurred between 02/01/10 and 14/08/10. Eligible articles were selected according to pre-determined criteria. Methodological quality was evaluated by use of the Quality Index as described by Downs & Black, followed by critical analysis according to outcome variables.The most widely reported measures of paediatric foot posture were footprints and measures of the heel angle and arch height. The current evidence suggests that the reliability of all measures of paediatric foot posture is highly variable and mostly poor to moderate. The only measures on which validity has been explored are navicular height and footprints.Whilst no definitive conclusions can currently be drawn from the existing evidence, the trend from the current literature indicates that static paediatric foot posture may be best-assessed using RCSP, NH or FPI-6. However the relationship between static measures and pain, static measures and gait function remain largely unsubstantiated in children. The direction of future research is to establish a universal method of assessment of paediatric foot posture, and the subsequent relevance of foot posture to pain and function across age groups. Continuation of research in this field will enable targeting of design parameters towards variables that are supported by evidence, and which may directly advance clinical decision-making.


Anaemia is a common haematologic side effect of dose-dense multi-cycle cytotoxic polychemotherapy requiring erythrocyte transfusions or erythropoietin (EPO) administration. To simulate the effectiveness of different EPO application schedules, we performed both modelling of erythropoiesis under chemotherapy and pharmacokinetic and dynamic modelling of EPO applications in the framework of a single comprehensive biomathematical model. For this purpose, a cell kinetic model of bone marrow erythropoiesis was developed that is based on a set of differential compartment equations describing proliferation and maturation of erythropoietic cell stages. The system is regulated by several feedback loops comprising those mediated by EPO. We added a model of EPO absorption after injection at different sites and a pharmacokinetic model of EPO derivatives to account for the effects of external EPO applications. Chemotherapy is modelled by a transient depletion of bone marrow cell stages. Unknown model parameters were determined by fitting the predictions of the model to data sets of circulating erythrocytes, haemoglobin, haematocrit, percentage of reticulocytes or EPO serum concentrations derived from the literature or cooperating clinical study groups. Parameter fittings resulted in a good agreement of model and data. Depending on site of injection and derivative , nine groups of EPO applications were distinguished differing in either absorption kinetics or pharmacokinetics. Finally, eight different chemotherapy protocols were modelled. The model was validated on the basis of scenarios not used for parameter fitting. Simulations were performed to analyze the impact of EPO applications on the risk of anaemia during chemotherapy. We conclude that we established a model of erythropoiesis under chemotherapy that explains a large set of time series data under EPO and chemotherapy applications. It allows predictions regarding yet untested EPO schedules. Prospective clinical studies are needed to validate model predictions and to explore the feasibility and effectiveness of the proposed schedules. Anaemia is a common side-effect of dose-dense multi-cycle polychemotherapies which may result in delays of treatment or reduction of chemotherapy dose We demonstrated in the past that mathematical models of granulopoiesis and thrombopoiesis can be used to identify optimal chemotherapy and growth factor schedules We present an ordinary differential equations model which combines four model concepts developed in different contexts: First, a cell kinetic model describing the development of mature erythrocytes from haematopoietic stem cells and its regulation by endogenous EPO was established by our group Rather than constructing a model from the scratch, it is our major intention to built it on these established structures. Hence, we adopt their assumptions, equations and parameters as far as possible. This approach takes into account that the developement of a comprehensive model of erythropoiesis under chemotherapy and EPO application in a single effort is difficult due to the complexity of the underlying biological processes. Our paper contributes to a second step of modelling by combining submodels which address single issues of the overall process of interest. However, since all submodels were developed on a limited data base and disjoint biological scenarios, a few adaptation are required to combine them. In the present paper, we describe these assumptions and modifications in detail. A complete set of equations can be found in Unknown parameters of the model are estimated by optimising the agreement