An introduction to the accompanying three papers. Osteoarthritis (OA) is a leading cause of disability in the United States. It is the most common form of arthritis and afflicts 13.9% of U.S. adults aged 25 and older and 33.6% 12.4 million) of those over 65—an estimated 26.9 million individuals in the United States in 2005 [ million Although our understanding of the underlying causes of OA is increasing, we are only beginning to appreciate differences in the disease that exist with respect to sex or gender. Studies sponsored by the Centers for Disease Control and Prevention and the National Institutes of Health have identified differences in the incidence and severity of OA between men and women as well as between racial and ethnic groups ,5. The bin vitro cell culture or animal models.Degenerative arthritis has been studied extensively in mice and other laboratory animals such as rats, guinea pigs, rabbits, dogs, and more recently, rhesus monkeys . Such stThat sex differences exist in OA was recognized as far back as 1956 . NonetheThe information provided in Table Cell culture studies are similarly limited with respect to the sex of the cell source. For those studies that use primary cells, whether from animals or humans, most do not directly compare the same set of experimental parameters for both sexes. Moreover, there is often a lack of a statistical design that provides sufficient power to adjust for inter-human variability. When cell lines are used, the sex of the cell is generally not provided. Thus, statements are made concerning underlying mechanisms that may not be accurate for the broader population of subjects.Diagnosis of knee OA is based on evidence of joint pain or reduced space between articulating bone surfaces due to thinning of the opposing articular cartilages. However, multiple tissues that comprise the knee joint appear to be compromised by the disease, including subchondral bone, articular cartilage, meniscus, anterior cruciate ligament, synovium and synovial fluid, and the innervation of these tissues. A change in any of these tissues can influence the distribution of load across the joint, with corresponding adaptations in the other tissues and ultimately the cartilages . Such paExperimental and computational data suggest that contact stress in joint cartilage is a significant predictor of the risk for developing knee OA. Limb alignment, a major determinant of mechanical stresses within the knee, can predict the development of radiographic signs of knee OA, but these data do not indicate how limb alignment could contribute to sex differences. Similarly, little is known about how sex-related changes in muscle function might contribute to the worsening of knee OA. Significant gaps in knowledge remain as to how changes in musculoskeletal traits, such as obesity, disturb the normal mechanical environment of the knee joint and contribute to sex differences in the initiation and progression of knee OA .Knee tissues are modulated by sex hormones during tissue development and throughout the life cycle in both males and females. Whereas menopause in women is associated with an increase in OA severity, systemic estrogen alone cannot explain the observed sex differences . Recent In addition to a greater prevalence of knee OA, women also often report greater pain and more substantial reductions in function and quality of life than men . OA painEpidemiologic studies have established that there are sex differences in the incidence and severity of knee OA. Therapeutic approaches to the treatment of OA, particularly regenerative medicine strategies, have not yet taken these sex differences into consideration ,22. EffeThe authors declare that they have no competing interests.This paper was written by the authors to provide an overview of the gaps of knowledge that exist in the study of sex differences related to osteoarthritis. BDB wrote the initial draft, which was then edited by the other authors. All authors read and approved the final manuscript.


Inflammatory bowel diseases (IBD) refer to a group of inflammatory conditions concerning colon and small intestine, which cause socially uncomfortable symptoms and often are associated with an increased risk of colon cancer. IBD are complex disorders, which rely on genetic susceptibility, environmental factors, deregulation of the immune system, and host relationship with commensal microbiota. The complexity of these pathologies makes difficult to clearly understand the mechanisms of their onset. Therefore, the study of IBD must be faced exploiting an integrated and multilevel approach, ranging from genes, transcripts and proteins to pathways altered in affected tissues, and carefully considering their regulatory mechanisms, which may intervene in the pathology onset. It is also crucial to have a knowledge base about the symbiotic bacteria that are hosted in the human gut. To date, much data exist regarding IBD and human commensal bacteria, but this information is sparse in literature and no free resource provides a homogeneously and rationally integrated view of biomolecular data related to these pathologies.Human genes altered in IBD have been collected from literature, paying particular interest for the immune system alterations prompted by the interaction with the gut microbiome. This process has been performed manually to assure the reliability of collected data. Heterogeneous metadata from different sources have been automatically formatted and integrated in order to enrich information about these altered genes. A user-friendly web interface has been created for easy access to structured data. Tools such as gene clustering coefficients, all-pairs shortest paths and pathway lengths calculation have been developed to provide data analysis support. Moreover, the implemented resource is compliant to the Galaxy framework, allowing the collected data to be exploited in the context of high throughput bioinformatics analysis.http://www.itb.cnr.it/ibd), a disease-oriented platform, which collects data related to biomolecular mechanisms involved in the IBD onset. The resource provides a section devoted to human genes identified as altered in IBD, which can be queried at different biomolecular levels and visualised in gene-centred report pages. Furthermore, the system presents information related to the gut microbiota involved in IBD affected patients. The IBDsite is compliant with all Galaxy installations , in order to facilitate high-throughput data integration and to enable evaluations of the genomic basis of these diseases, complementing the tools embedded in the IBDsite.To fill the lack of a reference resource for 'omics' science analysis in the context of IBD, we developed the IBDsite (available at Lots of sparse data exist concerning IBD studies, but no on-line resource homogeneously and rationally integrate and collect them. The IBDsite is an attempt to group available information regarding human genes and microbial aspects related to IBD, by means of a multilevel mining tool. Moreover, it constitutes a knowledge base to filter, annotate and understand new experimental data in order to formulate new scientific hypotheses, thanks to the possibility of integrating genomics aspects by employing the Galaxy framework. Discussed use-cases demonstrate that the developed system is useful to infer not trivial knowledge from the existing widespread data or from novel experiments. Inflammatory bowel diseases (IBD) are a group of inflammatory conditions concerning colon and small intestine, which cause socially uncomfortable gastrointestinal symptoms. In particular, Crohn's Disease (CD) and Ulcerative Colitis (UC) present the highest incidence in population and are associated with an increased risk for colon cancer .controlled inflammatory condition, caused by the physiological and irreplaceable presence of microbial flora in the bowel, to a pathological inflammation is still widely unknown [Understanding the causes of the IBD onset is not trivial, since they are complex polygenetic diseases that involve the contributions of genetic susceptibility  , which presents the IBDsite among the list of data sources, thus promoting analysis on IBD related genomic high-throughput users' data.Our approach was to develop the IBDsite, a disease-oriented platform, which collects data related to biomolecular mechanisms altered in IBD. This site records human and bacterial information related to CD and UC, and exploits data integration strategies and analysis tools to help inferring not trivial knowledge from the existing widespread data or for analysing novel experimental data. The IBDsite is freely available at the URL platform , as it cThe IBDsite aims at representing a first attempt to knowledge and discovery integration of IBD biomolecular aspects by collecting human genes involved in IBD  together with data about the involved commensal bacteria. To our knowledge, this represents an important and innovative reference in this field. On the other hand, the presented platform enables the integration with the Galaxy system, thus promoting the analysis of novel genomic experimental data (i.e. sequencing and microarray) in the perspective of enhancing the knowledge about IBD.The IBDsite relies on the MySQL database management system for data maintenance, which enables collecting and formatting heterogeneous data from different sources, in order to make them accessible to the scientific community through a unified query schema by means of a web interface Figure . InformaThe primary data collection about genes involved in IBD has been performed manually from literature, which in the authors' opinion is an added value of the implemented database, since it guarantees a high reliability of the resource. This manual process will be performed periodically, according to novel papers published in this field.Annotation data have been retrieved from diverse resources, differently formatted and organized, that includes: Gene Ontology  for starhttp links or ftp sections for resources without direct access to data. In both cases, the developed system is robust even in the case of changes to the original resources, since data are typically provided in structured formats. Retrieved information was parsed and formatted suitably to populate the database tables: this enables an easy data access (through sql query language) and the creation of a user-friendly interface. Monthly maintenance will be performed by the system developers to keep updated the IBD repository data. Users will be acknowledged of the latest updates.Different scripts for downloading annotation data regarding genes involved in IBD were implemented ad hoc for each resource, in a typical data warehouse fashion. Repositories that enable batch download modes, such as NCBI maintained databases, have been queried following standardized approaches (i.e. web services), while download has been enabled through suitable In order to exploit the IBDsite knowledge base for the analysis of biomolecular high-throughput experiments, the system was designed to be a source of data for the Galaxy platform, one of the most popular frameworks for genomic sequences management, their alignments, and functional annotation. This integration can be tested in our local instance of this framework, called Galaxy@ITB, through which all the examples in the following section were performed. The IBDsite was inserted among the data sources of Galaxy@ITB, by defining an XML configuration file, which instructs the system in loading the file provided by the external source. This configuration allows the user to select a set of genes of interest from the IBD database and to exploit it as knowledge base for analysing external datasets such as novel experimental results or literature datasets from recognised repositories.The web interface of the IBDsite Figure  has beenFor each human gene known to be involved in IBD a report is available. IBD genes are annotated by gene symbol, description, aliases, sequences and related molecular alterations with reference to the papers where are described. Collected evidence concerns genomic variations such as Single Nucleotide Polymorphisms (SNPs) occurring in genes and their flanking regions, gene expression level, and transcription and translation regulation factors. Gene expression information is supported by a link towards the Gene Expression Atlas  report pThe immunological aspect of the IBD onset must be evaluated in the perspective of patients' microbiome composition. Therefore, it is crucial to consider the presence (and the quantity) of commensal bacteria, which are known to deregulate the immune system in UC and CD. A whole section of the IBDsite is dedicated to the microbiome members involved in these pathologies. Data about bacteria can be accessed through the 'Bacteria' link in the left menu, where user can query the IBD involved bacteria and the human genes altered in disbiosys. The bacteria report lists the bacterial taxonomy information together with the reference papers about their involvement in the considered pathologies, and the correlated altered human genes, if known: in the latter case both these data are also visualized on the genes report page, thus providing a complete overview of the host/commensal relationship.GetData section (left menu). The interaction between the IBDsite and Galaxy allows to import information that enable the analysis of novel data in the light of the IBDsite knowledge content. Genomics data can be uploaded in the system either from the local file system or by downloading them directly from standardized remote repositories of high-throughput biomolecular data, such as SRA for short read sequences, or GEO and ArrayExpress for gene expression microarrays. This is possible in Galaxy@ITB by employing customized Galaxy tools (i.e. the genebank plugin and the Rgenetics package).IBDsite data can be accessed through the Galaxy platform, in particular, considering the Galaxy@ITB instance of this framework, by clicking on the suitable link listed under H. sapiens genes and gut bacteria genomics data, with particular concern to next generation sequencing data, both for DNA and RNA sequences, genotyping data, and gene expression microarrays.This integration strategy is oriented to The procedure for sequences analysis consists in different pipelines of semi-standardized steps, provided by the Galaxy framework and customizable for specific requirements. Generally, these steps include data quality evaluation, sequence trimming, data mapping on human and bacteria genomes, differential expression evaluation, and phylogenetic analysis. Relying on the features enabled by Galaxy@ITB, reads can be visualized on many different genome browsers  have been defined in order to point out real differences in the gene expression of affected and unaffected patients. Although the original analysis was performed with a different pipeline , resultThe added value provided by the IBDsite integration to these analyses is the possibility to reconsider data in the view of our knowledge base, by evaluating for example only specific pathways or biological processes involved in IBD. The whole perspective of the analysis can be reverted, by mapping expression of genes belonging to specific networks , instead of inferring annotation only of differentially expressed genes (like it is common to proceed).immune system pathway, although largely expected, comes out clearly when genes of this pathway are used to filter DEGs.Concerning biomolecular pathways and biological processes, the IBDsite can show genes involved in specific networks related to IBD. Dataset transmitted to Galaxy@ITB reports genes included into specific pathways: those altered in IBD are labelled and can be visualized as separate subsets. For example, we intersected the DEGs related to microarray data, achieved using fold change values calculated with the MAS5 algorithm, with genes of different pathways. Results are reported in Table http://www.itb.cnr.it/sup/ibd/v/colon-microarray).Data can be visualized in one of the genomic browsers available at Galaxy@ITB together with data extracted from the IBDsite, considering, for example, the whole set of genes maintained in the database and the subset related to the immune system. An example of data visualization is reported in Figure The immediate and complete vision provided by this integrative visualization on the whole genome allows a clear overview of the involvement of genomic regions in the pathology onset. For example, the chromosome 1 appears as one of the most involved in the four considered diseases , as it can be inferred by the correlation among DEGs found in the four pathology subgroups and the knowledge retrieved from the IBDsite. The possibility to visually integrate this information permits, for example, to recognise region chr1:140,000,000-170,000,000 Figure , as cruchttp://www.itb.cnr.it/sup/ibd/h/colon-microarray).The full set of data and analysis steps used for this use-case are accessible at Galaxy@ITB in the Published History under the name 'Colon Microarray'  describes the gut microbiome of different patients affected by UC, coming from gut biopsies collected in the frame of the multi-center Ulcerative Colitis Human Microbiome Project (UCHMP), which aims at examining the role of the enteric microbiome in causing human ulcerative colitis.Starting from the hypothesis that changes in microbiota will precede the onset of IBD, the main target of the study is to determine the role of the gut bacteria in the development of human inflammatory bowel disease using the whole metagenome sequencing technique. The microbial profile of the inflamed area will lead the choice of the most effective antibiotic therapy. Human tissue components have been removed from the samples, thus the aligned sequences should refer only to the bacterial components of the bowel. Data have been locally downloaded and analysed using a Galaxy workflow, which mainly relies on the annotation of sequences using Megablast  against Using Galaxy@ITB, two samples of commensal bacteria from UC patients (identifier: SRR059128 and SRR059129) have been analysed using the metagenomics workflow, and results about the obtained phylogenetic tree are shown in Figure Using the information available in the IBDsite, it is possible to annotate the phylogenetic tree computed by Galaxy@ITB in order to correlate information regarding the identified microbioma alterations and the gene correlated to this disbiosys. In particular, for each leaf of the phylogenetic dendrogram it is possible, by means of the IBD site knowledge base, to correlate the pathologies (and the PubMed identifier of the literature reference), the experiment type, the sample type, the involved human genes and additional notes when available. In the example, this annotation helps in correlating the disbiosys with mucosal cytokine and chemokine profiles, in particular for interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8) and TNF alpha -47.http://www.itb.cnr.it/sup/ibd/h/colon-metagenomics.Data and analysis steps used for this analysis are accessible at the Galaxy@ITB site in the Published History under the name 'Colon Metagenomic', through the link genebank plugin, available at our local instance of Galaxy.A last example is within the context of human RNA sequencing. In this case we chose to consider samples of patients affected by colon cancer versus normal samples, since no data are available concerning RNA-sequencing in relation to IBD affected patients. Nevertheless, the choice is well fitted with the research on IBD, since colon cancer onset is the most diffuse follow-up of inflammatory bowel diseases. RNA-sequencing data have been automatically downloaded from the SRA repository at NCBI using the http://www.itb.cnr.it/sup/ibd/h/colon-cancer).In detail, four runs have been downloaded for this example , which describe the expression profile by high throughput sequencing  of human colon cancer and normal samples of two affected patients. Data have been processed using a well-established workflow for next generation sequencing analysis, which mainly relies on the bowtie algorithm , in ordehttp://www.itb.cnr.it/sup/ibd/v/colon-cancer.Mapped results, both tumour affected and normally affected ones, have been visualized in the same track, together with the IBDsite data, to present the intersections among the differentially expressed genes and the set of genes known to be involved in inflammatory bowel disorders. Data visualization is available at the following link Some biologically considerations can be sketched immediately from these visualized data. An overview of the whole chromosomes set allows to identify a region with differential expression among normal and colorectal cancer affected patients: this corresponds to the SLC26A3 gene, known to be involved in gastrointestinal pathologies such as congenital chloride diarrhea  and ulceThe inflammatory microenvironment related to the IBD has a prominent role in the onset and progression of colon cancer, but also a plethora of growth factors and cytokines are involved in this process. For example, tumour-necrosis factor-α , interlehttp://www.itb.cnr.it/sup/ibd/v/micro-cancer). This hypothesis is confirmed by epidemiologic data that describe a higher risk to develop colon cancer in patients with UC with respect to CD [Combining different 'omics' data, although highly desirable, results a difficult task to accomplish, because of diverse experimental conditions and analysis inhomogeneity, which often lead to unmatchable data. An attempt to overcome this limitation is the use of a bottom-up approach to data, such as that proposed in the IBDsite. In the IBDsite the layer of manually collected genes allows to browse and integrate different types of data: this strategy is made possible by the interaction with the Galaxy framework, as shown in the previous discussion. By means of the IBDsite genes, information provided by IBD microarrays data with RNA-sequencing data of colon cancer can be combined, to formulate hypotheses about mechanisms underpinning the pathological inflammatory correlation of these diseases with colon cancer. Although RNA-sequencing data in patients affected by IBD would provide more consistency to the description of this process, the analysis performed shows a higher correlation of Ulcerative Colitis genomic patterns with colon cancer in comparison to Crohn's Disease. This aspect clearly emerges in Chromosome 6, Chromosome 10, Chromosome 11, Chromosome 12, Chromosome 16, where microarray derived expression profiles of IC and UC samples resembles the trend of sequencing data in patients with colon cancer much more then CD samples (ct to CD .Inflammatory bowel diseases represent a diffuse and disabling class of pathologies, affecting people that approach their life in a reasonably normal way. Regardless the high incidence and a variety of epidemiologic and genetic studies, causes of the onset and development of IBD remain unclear. The sparse and unstructured knowledge developed in this field neither correctly support the advances in the development of a pathology onset model, nor the identification of effective and personalised therapies for these complex pathologies.The main targets in the field are the comprehension of the mechanisms underlying the pathology development, the identification of the bacteria involved in the interactions with the human genome, and the identification of the biomolecular hubs where it is possible to intervene with therapies, especially considering the great interindividual variations that can modify the enteric microbial communities.The presented work tries to fill the lack of a curated IBD knowledge base. The IBDsite represents both an infrastructure to analyse high-throughput experimental data and an integrated collection of homogeneous knowledge, that remains crucial for evaluating new information and to formulate novel scientific hypotheses. The IBDsite maintains, in a structured format, the available biomolecular knowledge about the human genes altered in IBD and the bacteria found involved in their onset, thus acting as a non redundant, consistent and homogeneous container for CD and UC known biomolecular information.The IBDsite, having the ability of working in connection with the Galaxy platform, provides a crucial possibility of handling new experimental data and exploiting the collected knowledge base information, in order to analyse new samples in the light of the present understanding for discerning the multifactorial mechanisms of the IBD onset.The proposed infrastructure is capable of handling different types of IBD data and experiments, providing them the appropriate homogeneity in order to be integrated. The possibility of exploiting data from the IBDsite in order to correlate different experiments is an added value to the work: as in all complex pathology, the collection of data at all 'omics' levels is the only affordable way to achieve a complete vision on the involved biomolecular mechanisms.The authors declare that they have no competing interests.IM participated in the design of the study, performed literature analysis and developed the Galaxy interaction of the system. FV participated in the design of the study, performed literature analysis and developed the reference database, developed the web interface and drafted the manuscript. LM coordinated the project and provided access to the computational facilities for maintaining the bioinformatics resources. All authors read and approved the final manuscript.