of model predictions with data of a large variety of published clinical data sets comprising four different EPO derivatives . These scenarios are accompanied by modelling clinical data obtained from randomised clinical chemotherapy trials of cooperating national study groups. We also validate the model by predicting data not used for parameter fitting and present some model predictions of yet untested combinations of chemotherapy and EPO.Haemoglobin data were taken from published studies of the German High Grade Non-Hodgkin-Lymphoma Study Group We constructed a model of human erythropoiesis under chemotherapy and EPO applications by combining a cell kinetic model of bone marrow erythropoiesis An ordinary differential equations model of bone marrow erythropoiesis was developed in the past see . We brieMaturation is modelled by a set of concatenated first order transitions of the formThe differential equations system is regulated by several feedback loops. Loop 1 and 2 are feedbacks regarding the stem cell self-renewal and proliferation Quantities See also , p. 69.A further model assumption is that in steady state most erythrocytes die dependent on age (i.e. similar to a first-in-first-out kinetic), but under stimulation, erythrocyte degradation occurs more randomly  This model of bone marrow erythropoiesis was established on the basis of several experimental results for example on irradiation, bleeding, hypoxia and hypertransfusion in mice A pharmacokinetic model of EPO was developed by Krzyzanski & Wyska Coupling this pharmacokinetic model with our cell kinetic model is achieved by three mechanisms:Model assumption 1: There is an additional influx of endogenously produced EPO into the central compartment. Endogenous EPO is produced in accordance to the cell kinetic model.Model assumption 2: The production of EPO receptors depends on the bone marrow cellularity, i.e. the compartments BE, CE, PEB, MEB, RET of the cell kinetic model.Model assumption 3: Internalised EPO serves as the argument of all Z-functions of the cell kinetic model representing EPO regulations.All three assumptions are natural extensions of the model. A schematic illustration is shown in Dynamics of the peripheral compartment and the EPO-receptor complex are described by the following equations The specific elimination of EPO depends on the number of EPO receptors. According to assumption 2, we assume that the dynamics of EPO-receptors We now derive the initial values of the differential equations. At first, we set.Initial values of the other equations are also derived from steady-state conditions:According to The coupling of the pharmacokinetic model and the cell kinetic model is completed by using the relative internalised EPO  which is modelled by a factor Different cytotoxic drugs damage independently of each other These assumptions are extensively discussed in The output-function Implementation and simulation of the model were performed using MATLAB 7.5.0.342 (R2007b) with SIMULINK toolbox . Numerical integrations of the equation system were performed using the variable step solver from Adams and Bashford included in the SIMULINK toolbox (ode113).Data on bone marrow erythropoiesis are hardly available for humans. Consequently, one can find only a few hints regarding values of certain model parameters in the literature. The majority of model parameters must be estimated indirectly via fitting the predictions of the model to clinical data after various therapeutic interventions. This applies for parameters of all submodels: the cell kinetic model, the PK model, the injection model and the chemotherapy model.Even if parameter estimates of the submodels were provided in general, it was necessary to adapt these parameters in view of the greater data base to be explained with our model. For example, the injection model was developed for sheeps and must be re-parametrised for humans. The original PK model The effects of chemotherapy on this cell kinetic model were also not studied in the former publications. Modelling different chemotherapy regimens is of particular concern since it requires estimation of corresponding toxicity parameters. In general, these parameters describe the toxicity of a drug combination, since cytotoxic drugs are usually applied as polychemotherapy including several drugs at the same time. However, by comparing different chemotherapies, it is sometimes possible to estimate the toxicity of single drugs.To reduce the number of parameters to be fitted, we used the following approach:We made simple model assumptions to reduce the complexity of equations, and with it, the number of unknown parameters.Parameters for which experimental data are available were taken from the literature, either directly or by defining plausible ranges.Parameter values of the cell kinetic model and the chemotherapy model which were determined by previous modelling works were kept constant. This applies for parameters of the stem cell