The seas along the western European margin encompass a vast geographical area comprising numerous different habitats, and are home to more than 10,000 metazoan species. Although research in this extensive region has been undertaken since the early 1800s, many new species are being described and distributional patterns identified. Recent studies incorporating the most extensive data series ever used in such European studies have failed to find any relationship between latitude and infaunal shelf biodiversity. Along the European shelf, species richness generally increases to a depth of 200 m and then decreases from 300–500 m. In the deep Northeast Atlantic, a unimodal curve illustrates how macrofaunal species diversity changes with depth whilst the megafauna appear to have a bimodal distribution. Regional studies are equivocal in that poleward increases in species diversity have been observed in some studies or taxa, but not in others. In the North Sea, arguably the best studied system in European waters, there appears to be a distinct increase in diversity with increasing latitude. Since this trend is confounded by similar latitudinal gradients in depth and trawling intensity, there is no clear explanation for the biodiversity pattern. Climatic shifts in diversity patterns and species ranges have recently been observed. Here we report previously unpublished data on changes in species richness that have been observed along the Norwegian coast over the past two decades, with the most northerly region seeing more than a 15% increase in the number of species being discovered there. This review synthesizes published and new biodiversity data across multiple spatial and temporal scales, and from the coast to the deep-sea, to provide an overview of what is known along the western European margin. Threats to the biodiversity of the region are highlighted, as well as identifying where there are still gaps in our knowledge.As anthropogenic disturbance and climate change threaten biodiversity in terrestrial and marine habitats, one of the urgent challenges in ecosystem research is to identify causal links between biodiversity and ecosystem functioning (BEF) and services The regional focus of this paper is from ca. 35° N (approximately the same latitude as the entrance to the Mediterranean) to the Arctic and from ca. 25° W (the Mid-Atlantic ridge as the western boundary) to ca. 30° E , encompassing the shallow and deep waters of the Northeast Atlantic, the North Sea, and the Arctic.2, but it has a 45,000 km long coastline, compared with the 112,000 km coastline for the entire Atlantic Ocean. The Eurasian Basin is divided by the Gakkel Ridge to form the Nansen and the deeper Amundsen basins the latter also including the North Pole. Although the basins are relatively deep , the average depth of the entire Arctic Ocean is not more than about 1,330 m, reflecting the fact that more than half of the area is continental shelf. The North Sea (2.The deep-water areas of the Northeast Atlantic  contain orth Sea  is part www.hmapcoml.org). These large-scale circulation patterns structure the marine  climate and habitats of the entire area and extend across more than 60 degrees of latitude, and are therefore a major consideration when looking at potential latitudinal gradients in biodiversity. Our study area is largely influenced by the NAD and its northern extension, the West Spitsbergen Current (WSC), along with water exiting the Arctic throught the Fram Strait as the East Greenland Current (EGC) and coastal waters modified by their respective nearshore processes (e.g. waters exiting the North Sea). This leads to dramatic spatial gradients on a variety of scales with potentially strong, but poorly studied, impacts on macrofaunal diversity patterns.One of the most conspicuous and important factors influencing marine ecosystems in the North Atlantic and the Arctic is the Gulf Stream System (GSS). Together with its northern extension toward Europe, the North Atlantic Drift (NAD), the GSS is a powerful, warm ocean current that originates in the Gulf of Mexico, follows the eastern coastlines of the United States and Newfoundland, and crosses the Atlantic Ocean toward Northwest Europe. The Gulf Stream not only dramatically warms the climate of countries such as Norway and Great Britain but also has a major influence on the marine life in this region. The warm water of the NAD mixes with the colder waters found in the North Atlantic, increasing turbulence and thus nutrient availability, which in turn resulted in some areas of the North Atlantic becoming the most productive fishing grounds in the world, until overfishing led to a dramatic decline  gives rise to a distinct coastal water mass with low salinity and high turbidity. Seafloor topography largely drives water circulation within the North Sea. The combination of current speed, depth, wave height, and density structure control the heat transfer through the water column and, consequently, stratification. In addition, borders (fronts) between thermally stratified and mixed areas play an important role in the ecology of the North Sea. The presence or absence of stratification also has major consequences for the temperature regime near the bottom and notably the maximum temperatures in summer. Bottom water temperatures in the southern North Sea vary on average between 5°C and 16°C, with more extreme values in shallow coastal waters. These temperatures are relatively high with respect to latitude because of the combination of depth and water supplied by the warm NAD. In the northern North Sea the temperature range is much smaller, with a variation between 6.5°C and 9°C.Natural History of European Seas, in which he divided the European seas into so-called faunal provinces on the basis of the benthic fauna. Because of the economic importance of fisheries, scientific institutions for fishery research were founded around the North Sea, for example, in Germany in 1885 and in the Netherlands in 1888. Their scientific activities also led to more knowledge about the invertebrate fauna Systematic study of the North Sea fauna has a long history dating back to inventories of the tidal zone. The successful use of dredges by Audouin and H. Milne-Edwards in France (1826–28) and Michael Sars in Norway (1829) opened the way to study the deeper waters of the North Sea. From 1839 onward the British began to systematically investigate their marine fauna, and Edward Forbes was one of the main instigators. Results of dredging campaigns led in 1859 to Forbes's book on the In founding the International Council for the Exploration of the Sea (ICES) in 1902 the participating countries  were committed to regular surveys of the fish fauna in the Northeast Atlantic. This resulted in more vessels becoming operational and more data on various aspects of the North Sea ecosystem becoming available.At the end of the nineteenth century, the first marine laboratories were founded. In Scotland in 1884, laboratories were opened in St. Andrews and Granton, the latter being the precursor of the Scottish Marine Biological Association (1897) in Millport, now the Scottish Association for Marine Science in Oban. A second laboratory was opened in the UK, the Plymouth Marine Laboratory (1888) following the foundation of the Marine Biological Association of the United Kingdom (1884). In the Netherlands, the Zoological Society (1872) erected a mobile station in Den Helder (1876) that became the precursor of the Netherlands Institute for Sea Research, now based at Texel. The first complete survey of the North Sea was only conducted in 1986 by a concerted action of the North Sea Benthos Group of ICES Lightning and Porcupine expeditions in 1868-70 highlight the historical importance of this area The Northeast Atlantic deep sea is one of the most well studied deep-water areas in the world and has often been described as the cradle of deep-sea biology One of the primary goals of the MarBEF program was to compile existing data and facilitate analyses so a more holistic view of biodiversity in European waters can be obtained. The initial effort focused on soft-sediment benthos and resulted in the “Macroben” database The Census of Marine Life (Census), European Census of Marine Life (EuroCoML), and affiliated programs have been extremely active in the Northeast Atlantic, North Sea, and Arctic region. There have been a variety of Census field projects operating in the areas of interest, looking at a number of different environments ranging from the coastline down to the deep sea. An important aspect of the program is to ensure that all the data collected are housed in a central location and preserved for future reference. There is a dedicated online open access system within Europe, EurOBIS, which like the Ocean Biogeographic Information System, not only houses new data that have been collected but aims to collect and store historical datasets from around Europe.EuroCoML decided that four separate review papers were needed to cover the vast area of European Seas. The regional focus of this paper is from about 35° N to the Arctic and from about 25° W to about 30° E, encompassing the shallow and deep waters of the Northwest European margin, the North Sea, and the Arctic. Separate reviews relating to marine biodiversity in the Baltic Sea Generally, biodiversity estimates of soft-sediment macrofauna in shallow-water areas are based on counts of species or higher taxa in discrete grab or core samples after sieving over a mesh size of usually 1 mm. Over the past century, many types of grabs and corers have been devised, and the most common ones used today are the van Veen grab and the Reineck boxcorer The collection of macrofauna from the deep waters of the Northeast Atlantic is undertaken using different types of corers, namely, box corers Another problem to be considered is whether the samples should be fixed in formalin prior to, or after, sieving. Sieving after fixation is thought to lead to i) a reduction in the number of animals lost and ii) more intact specimens n individuals are randomly drawn from the sample. For example, ES50 is the number of species expected to be found in 50 randomly selected individuals from a sample. Hurlbert's index is one of the most commonly used indicies in benthic communities, where samples are of an uneven size, and thus is appropriate for the continental- and regional-scale comparisons we make here, even though it may over-estimate the number of species present How to measure biodiversity is the subject of many past and ongoing scientific discussions involving issues of scale and organization level  e.g., . MargurrDue largely to local and regional environmental monitoring efforts, nearshore benthos  is the best studied component of European marine biodiversity. Many of these studies cover a relatively small scale, but recent integrative efforts by MarBEF have resulted in analysis of data compiled from across the European seas, and it is estimated that there are more than 10,000 species in the areas of interest . SpatialEarlier studies have noted several phenomena pertinent to our region of interest. First, fewer species are recorded in the Arctic than in boreal areas Systems of geographical delineation of faunal regions may be useful both for investigation of underlying ecological or environmental processes, and for developing meaningful management policies at different spatial scales. While multiple systems of drawing biogeographical boundaries have been proposed, empirical data have rarely been sufficient to test their rigor. Arvanitidis et al. Two of the more prevalent biodiversity patterns described in the literature are the decrease in richness with increasing latitude  and uniTwo approaches used Macroben to evaluate biodiversity patterns across the whole of European waters, albeit restricted to continental shelf (0–475 m) depths. Whereas an increase in richness to 200 m depth was observed in both studies, no evidence was found to support a LSDG once sampling effort  and depth covariates were removed Novel statistical techniques were used to explore mechanisms that may generate the observed patterns. Processes invoking isolation of subpopulations and subsequent expansion from multiple refuges were more strongly supported than expansion from a center of origin The use of new mathematical models to predict species richness based on sampling intensity and knowledge of habitat characteristics offers promise for both research and management of European biodiversity. Mid-domain effect modeling Standard ecological questions are often best addressed by studies across small- or regional-scale gradients. Macroecology, however, asks questions on large and multiple scales, often spanning several ecosystems Interest in large-scale patterns of benthic communities and their biodiversity was stimulated around 1986 by the growing recognition that the intensive bottom- and beam-trawl fisheries in the North Sea were posing a threat to benthic communities, biodiversity, and habitat. Subsequent investigations such as IMPACT have largely confirmed these negative trawling effects In an earlier analysis of the NSBS data from the southern and central North Sea, Duineveld et al. 2 van Veen grab and a 1 mm mesh Recently an effort has been made to compare patterns found in the 1986 NSBS survey with newer data from 2000–2001 Discriminating which variable is responsible for the observed richness trend, given that a single variable exists, is problematic considering that most variables are correlated . Nevertheless, the common message in the papers above is that hydrographical variables strongly influence the latitudinal trend in macrofauna species richness, and bottom water temperature seems the most influential. One factor not accounted for in explorations of latitudinal diversity trends in the North Sea is the effect of trawling, and especially beam- trawling, on benthos. Intensity of beam trawling is highest in the southern North Sea Quantitative investigations of soft-sediment epibenthos for the entire North Sea were undertaken, initially by Frauenheim et al. On the European continental shelf, LSDGs of soft-sediment fauna have been studied over a range of scales, from 15 to 45 degrees of latitude, and with variable results. At distance ranges of about 15 degrees in the Arctic Ocean, diversity of macrofauna and nematodes was found to decrease with increasing latitude, after controlling for depth  [95], [9Fish species richness has been shown to decline with increasing latitude both globally There has been an order of magnitude fewer studies examining deep-sea diversity latitudinal gradients. Both ecological and evolutionary processes appear to influence global-scale deep-sea diversity patterns, although the patterns themselves are still not fully understood. In studies investigating the deep waters of the Northeast Atlantic and up into the Arctic, there does not appear to be a strong latitudinal diversity gradient. The Greenland-Iceland-Faroe Ridge acts as a physical barrier and thus has an effect on the overall diversity. Work published by Russian researchers in the late 1980s found that the species richness of many different faunal groups, including cumaceans More recent large-scale studies and analyses have investigated the influence of latitude on diversity, most notably Rex et al. A large-scale study undertaken in the UK Atlantic Margin highlights the difference in macrofaunal diversity between the Rockall Trough (Northeast Atlantic) and the Faroe-Shetland Channel (FSC), part of the Nordic Seas region One of the biggest problems in trying to determine whether a latitudinal species-diversity gradient exists is that data are limited for the deep sea. Further, data are almost never collected for the specific use of investigating these gradients, and thus interpretation of results is somewhat difficult. There are also potential problems in making comparisons among the different studies, as there are often variations in the way that the fauna were collected, even within the same size groups. For example, the macrofauna may be collected with grabs, box corers, or megacorers as well as by using different diversity analyses Water depth is a parameter that covaries with a variety of environmental characteristics, and not always in the same manner. This makes correlations of biodiversity (or any measurement) with depth open to considerable interpretation, because it is rarely depth per se  that is the cause of any relationship revealed. Despite this, depth is a useful, and easily measured, aggregate parameter that has been linked to spatial patterns in biodiversity. Over the range of ocean depths, data from a variety of invertebrate groups suggested a trend of increasing species richness to depths of about 2,500 m, and a subsequent decline at greater depths In many regional studies, latitude is a common covariate with depth. Depth gradually increases with latitude from about 20 m to 100 m for the area covered by the 1986 North Sea Benthos Survey (NSBS) We have used the same North Sea dataset (as highlighted earlier) and have plotted the ES50 results against depth  and findAt shelf depths, there is little theory to suggest how diversity should vary with depth, and again, multiple factors may covary with depth. These factors include light, disturbance, sediment grain size, current speed, oxygen concentration, salinity, and ecological interactions. In shallow waters (under 50 m), there is some evidence of depressed biodiversity among epifaunal and infaunal organisms in the shallowest areas, and increasing richness with depth thereafter . ExampleIt is generally assumed that in the North Atlantic deep sea the relationship between macrofaunal diversity and depth fits a parabolic curve Kelliella atlantica  and of the asteroid Hymenaster membranaceus, also at a depth of about 2,600 m, lead to the noticeable decline in diversity at this depth Benthonella tenella, in association with diversity indices that are size dependent, may have accentuated the decline in diversity between 1,400 and 1,600 m. However, the peak in diversity at about 4,000 m is similar to that found by Flach and de Bruin For the megafauna, peaks in diversity appear to vary depending on location and the taxonomic group being investigated. For example, Sibuet Levin et al. www.arcodiv.org), and will be reviewed elsewhere. Here we will, however, mention several general results especially pertinent to patterns of biodiversity in European Arctic waters.Whereas some of the discussion presented here has referenced studies performed in Arctic areas, the majority of Census activity related to the Arctic has been performed under ArcOD  in the high Arctic . The first Norwegian deep-sea sampling expedition, known as the Norwegian North Atlantic Expedition 1876-78, had sampling stations in some Norwegian fjords, the Norwegian shelf north of Bergen, the deep Norwegian Sea, the western Barents Sea and Svalbard ( =  Spitsbergen) up to 80° N. Since then, there have been no national benthic surveys conducted, and most of the data that have been collected have been associated with individual research projects and monitoring efforts around petroleum installations.Norwegian naturalists and scientists have been sampling the marine inshore fauna along the Norwegian coast since the 1740s. The first publication containing information on marine species from the Norwegian coast is Bishop Erik Pontoppidan's work In 1995 a compilation of reliable information  on the presence of marine benthic macroorganisms was performed for the Norwegian coast Brattegard has created an updated dataset on the distribution of benthic invertebrates and included shifts in distribution range for many of the species  parallelled a rise in temperature and Atlantic inflow. In concert with this so-called regime shift Ongoing surveys since 1948 with the Continuous Plankton Recorder across the North Sea carried out by the Sir Alister Hardy Foundation for Ocean Science (SAHFOS) revealed periods with dramatic changes in the amount and composition of phyto- and zooplankton. One such period was in the late 1980s when a peak in phytoplankton color and oceanic zooplankton , these include sediment parameters, salinity, temperature, and perhaps trawling pressure. Whereas the details of how variations in identified factors can produce diversity patterns requires further study, care needs to be taken when placing regional results into a latitudinal context, as this may hinder exploration of actual causal factors.Whereas this may seem to be an academic question, it is important when evaluating the robustness of theory and in suggesting mechanisms responsible for the diversity patterns we observe. Renaud et al. Semibalanus to slower growing chthamalids and that there was an increase in diversity of grazers such as limpets in northern parts of Europe Biodiversity is known to play a critical role in ecosystem function and, perhaps, system resilience Mathematical/statistical modelling has not been widely used in biodiversity research but the studies that have been cited indicate value for identifying and perhaps explaining biodiversity patterns, for mapping hotspots or other specific areas for management purposes, and for predicting how system change due to climate variability or human activities may alter marine biodiversity. This can also help biodiversity researchers construct testable hypotheses from theory and ecological understanding, and allow us better understand mechanisms responsible for biodiversity patterns on different scales of time and space.Throughout this paper, we have compiled and analyzed trends and patterns in biodiversity that have been observed and reported in the scientific literature. However, a comparative way of synthesizing and expressing the current state of knowledge is to identify and highlight gaps in knowledge. In which areas do we still lack vital knowledge? Based on the existing literature, spatial trends have been relatively well studied, however, there are still obvious gaps in knowledge related to e.g. large scale patterns in the hyperbenthos or general knowledge across habitat boundaries. Temporal trends, however, are scarce. Pending co-ordinated multi-national and possibly multi-decadal efforts that provide long time-series data and baseline values on which spatial variability may be interpreted; our understanding of the marine environment will remain limited. Furthermore, the organisms for which there exist time series data are predominantly either economically important or ecologically dominant species To reduce our gaps in knowledge there is a need for greater coordination between European research programs, particularly in standardization of sampling techniques and analysis of data, for large-scale spatial and temporal patterns to be fully understood. Programs such as the Census of Marine Life and the EU-funded programs such as MarBEF and HERMES have instigated greater collaboration among researchers, and this should continue if we are to fully understand the influence that latitude, depth, and other environmental variables have on species diversity. Only then can we appreciate and comprehend the extent to which anthropogenic impacts, such as climate change and trawling, affect biodiversity.File S1Translation of the abstract into French, German, Italian and Spanish.(0.04 MB DOC)Click here for additional data file.