regulation and chemotherapy toxicity.Different EPO derivatives and injection sites were grouped according to expected similarities regarding absorption kinetics. Parameters of the injection model were assumed to be constant within these groups but may differ between groups. Nine such groups were identified (see below). PK parameters were assumed to be constant for endogenous EPO and the derivatives Alfa, Beta, Delta but are assumed to be different for Darbepoetin.Parameters of our model were determined by a stepwise fitting procedure. First, pharmacokinetic and -dynamic parameters (PD) of EPO and parameters of the cell kinetic model were estimated by comparing the predictions of the model with available time series data after application of EPO into healthy volunteers. Then, data of different chemotherapy regimens are used to identify corresponding toxicity parameters. Parameters are kept constant in the following fitting process. Fitting is achieved by optimising the agreement of model and data via the condition.We used (1+3)-evolutionary-strategies with self-adapting mutation step size to find optimal parameter sets as good as possible ,  see . For theA complete parameter list for our model is provided in the German High Grade Non-Hodgkin's-Lymphoma Study Group and the German Hodgkin's Lymphoma Study Group. For our purposes, we censored patient data at the time of first erythrocyte transfusion. Patient data of the same chemotherapy arm, cycle number and cycle day were pooled in order to obtain median time courses under the different therapies. Data sets of patients with anaemia due to kidney failure were intentionally not considered so far since this clinical situation requires other model assumptions. An overview of all available data sets is presented in Available data sets comprised time course data of haemoglobin, haematocrit, reticulocyte counts, percentage of reticulocytes, red blood cell counts or serum concentration of EPO after single or multiple applications of four different EPO derivatives  to healthy volunteers at a large variety of injection sites. These data were taken from the literature. Automated tools were used to extract the data from the figures as precisely as possible . Since our model describes the dynamics of reticulocytes and erythrocytes, a set of transformation formulas are required to compare model results with available dynamics of haemoglobin (HB) or haematocrit (HCT). Baseline HB values showed a large variance between the heterogeneous populations in the data sets. Since relative compartment sizes were modelled, comparison with data can be achieved by normalising values to baseline. We assume proportionality of HB and the numbers of reticulocytes and erythrocytes. Thus,Analogously, we use the baseline RBC as normalisation factor Percentages of reticulocytes and the haematocrit were determined byWe first study the qualitative behaviour of our model on the basis of our parameter estimates.Model dynamics after single perturbations were investigated. A constant influx of 10 IU EPO per day  results in a new steady state after a damped oscillation see . The comNext we studied the behaviour of the relative cell counts of all compartments after a single application of CHOP chemotherapy. After a damped oscillation, the system returns to steady state in about 20–40 days see .The injection model, the cell kinetic model, and the pharmacokinetic model were parameterised in one step. Nine different groups of subcutaneous injection modes were distinguished according to available data sets in the literature, namely injections of EPO Alfa into thigh, injections of EPO Alfa into shoulder, injections of EPO Alfa into forearm, injections of EPO Alfa into upper arm or abdomen, injections of EPO Beta into forearm, injections of EPO Beta into arm or abdomen, injections of EPO Beta into thigh, injections of EPO Delta, injections of Darbepoetin Alfa. While the injection parameters differ between groups, the PK parameters are the same for all EPO derivatives and equal to those of endogenous EPO except for Darbepoetin. Parameters of the injection and pharmacokinetic model were determined by fitting the predictions of the model to data of EPO applications into healthy volunteers.We simulated 42 scenarios differing in EPO derivatives applied, injection sites, doses and schedules and compared the predicted EPO serum concentrations with the data. Parameter estimates of the injection model and the pharmacokinetic model resulted in a good agreement of model predictions and data of EPO serum concentration for almost all scenarios. Examples are shown in To illustrate the differences between derivatives and injection sites, we calculated the bioavailability and the percentage of drug absorbed via the lymphatic system. Here, we define bioavailability as the ratio of integrated influxes into the central compartment under subcutaneous and intravenous injection respectively , table 2Regarding PK parameters, we