A case of implant-bone prosthetic rehabilitation, after the fracture of the maxillary central incisors, which had been treated with grafting of a bone substitute, is reported. This case was followed by the normal procedures of implantology within the traditional timeframe for bone regeneration. However, a barrier membrane was not used which shows that even along with the use of graft material a sufficient amount of bone could be achieved for a subsequent rehabilitation. Therefore, after a five-year follow-up period, osseointegration was maintained with no marginal bone loss. Dental trauma could be the most common uncomfortable distress of the facial region. Dental coronal fractures and dislocations occur frequently, and because of their position, the teeth of the anterior maxillary area are therefore more usually subjected to this type of injury . FollowiA 39-year-old caucasian man was referred to the Dental School of the University Federico II of Naples in 2008. His main complaint was localized pain in region 1.1 and 2.1 during the function, the clinical examination showed that either the two fixed prosthesis elements where of ceramic metal as well as the presence of a secreting fistula in correspondence of the mucosa in the apical area of 1.1. A periapical radiograph was performed and the medical history was documented which has no relevance with regard to the disease in question. Dental history reports that prior treatments and retreatments on these teeth have been completed before the prosthesis.The clinical and radiographic findings showed a probable root vertical fracture of the tooth 1.1 as well as a possible vertical fracture of the tooth 2.1 .For aesthetic reasons, an alginate impression was taken and a plaster cast was made to construct an interim prosthesis.Therefore, a decision was made to place two commercial teeth in a row on a steel wire to construct a prosthesis similar to the Maryland Bridge.Probable causes were an inadequate apical seal and/or one or more vertical root fractures that were not captured via periapical radiograph.The periapical radiograph from 1.1 revealed an osteolytic area in the middle third of the root. This osteolytic area also enveloped apical third of the root. A beta-lactam antibiotic (Amoxicillin) was given orally 2 gr one hour before the surgery, and a diagnostic flap surgery was planned, which allowed to establish the correct treatment plan.Previous to local anesthesia, a diagnostic flap was opened, respecting marginal soft tissues. The root's vertical fracture of 1.1 was immediately evident, while curettage of the exposed portion of the root of 2.1 also revealed a further fracture in the apical mesial portion of the root. The teeth were gently extracted with particular attention to the preservation of the hard and soft tissues at the sites. The extraction sockets were debrided using piezosurgery devices and alveolar surgical curettes to remove the granulation tissue . The socThe Vicryl polyglactin  absorbable suture was used to close the flap. The interim prosthesis was delivered by using an adhesive system to attach to the adjacent teeth. This prosthesis allows to achieve an acceptable esthetic outcome, as well as a good phonatory function and an initial tissue conditioning for the pontic areas  8]. . 8]. The postoperative therapy requires good oral hygiene, rinsing with mouthwash containing 0.2% chlorhexidine solution twice a day, and an evening application of the same product in gel form, as well as the administration of a nonsteroidal anti-inflammatory aid (Ketoprofen 80 mg) for three consecutive days.The patient was asked to be seen for regular followups for the next three months.At the end of the 3 months, the patient showed remarkable healing of the soft tissues, and the gingiva appeared with an excellent color and texture of the tissue too. It also began to outline the proper and harmonious design of the facial mucosa curvatures, which were conditioned by the interim prosthesis .At six months, from the socket preservation, a periapical radiograph was taken and showed limited bone loss, which was related to the steel wire of the interim prosthesis .Moreover, radiographically, the bone appeared homogenous and with a good radiopacity to show a sufficient remineralization.An alginate impression with the interim bridge in situ was taken, to construct a surgical template. The patient was satisfied with regard to the esthetic outcome. A thermoplastic vacuum-formed template was then built and two holes corresponding to the cingulum of the maxillary central incisors were performed. The temporary bridge was removed to place two implants. A bone trephine was used to obtain a bone specimen to assess the bone quality and structure. The implant phase, however, was necessary to find the maximum stability in the apical zone . In the After the initial surgical phase, two resin temporary crowns were applied leaving a sufficient space to allow for any swelling. Only after three weeks, the interim crowns were modeled with a slight contact to the soft tissue.At six months, the implants were uncovered and a resin screw retained prosthesis was constructed with an emergence profile which was suitable for supporting the buccal soft tissue. It also important to remember to avoid any excessive compression, which could lead to a soft tissue shrinkage in the subsequent months of maturation. The occlusal contacts were verified for the presence of a slight contact during maximum intercuspation using shimstock  in order to protect the implants from any intense forces. Of course, functional loads resulting from lips, tongue, and food bolus will remain , 11.After waiting for another 6 months, we proceeded to the prosthetic finalization. The patient has a good soft tissue maturation induced by the design of temporary crowns. A periapical radiograph was taken to evaluate the proper bone remodeling.A custom impression of the healed soft tissue, recording and transferring the soft tissue contour with a gingival outline, was taken according to the technique of Hinds, 1997 .Therefore, after trying all the components, the definitive gold-ceramic crowns were delivered with good accuracy as well as a proper emergence profile to support the tissue. The patient was instructed to maintain good oral hygiene by brushing and flossing.A year later, the patient was seen to observe overall tissue healing, aesthetics, and radiographic osseointegration of the implants.This type of rehabilitation used integrates in the morphological and occlusal context of the oral cavity. In this way, this procedure, appropriately managed, is capable of producing the desired implant-prosthetic outcome, ensuring comfort and patient satisfaction  13]..13].We recommended the patient a thorough oral hygiene through the use of brush, dental floss, and pipe cleaner.The patient was recalled for a clinical control after a three- and five-year period. A new periapical radiograph was taken which showed an excellent osseointegration of the implants with only a minimal loss of bone height according to the Albrektsson criteria and ICOI Consensus Conference, 2007  14, 15], 1514, 1Overall the patient was quite satisfied with the esthetic outcome and had no clinical issues.With regard to implant-prosthetic rehabilitation after fracture of the upper central incisors, various techniques have been proposed, which include the postextractive immediate loading implant. In this particular case, however, we felt it was necessary to wait for bone healing after the use of a substitute bone graft, without a barrier membrane. In addition to chemical and physical properties of the material, the aesthetic success must not be underestimated.Moreover, the primary stability of implants was apically searched in the residual native bone and the major axes were placed palatally, in correspondence with the cingulum to maintain the possibility to build a screw-retained crown and to preserve the height and thickness of the facial bone wall. Therefore, the implant shoulder was positioned about 1 mm palatal to the point of emergence at the adjacent teeth. When the implant is placed too facially, a resorption of the facial bone wall could occur with a subsequent recession. With the implant positioned too palatally, an implant crown with a ridge-lap design might be needed.The position of the implant must be chosen by using a 3D position, which respects the comfort zone, as stated by Belser et al. . TherefoTherefore, prosthetic phase is extremely important.In this case, we could ask if a barrier it is necessary for the alveolar ridge preservation and the answer is indeterminate at this time. The results between the studies regarding the use of any barrier are almost similar; no clear benefit was demonstrated with or without a barrier. Use of a barrier may prove beneficial in cases where extraction socket walls are partially or completely missing , 20.To make sure of obtaining a good bone growth, we have performed a sample of bone from the site analyzing it histologically. Microscopic findings of implanted bone showed fragments of bone embedded in fibrous tissue; the same fragments at higher magnification showed fibroblasts, calcifications, fragments of bone tissue, and traces of grafted material  21]..21].However, additional controlled and comparative studies are needed to confirm or refute these findings.Another important matter is whether the addition of growth factors could provide a benefit for alveolar ridge preservation procedures .However, a small number of studies have evaluated the use of growth factors for alveolar ridge preservation after tooth extractions, and there was significant heterogeneity in the condition of the host extraction socket and bone at time of graft placing . Use of In this report, it has been showed how graft material can be used in extraction sockets without a barrier membrane ensuring nevertheless a good support for the implant prosthetic rehabilitation. Inspite of the nonuse of membranes, the target was obtained, so the question we asked for is that using a grafting material not completely resorbable or with a slow resorption as Bio-Oss, in this technique of resorption prevention, might suggest that the use of membrane does not affects the result with the biological and financial savings of a membrane.In agreement with literature, our case also shows, that with regards to socket preservation, bone loss was limited without affecting the clinical outcome. Therefore, this specific technique could also be used in an aesthetic area that may be a challenge. Our results show that over a period of time the clinical outcome remained stable.