distinguished between EPO Alfa, EPO Beta, EPO Delta and endogenous EPO on one hand, and, Darbepoetin-Alfa on the other hand. Corresponding sets are valid for all application modes and doses considered. Unspecific elimination of Darbepoetin (The same pharmacodynamic (PD) parameters were assumed for endogenous EPO and the first generation EPO derivatives . But for Darbepoetin, PD parameters were allowed to differ which results in two parameter sets for loop 3.Parameter estimates resulted in a good agreement of model and data for almost all scenarios. Examples are shown in After establishing parameter sets of our injection, PK and cell kinetic model, we aim to apply our model to different chemotherapy scenarios. This requires estimation of toxicity parameters which were determined by fitting the predictions of the model to data of patients treated with either BEACOPP-21, BEACOPP-21 escalated, CHOEP-14,21, CHOP-14,21, the sequential chemotherapy schemes EC-T , E-T-C , E-T-C plus EPO, highCHOEP-21, Platinum plus Etoposide or Platinum plus Etoposide with Darbepoetin. For most of these scenarios, only haemoglobin dynamics are available. Toxicity parameter estimates resulted in a good agreement of model predictions and data in the sense that for almost all scenarios and time points, the model curve is in between the 25th and 75th percentile of patients data et al. with weekly injections of 600 IU EPO Alfa into the thigh A few data sets were not utilised for parameter fitting, but were used for model validation. Data sets can be used for validation if no additional parameters must be fitted, i.e. the chemotherapy toxicities and the injection model parameters are known from a different data set. These data comprise the following scenarios: EPO serum concentration from athletes receiving a single dose of 50 IU/kg or multiple application of a high dose of 200 IU/kg EPO Alfa on day 0, 2, 4, 7, and 10 into the upper arm After calibration and validation of the model, it can now be used to make clinically relevant predictions. As an example, we study the effect of anaemia prophylaxis during 8 cycles of BEACOPP-21 escalated or CHOP-14 chemotherapy by weekly injections of Darbepoetin 150 IU/kg. Predictions are presented in et al.In the present paper, we developed a comprehensive ordinary differential equations model of human erythropoiesis under chemotherapy and EPO applications. For this purpose, we combined a former cell kinetic model of bone marrow erythropoiesis et al.et al.The proposed model is not the first attempt to model erythropoiesis: Ordinary differential equations based models of the cell kinetics of erythropoiesis in mice and rats were developed by our group in the past The cell kinetic model of erythropoiesis proposed by Wichmann & Loeffler et al. was attached in a modified version. As in The injection model proposed by Kota For chemotherapy modelling, we used exactly the same assumptions and equations as for our granulopoiesis model The majority of model parameters were determined by fitting the predictions of the model to clinical data sets. For this purpose, we extracted virtually all available clinical data of EPO serum concentration, haemoglobin, haematocrit, reticulocytes and red blood cells under various scenarios such as EPO application, chemotherapy and combinations of it from the literature. We also used the raw data of cooperating clinical chemotherapy trials groups. We aimed at modelling this large variety of scenarios in order to test whether our model assumptions are valid in a broader range of clinical applications.We used the same cell kinetic parameters for all scenarios except for those quantities regulated by EPO. For these parameters, we assumed differences between Darbepoetin Alfa and other EPO derivatives. This was motivated by observed pharmacodynamic differences between Darbepoetin and conventional EPO possibly due to additional glycosylations of Darbepoetin For parameterisation of the pharmacokinetic model, we assumed differences between Darbepoetin and conventional EPO or endogenously produced EPO again. We reparameterised the model proposed by Krzyzanski & Wyska Since there was a large variation in injection sites for subcutaneous EPO applications, it was necessary to assume nine different parameter sets for the injection model in dependence on the site and the derivative. We aimed at keeping the number of these parameter sets as small as possible by grouping the different injection sites appropriately.To simulate chemotherapy scenarios, it was necessary to estimate corresponding toxicity parameters. Even though the toxicity to stem cells strongly influences the model behaviour, we decided to use exactly the same estimates as for our model of granulopoiesis Parameter estimates appeared to be plausible in general. For the injection model, we estimated that EPO Beta and Darbepoetin are both characterised by a high bioavailability of about 80% Click here for additional data file.


Estimates of age-specific fertility rates based on survey data are known to suffer down-bias associated with incomplete reporting. Previously, William Brass  proposed a series of adjustments of such data to reflect more appropriate levels of fertility through comparison with data on children-ever-born by age, a measure of cohort-specific cumulative fertility. His now widely-used Parity/Fertility or PF ratio method makes a number of strong assumptions, which have been the focus of an extended discussion in the literature on indirect estimation. However, while it is clear that the measures used in making adjusted age-specific fertility estimates with this method are captured with statistical uncertainty, little discussion of the nature of this uncertainty around PF-ratio based estimates of fertility has been entertained in the literature. Since both age-specific risk of childbearing and cumulative parity (children ever born) are measured with statistical uncertainty, an unknown credibility interval must surround every PF ratio-based estimate. Using the standard approach, this is unknown, limiting the ability to make statistical comparisons of fertility between groups or to understand stochasticity in population dynamics. This paper makes use of approaches applied to similar problems in engineering, the natural sciences, and decision analysis—often discussed under the title of uncertainty analysis or stochastic modeling—to characterize this uncertainty and to present a new method for making PF ratio-based fertility estimates with 95 percent uncertainty intervals. The implications for demographic analysis, between-group comparisons of fertility, and the field of statistical demography are explored. Population dynamics are driven by the often complex interplay of demographic components of change including births, deaths, immigration, and emigration level of fertility achieved by a specific age level of achieved fertility captured in the cohort measures As early as 1964, William Brass suggested the possibility that given an assumption that underreporting is equivalent across age groups, period measures of age-specific fertility could be adjusted by leveraging information on cohort parity such as the average number of children ever born to a woman of a given age While there is no set algorithm for determining which PF ratio to use The simplicity and ease with which the Brass PF ratio method is implemented, its apparent ability to produce much more reasonable estimates of age-specific fertility rate and total fertility rate than unadjusted ones, and its promotion by the leading demographic research units involved in training demographers around the world uncertainty analysis or stochastic modelinginputs formulated with uncertainty affect uncertainty in an output of interest This issue of statistical uncertainty in estimation of fertility using the PF ratio method has received little attention in the demographic literature. Given the data sources used in its construction, however, it seems an unavoidable fact that such statistical uncertainty is associated with the ratio. Estimates of age-specific fertility risk and children ever born by age are both measured through survey data in the case of variant B3, and sampling variability is contained in both. If we think of both estimates as inputs to the PF ratio estimate, then the adjusted age-specific fertility schedules represent an output into which the uncertainty associated with each input is propagated. This uncertainty has not received an adequate amount of attention within the demographic literature, perhaps because demographers remain unfamiliar with potential solutions to the challenge and often make little use of statistical theory in their work as well as an estimate of the 95% upper and lower confidence bounds for the estimate. The end result of the procedure is a stochastic estimate of age-specific fertility that both adjusts for under-reporting while incorporating statistical information on uncertainty into the estimates. It provides a statistical distribution of ASFR to assess quality as well as to incorporate into stochastic projection models if desired The conceptual application of uncertainty analysis to the estimation of PF ratios with uncertainty intervals is illustrated in www.measuredhs.com) and survey-based estimates of age-specific fertility from the Sample Registration System (SRS) of India (www.census.in). These data are utilized to illustrate the stochastic approach to the Brass method and provide point estimates and 95% upper and lower uncertainty bounds on these estimates using the exact process described in The purpose of this paper is to illustrate this novel approach for producing stochastic estimates of ASFR within the Brass PF ratio framework. The paper utilizes data from 5 randomly-selected Indian provinces and models age-specific fertility using data on children-ever-born from the Demographic and Health Surveys (http://censusindia.gov.in). Data on children ever-born was obtained for each province using microdata from the associated Demographic and Health Surveys (www.measuredhs.com) for 1998. The one year gap between these datasets was considered trivial and unlikely to be of sufficient temporal duration to introduce bias into these estimates. Average children ever born by age were computed from the DHS data by the authors, including estimation of variance associated with each these estimates. The uncertainty associated with the SRS estimates is not publicly-reported, presenting a challenge for evaluating the uncertainty in PF ratio estimates associated with these data. As a reasonable substitute, we estimated variances associated with the binomial proportion of births in the last twelve months captured in the Demographic and Health