In vivo measures of efficacy and potency require an appreciation of the overall pathophysiology of the model and standardization of outcome measures. The new concepts of how MSC’s participate in the tissue regeneration and wound repair process and further, how this is impacted by estimates of efficacy and potency are important new topics. In this regard, this chapter will review some of the in vitro and in vivo assays for MSC function and activity and their application to the clinical arena.The advent of mesenchymal stem cell (MSC)-based therapies for clinical therapeutics has been an exciting and new innovation for the treatment of a variety of diseases associated with inflammation, tissue damage, and subsequent regeneration and repair. Application-based ability to measure MSC potency and fate of the cells post-MSC therapy are the variables that confound the use of MSCs therapeutics in human diseases. An evaluation of MSC function and applications with attention to detail in the preparation as well as quality control and quality assurance are only as good as the assays that are developed.  The priin vivo in terms of tissue regeneration, wound repair, or immune-modulation. The difficulty arises in the availability of testing MSCs for phenotypic predictors in vitro, which would define the in vivo ability to function and generate the desired effector responses. This chapter will discuss the current models for the activity of MSCs and how they are correlated with the in vivo effectiveness of MSC function and activity.The fact that every MSC-donor is different  clearly becomes the most important of the uncontrolled aspect of both allo- and autologous MSC-based therapies. Published information suggests that the secretion of powerful bioactive molecules by MSCs may differ by a factor of 10 between different donors of matched age and gender . Thus, pMesenchymal stem cells actively respond to stress or injury in a manner that is very similar to how the adaptive and innate immune system cells respond to pathogen exposure or apoptosis. When supplied exogenously, MSCs home to sites of injury primarily inflamed or broken blood vessels. There is an intricate array of soluble mediators generated by MSCs in a milieu specific manner, almost “sensing” the requirement of the environment. These products can promote angiogenesis, regeneration, remodeling, immune cell activation or suppression, and cellular recruitment. At the same time the MSCs can also actively participate in bactericidal activity. MSCs are perivascular cells, pericytes, and as such, they will not respond the same given that the microenvironments of different tissues are not the same, this will also impact efficacy and/or potency. The functionality of the MSC preparation is based upon isolation and expansion procedures and the injury-specific environment that they find themselves in. The ability MSCs to respond to the given environment is attributed to the ability of them to respond to changes or requirements of the milieu through transcriptional regulation and translation of appropriate responding mediators. This responsiveness is due to receptor recognition and signal transduction followed by the release of specific protein mediators that influence the milieu for repair, control of inflammation and infection. The repair process entails regulating extracellular matrix deposition, collagen synthesis, fibroblast proliferation, platelet activation, fibrinolysis, and angiogenesis. The immune process often involves suppressing T-cells, activating macrophages, and potentially recruiting neutrophils. Studies have also suggested that immune regulation by the MSCs is defined by the soluble products generated by the MSCs facilitating changes in immune cell activation status. The products produced by the MSCs are not different from those of the immune system and can include cytokines, inflammatory mediators, and antimicrobial proteins. Table What is efficacy? Efficacy as defined as “the power to produce an effect” . The “ef6 cells/mouse post-challenge. The difference between these observations has to do with the localization of inflammation and injury in the context of allergen challenge. These studies further showed that the MSCs are potent in the context of pre-existing airway disease. Similarly, the intensity by which the MSCs induced the response is shown by the variability of the change in lung differential between study sets. The investigators in these studies further define this variability based upon the separately assayed ability of the MSCs to induce bone formation in an in vivo implantation efficacy assay. These studies showed that the assay for bone formation mirrored the in vivo decrease in cellular recruitment even in the context of allergen challenge. The connection between bone formation and lung efficacy is not obvious to us and likely somehow reflects the functionality of MSCs in two different microenvironments.Potency, or the ability or capacity to achieve or bring about a particular result, is a major component which defines the ultimate efficacy or the power to achieve a particular event . The difin vitro using very tightly controlled environments in terms of medium, fetal calf serum (FCS), supplements, and buffering systems  for 25 days –22. To dFor adipocyte differentiation, MSCs are cultured in a defined medium including FCS, dexamethasone, ascorbic acid, and 1-methl-3-isobutyl-xanthine. Growth occurs for at least 14 days followed by Oil-Red O staining to show adipocyte differentiation , 24.For osteoblastic differentiation, MSCs are cultured in DMEM supplemented with 10% FBS, β-glycerophosphate, dexamethasone, and ascorbic acid. After 21 days, the cells can be stained with van Kossa’s stain to reveal osteogenic differentiation or chemically analyzed for calcium accumulation –27.A variety of functional assays can also be used to measure MSC efficacy. The point of these assays is to measure the ability of the MSCs to induce a specific effect. MSCs have been shown to have an immunosuppressive effect on T-cells  both as in vivo experimental studies have shown the value of allogeneic or xenogenic MSCs for a variety of disorders including: sepsis, hepatic failure, acute renal failure, and myocardial infarction . Changes in the scoring of the MSC-treated mice can then be used as a measure of efficacy. For potency, the same model can be used comparing single dosing versus multiple dosing for selected time points , mice are injected with myelin protein to induce experimental autoimmune encephalitis EAE . The aniin vivo models such as hepatic failure, kidney failure, and myocardial infarction but these require more time, are even more expensive, and at the end, probably do not provide any more detail than the short term in vivo efficacy assay described above accept for the end-point evaluation of investigation  to improve overall survival of MSCs in vivo  dependent. Thus, MSCs genetically enhanced with Akt1 can repair infarcted myocardium, prevent remodeling, and nearly normalize cardiac performance. Other studies have started to focus on the use of targeted MSC in other diseases. In one set of studies, an adeno-associated virus vector was used to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta  or minimally invasive . Although MSCs obtained from different regions of the body show similar potential for differentiation and therapeutic effects, they each express different cellular markers. For example, adipose-derived MSCs express higher levels of CD49d, CD34, and CD54 whereas bone marrow-derived MSCs have higher levels of CD106 , 57. TheIn vitro and ex vivo assays are available to define the relative efficacy of several in vitro and in vivo assays which can ultimately be used to define MSC efficacy and potency, but these are going to be tissue-specific and disease-specific. In the end, correlating a specific type of disease process (inflammation versus remodeling) must be used to define the activity of the MSCs. This will become especially true as new sources besides bone marrow-derived MCSs become clinically available and are approved for clinical applications.Mesenchymal stem cells have become a major focus for a potential resource in therapeutic cell-based therapies. MSCs are multipotent cells derived from stromal tissue, which have the capacity to differentiate into mesodermal and endodermal types of cells. Not only do MSCs have the capacity to differentiate into different types of cells depending on the tissue matrix, they also actively contribute to their milieu by secreting soluble products that actively participate in MSC and surrounding cell phenotype. The issues related to therapeutically translating MSCs into the clinical arena are based upon the ability to predict the host response, the intensity of the response, and the duration of the response. These parameters are associated with the potency and efficacy of the stem cell preparation. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The current study investigated the role of cultural norms on the development of face-scanning. British and Japanese adults’ eye movements were recorded while they observed avatar faces moving their mouth, and then their eyes toward or away from the participants. British participants fixated more on the mouth, which contrasts with Japanese participants fixating mainly on the eyes. Moreover, eye fixations of British participants were less affected by the gaze shift of the avatar than Japanese participants, who shifted their fixation to the corresponding direction of the avatar’s gaze. Results are consistent with the Western cultural norms that value the maintenance of eye contact, and the Eastern cultural norms that require flexible use of eye contact and gaze aversion. A brief look at another person’s face would tell you a lot about who they are . Faces are also the “window to the soul”, because facial expressions signal their emotional states, and gaze direction would tell you what they see and what they know. Having such significance in human social communication, it is not surprising that faces attract attention from very early in infancy. Even newborns preferentially orient to faces , especiaAn important question about the development of face gaze is the role of postnatal environment. Several major theories of social skills development emphasize the role of input from their parents (or caregiver) as well as those from other members of the society, which are essential for the infant brain to learn the social world and become an “expert” . For exaTwo independent lines of research contrasted face gaze between Western European/North American culture and Eastern Asian culture, and found clear differences in the face gaze between the two cultures. First, a series of studies , 2008 reThe current study aimed to bridge these gaps in knowledge by investigating how cultural background (British or Japanese) affects the face gaze when they observe dynamic face stimuli. We also examined whether the gaze direction of face stimuli (looking toward or away from the observer) would affect cross-cultural differences in face gaze. We predicted that British participants would maintain longer and sustained eye contact and make a triangular fixation , whereas Japanese participants would show shorter and flexible eye fixations and more central fixations. We also predicted that the response to direct and averted gaze would be modulated by the cultural background because of the different cultural norms on the use of eye contact, but no further specific predictions have been made due to the exploratory nature of the manipulation.Nineteen British adults  and 22 Japanese adults  participated in the study.  British adults were recruited in central London, and Japanese adults were recruited in central Tokyo. All the participants have normal or corrected-to-normal acuity.Four computer-generated faces  were selected from the library of Poser 7 , and were used to create 7-second animations with the same software. All the animations started with a face presented upright, facing 30º to the left or to the right and gazing forward, followed by a mouth movement (1 second after the start) and an eye movement (2 seconds after the start). Mouth movements were either smile , 1b or mTwo Tobii T120 eye-trackers , equipped with an integrated 17-inch display, were used to present stimuli and record eye-movement in London and in Tokyo. Tobii Studio software was used to control stimulus presentation and to analyse the gaze data.Recordings were conducted in a quiet and soundproofed room within each research institute. Participants were instructed to watch the movies of the faces. The same experimenter (AS) conducted the recording in both the UK and Japan, to maintain strictly similar experimental conditions such as instructions. A 9-point calibration was conducted using Tobii Studio software before the recording. Recording consisted of two blocks, and each of 32 animations was presented twice (once in each block), in a randomized order. An experimenter also sat in the same testing room, out of sight of the participant, and monitored the recording with Tobii studio software. Viewing distance was approximately 60–65 cm from the display.The gaze data were initially processed with Tobii studio software to calculate the total visit time. Then, we calculated the fixation duration for each stimulus, for the following areas of interest (AOIs); front eye, back eye, bridge, centre and mouth. Note that faces are tilted either to the right or to the left, one of the eyes is always closer to the observer  than the other eye , the latter which is off to the side . The AOIThe gaze data for different head orientations and the blocks were averaged together. The visiting duration for each AOI were then divided with the total visiting duration of the whole face, to calculate the relative visiting duration. The relative visiting duration was analysed with mixed-design ANOVAs to test the effects of cultural background (British or Japanese) and the sex  of the participants, as well as the ethnicity (Caucasian or Eastern Asian), gender , gaze direction (direct or averted), mouth movement (smile or mouth opening) and the AOI  of the stimuli. An initial ANOVA was conducted on the whole 7-second data, which were then followed up by the analyses of seven 1-second bins of the data. For the significant interactions, post-hoc analyses were conducted on each contrast with Wilcoxon sign rank tests with the Bonferroni correction for multiple testing, to provide robust statistics.F = 4.684, p < .01, p2η = 0.11, demonstrating that British and Japanese participants fixated differently to the face, depending on whether the face was with direct or averted gaze. The effect was modulated by the sex, F = 4.142, p < .01, p2η = 0.10 , but not with other factors such as the mouth movement, the gender or the ethnicity of the face stimuli. These interactions remained significant when we excluded British participants of non-Caucasian ethnic origin , and when we excluded British participants who had stayed two years in East Asian countries  and East Asian participants who had stayed 6 months in the USA .As predicted, the interaction between the cultural background, gaze direction and the AOI was significant, Follow-up analyses revealed that Japanese participants fixated longer on the back eye than did British participants, in both direct gaze and averted gaze conditions. British participants, by contrast, fixated longer on the mouth than Japanese participants, in both direct gaze and averted gaze conditions. British participants also showed longer fixation on the centre, which was only significant in the averted gaze condition .The two groups showed similarities and differences in the response to different gaze directions. Both groups showed longer fixations on the front eye in the direct gaze condition than in averted gaze condition. However, only Japanese participants showed differential fixations on the back eye, with longer fixation in averted gaze condition . No otheFurther analyses were conducted on the seven 1-second bins of the data, to explore the time-course of the differential fixations on the four AOIs showing the gaze and cultural background interactions; front eye, back eye, mouth and centre.No effects reached significance for the first, second and third bins. In the fourth bin, the point right after the gaze shift of the stimuli, both groups showed longer fixation in direct gaze condition than in averted gaze condition. Interestingly, this effect was exaggerated in Japanese participants, who showed even longer fixation than British participants in response to direct gaze and even shorter fixation than British participants in the averted gaze condition . The samIn the first, second and third bins, the only significant effects were the differences between groups, with Japanese participants fixating longer than British participants. From the fourth bin onward, however, Japanese participants fixated longer than British participants only in the averted gaze condition, but not in the direct gaze condition .The group difference in mouth fixation remained from the first to the fourth bin , which tThe group differences were significant in the fourth and fifth bins, only in averted gaze condition .The current study is the first to investigate how the cultural background of the observer affects the face gaze when they observe dynamic faces looking toward or away from the observer. The results clearly demonstrate the cultural difference between British and Japanese participants in the way they look at different parts of the faces, and how they respond to different gaze direction. First, British participants fixated more on the mouth than did Japanese participants, replicating previous studies using static images of faces . Second,Interestingly, these two groups responded differently to the gaze shift of the stimuli. Second-by-second analyses revealed that both groups of participants fixated longer on the front eye immediately  after they saw the gaze shift toward them, but such a change was more exaggerated and lasted longer with Japanese participants than British participants. Moreover, the initial cultural difference in the fixation on the back eye was overridden by the effect of gaze shift, in which Japanese participants fixated longer on the back eye only when they saw an averted gaze. By contrast, the fixation on the back eye was not affected by the gaze direction in British participants. The results showed that Japanese participants shifted their own fixation to the corresponding direction of the observed gaze shift , as if they “followed” the direction of the face gaze. British participants did not show such a change of fixation following observed gaze shift. It might suggest that the eye fixation of British participants reflects the cultural expectation to maintain eye contact, but the eye fixation of Japanese participants reflects the cultural norm to conform to others’ behaviour. Note that it is not a general difference in the sensitivity to facial motion, because mouth movements did not exaggerate or diminish the cultural differences in mouth fixation. The effect was more prominent in males than in females , suggesting that the effect of cultural norm on face and gaze processing manifests more strongly in males. Further studies will be required to see how the differences in gender-related cultural norm interact with face-scanning behaviour.The current study clearly demonstrates the cultural differences in face gaze in adults, but it cannot tell us how it develops. For example, To summarize, the current study revealed that the cultural background of the participant affects how they look at another person’s eyes and mouth, and how they modulate eye fixation in response to the gaze direction of others. These differences are consistent with the culturally-relevant strategy of perceptual analyses, as well as the cultural norms on the use of eye contact in face-to-face communication. These results highlight the new frontier of the research about how cultural norms can affect behavioural, cognitive and neural development, which would provide a great opportunity to study the effect of postnatal environment on human behavioural and cognitive development.


Complaints of arm, neck and shoulder are a major health problem in Western societies and a huge economic burden due to sickness absence and health-care costs. In 2003 the 12-month prevalence’s in the Netherlands were estimated at 31.4% for neck pain, 30.3% for shoulder pain, and 17.5% for wrist and hand pain. Research data suggest that these complaints are increasingly common among university students. The aims of the present study are to provide insight into the prevalence of complaints of arm, neck or shoulder in a university population, to evaluate the clinical course of these complaints and to identify prognostic factors which influence this course.The present study is designed as a prospective cohort study, in which a cross-sectional survey is embedded. A self-administered cross-sectional survey will be conducted to gain insight into the prevalence of complaints of arm, neck or shoulder among university students and staff, and to identify persons who are eligible for follow up in the prognostic cohort study. Patients with a new complaint of pain and discomfort in neck and upper extremities between 18–65 years will be asked to participate in the prognostic cohort study. At baseline, after 6, 12, 26 and 52 weeks individual patient data will be collected by means of digitized self-administered questionnaires. The following putative prognostic determinants will be investigated: socio-demographic factors, work-related factors, complaint characteristics, physical activity and psychosocial factors.The primary outcome is subjective recovery. Secondary outcomes are functional limitations of the arm, neck, shoulder and hand, and complaint severity during the previous week.To our knowledge, this is the first prognostic study on the course of complaints of arm, neck or shoulder that is conducted within a university population. Moreover, there are hardly any studies that have estimated the prevalence of these complaints among university students. The results of this study can be used for patient education and management decisions, as well as for the development of interventions. Moreover, identification of high risk groups in the population is needed to generate hypotheses or explanations of health differences and for the design of prevention programs. Complaints of the arm, neck and shoulder (CANS) are a major health problem in Western societies ,2. ReporA population that receives relatively little attention with regard to CANS is the university student population. Although there are only few studies that estimated the prevalence of upper extremity symptoms among graduate or college students ,6, reseaThe high prevalence rates and the consequences of these complaints on well-being, career and productivity highlight the importance to gain insight into the frequency of these complaints among specific risk groups such as university students, as well as into the course of these complaints and their prognostic factors. However, studies on the course of CANS and the prognostic determinants are scarce, especially in a working population and among students.Until now, studies on course and prognosis of upper limb musculoskeletal complaints show a large variety in patient characteristics and settings. Moreover, most studies have focused on a single site in the upper limb  . AlthougOccupational factors, such as job demands and decision authority, have not often been taken into account although these factors have been shown to be related to outcome in research of work-related risk factors for low back pain . KnowledTherefore, the primary aims of the present study are:1. To provide insight into the prevalence of CANS among a population of university staff and students.2. To evaluate the clinical course of non-traumatic arm, neck and/or shoulder complaints in university staff and students and to identify prognostic factors which influence the course of non-traumatic arm, neck and/or shoulder complaints in this population.The present study is designed as a prospective cohort study (CANS Cohort Study), in which a cross-sectional survey is embedded. This study will be performed in the South region of the Netherlands. Recruitment of the study population will take place at two universities in this region: Maastricht University and Zuyd University of Applied Sciences. The base population (employees and students) of Maastricht University and Zuyd University of Applied Sciences consists of approximately 40,000 people . Study design and participant flow are shown in Figure +, the Netherlands, has approved the study protocol, procedures and informed consent. Written informed consent will be obtained from all patients.With regard to the prospective cohort study, at baseline, and after 6, 12, 26 and 52 weeks, individual patient data will be collected by means of digitized self-administered questionnaires on patient level. The Medical Ethics Committee of the Maastricht University Medical CentreEmployees or students with a new complaint or new episode of complaints of pain and discomfort in neck and upper extremities  between 18–65 years are eligible for participation in the study. An episode of complaint is considered ‘new’ if patients have not visited their health care professional for the same complaint during the preceding 6 months. Exclusion criteria are: complaints caused by acute trauma or by any systemic disease, malignancy, prosthesis, amputation, congenital defect or a co-morbidity causing severe disability in daily activities, and not being able to fill in Dutch or English questionnaires. Patients who are pregnant will also be excluded from the study. Patients can be included in the study only once.To identify persons with CANS for participation in the prospective cohort study, all employees and students of both universities receive a survey questionnaire. The content and procedure of this survey will be explained in the following paragraph.The survey questionnaire is accompanied by an invitation letter, in which the respondent is informed concerning the purpose of the survey and about the possibility to participate in the prospective cohort study, if appropriate. Respondents who report to experience CANS in the survey questionnaire are requested to fill out an additional question in which they are asked whether they are interested in participation in the prospective cohort study. If they show interest, permission is asked in the survey questionnaire to be approached by the researchers. Subsequently, the researcher checks the inclusion criteria, which will be obtained from the survey questionnaire. If the respondent meets the inclusion criteria, a patient information leaflet about the study and an informed consent form will be sent by post, including a free post return envelope. A non-response card is also included.Patients are asked to fill out the informed consent form within one week, if they decide to participate in the follow up study. Patients who are unwilling to participate are asked to return the non-response card.In order to increase the response rate, patients who have not returned the informed consent form or the non-response card within two weeks will receive a reminder by e-mail. After returning the informed consent form, the patient obtains a personalized code by e-mail and is able to login to the web-questionnaires. Patients are asked to fill out the baseline questionnaires within 10 days.A self-administered cross-sectional survey will be conducted to gain insight into the prevalence rates of CANS among university staff and students, and to identify persons with CANS who are eligible for follow up in the prospective cohort study. All employees and students of both universities receive the survey questionnaire, accompanied by an invitation letter, by e-mail. In the invitation letter, employees and students will be asked to provide written informed consent, which covered the usage of the questionnaire data. To realize smooth processing of the large amount of questionnaires and to guarantee smooth enrolment of participants in the cohort study, questionnaires will be sent in separate batches, each containing approximately 2500 questionnaires. After 3 weeks a reminder e-mail will be sent and after 6 weeks a second reminder will be sent by e-mail.The survey questionnaire consists of general questions and health questions about musculoskeletal conditions in: (1) neck; (2) shoulder; (3) upper arm(s); (4) elbow(s); (5) lower arm(s); (6) wrist(s); (7) hand(s)/ finger(s). The questions about each area start with the following question: “Did you experience pain/ symptoms in ‘anatomic area’ for at least one week during the past 6 months?” If a person replies positively, the person is asked to answer additional questions concerning the complaints at the relevant anatomic area: questions on the duration and severity of the pain/symptoms, consequences for health care utilization, questions on work status and sickness absence. Moreover, a list of the complaints that are classified as specific CANS, according to the CANS model  will be In the questionnaire which will be sent to the student population, specific questions on employment status are replaced by questions on study load and additional part-time jobs.The items included in the survey questionnaire are partly derived from the Maastricht Upper Extremity Questionnaire (MUEQ), which has been found to be a valid questionnaire to assess the occurrence and nature of CANS and work-related physical and psychological risk factors . Items oA computer-based patient record system (CPR), has been custom-built by Fastguide® . The CPRBased on information derived from preceding studies -12, the After baseline assessment, follow-up assessments will be conducted 6, 12, 26 and 52 weeks.The primary outcome measure, subjective recovery, will be measured with the Global Perceived Effect (GPE) scale ,25. PatiThe course of the complaints will be described by means of descriptive statistics. Determinants that might influence the clinical course will be established by means of multivariate logistic regression analysis and analysis for repeated measurements over time . In univariate analyses the putative prognostic variables for the persistence of complaints will be selected. These analyses will be done separately for the working and non-working subpopulations.Outcome measures will be analyzed separately in different multiple regression analyses. Pearson or Spearman correlation coefficients will be used to calculate correlations between potential determinants. When a high correlation between two determinants is found, only the most predictive determinant in the univariate analyses will be included in the multiple regression models.Prevalence rates of CANS over the past twelve months that lasted for at least one week will be computed including 95% confidence intervals (CI).A recruited sample of 500 patients will be sufficient to detect prognostic indicators with at least 80% power, assuming a maximum of 20% loss to follow-up and 5% two-tailed significance level. The sample size is likely to be adequate for exploring the independent association of at least 15 prognostic indicators for better outcome. The base population (employees and students) of Maastricht University and Zuyd University of Applied Sciences consists of approximately 40,000 people. We need an enrolment of 0,8% of the total population for the prospective cohort study. Regarding the already mentioned prevalence rates of the complaints in the introduction it seems a realistic goal.Little information is available on the course and prognostic factors for recovery in patients with CANS, as well as on the prevalence of CANS in a population of university staff and students. This study is designed to provide insight in the prevalence of CANS, its course and the long term prognosis in a university population.An important strength of this study is the ability to present the natural (untreated) course of CANS, because not all employees and students that will be followed up in the prognostic cohort study, are under treatment for their complaints. Another strength is that the use of three domains of recovery  allows to describe a broader perspective of relevant health outcomes for patients with CANS.The study also has some limitations. Sample selection might have implications for the internal validity of the study, especially if there is selection bias at the moment of the survey. Because the goal of the present study is to invite all employees and students to complete the survey questionnaire , selective non-response is not likely to occur. A possible threat for the internal validity of the study concerns the issue of selective withdrawal. Selective withdrawal might occur in case of the self-administered survey questionnaire or the questionnaires of the cohort study. This might be due to the possibility that respondents or participants with relatively severe CANS are not able or willing to complete digitized questionnaires because of their pain or symptoms. Furthermore, employees or students with severe CANS may have already left the labour force or discontinued their study. This may lead to an underestimation of severe CANS in the university population. Consequently, a higher prevalence of people with severe CANS would possibly be observed in the general population.On the contrary, it is also possible that people with complaints are more willing to participate than those without problems. This may lead to a slight overestimation.Moreover, in this cohort study the complaints will be divided into specific and non-specific diagnosis based on the CANS-model . AccordiThe results of the prospective Cohort Study (CANS cohort study) can be used for patient education and management decisions, as well as for the development of interventions for these complaints. Moreover, identification of high risk groups in the population is needed to generate hypotheses or explanations of health differences and for the design of prevention programs .To our knowledge, this is the first prospective study on the course of CANS, and the factors that influence this course, that is conducted in a university population. Furthermore, there are hardly any studies that have estimated the prevalence of CANS among university students.The results of this study will be presented as soon as they are available.The authors declared that they have no competing interest.VEJB conceived of the project, led the design and co-ordinates the study, and wrote the draft of the manuscript. RADB and CHGB participated in the study design and commented on drafts of this paper. CHGB provided advice for the method section. All authors have reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/8/prepub