Surveys, using the normal approximation A random sample of five Indian provinces was taken to include Bihar, Uttar Pradesh, Tamil Nadu, Madhya Pradesh, and Goa . ReporteUsing these data, PF ratios were computed, first using the standard procedure , then thThe base results suggest that fertility underreporting is greater in younger age intervals and varies across provinces . A consieven higher estimates of the overall level of fertility than were observed in the traditional application of the Brass method. The 95 percent uncertainty intervals presented in The main contribution of this research has been to illustrate how the Brass PF ratio method may be extended to incorporate statistical uncertainty associated with data inputs using simple methods from stochastic modeling and uncertainty analysis. The results of the procedure provide point estimates and 95% uncertainty intervals for age-specific fertility that adjust survey-based estimates for underreporting of births. The approach represents a potentially significant improvement in stochastic demographic modeling, suitable for use in making between-group fertility comparisons as well as in demographic estimation or projection models for which a desire to incorporate statistical uncertainty exists. In the latter vein, there are natural links between this work and recent developments within the field of stochastic population forecasting From a practical point of view, it is clear that the results of the stochastic Brass PF ratio method differ in important ways from those found using the traditional procedure. First and foremost, the stochastic PF ratio-based estimates of age-specific fertility suggest much higher total fertility rates than those calculated using the deterministic procedure . On averNo gold-standard reference fertility levels exist against which we may benchmark the performance of the stochastic method—or the traditional application for that matter. A number of possibilities exist with respect to the accuracy of the proposed method. First, it is possible that the traditional PF ratio based estimates of age-specific and total fertility are accurate and that the stochastic variants do represent over-estimates of fertility levels. A closer correspondence between the DHS-reported parity of women 45–49 and the traditional Brass-based estimates is supportive of this proposition, but this is likely a mechanistic artifact because it is the DHS data itself which drives the P3/F3 ratio used in the deterministic version of Brass' model. It seems more likely that some aspect of the error propagation associated with the simulation of the distribution of the P3/F3 ratio is responsible for the increased values. Along these lines, inflated values of the P3/F3 ratio could occur if the distributions associated with ASFR estimate are mis-specified. One way this could have occurred in the current analysis is associated with an assumption that the DHS distributions of fertility during the past 12 months would be reflective of the variation associated with the SRS-based estimates—introduced by a lack of available data from the Indian Census on standard deviations of these measurements. In this case, wider than estimated margins of error associated with mis-specified standard deviations could produce the higher than expected stochastic Brass PF ratio-based estimates reported here.While possible, this shortcoming does not invalidate the thrust of this paper. The focus of here has been illustrative: it provides a clear example with readily-available data of the proposed application. In practical application, any appropriate distribution might be applied to estimating the first-step inputs driving the procedure. Moreover, direct estimates of standard deviations with appropriate sample sizes—rather than surrogate inputs to approximate them—should produce less-inflated estimates of age-specific and total fertility rates. It remains possible that incomplete reporting in the DHS could result in mis-estimation of the surrogate standard deviations utilized in this study—just as easily as the case with the SRS-based estimates. In that case, the inflation could result from the same systematic incompleteness associated with this dataset. Systematic underreporting of fertility can lead to mis-estimation of variance just as easily as the proportion, suggesting that this inflation could as easily be real as artifactual. Without a gold-standard for comparison, this issue will remain unresolved in the current research.It is clear that the potential inflation of these estimates, however, is not a basic shortcoming related to the use of monte carlo simulation for fitting distributions, which has a long history in statistics, uncertainty analysis, and decision modeling. It is intimately associated with many forms of hypothesis testing in these disciplines The approach presented in this research appears to be a fruitful direction for further development of a stochastic methods for indirect estimation in demography. The paper presents a valid method for estimating the uncertainty associated with Brass PF ratio-based age-specific and total fertility rates. Future evaluations should explore the properties of this method across a larger variety of settings and attempt, where possible, to investigate further whether the method is prone to upward-biasing of these estimates or, in fact, produces more accurate ones. In either case, demographers in need of incorporation of statistical uncertainty into indirect estimation procedures may find the described method here to be a fruitful avenue for application.Figure S1Glossary of Terms.(DOCX)Click here for additional data file.Figure S2Annotated Sample R Code to Perform the Stochastic Brass PF Ratio Method(DOCX)Click here for additional data file.