Arenaviridae is a diverse and growing family of viruses that includes several agents responsible for important human diseases. Despite the importance of this family for public health, particularly in Africa and South America, much of its biology remains poorly understood. However, in recent years significant progress has been made in this regard, particularly relating to the formation and release of new enveloped virions, which is an essential step in the viral lifecycle. While this process is mediated chiefly by the viral matrix protein Z, recent evidence suggests that for some viruses the nucleoprotein (NP) is also required to enhance the budding process. Here we highlight and compare the distinct budding mechanisms of different arenaviruses, concentrating on the role of the matrix protein Z, its known late domain sequences, and the involvement of cellular endosomal sorting complex required for transport (ESCRT) pathway components. Finally we address the recently described roles for the nucleoprotein NP in budding and ribonucleoprotein complex (RNP) incorporation, as well as discussing possible mechanisms related to its involvement.The  Arenaviridae is a diverse and growing family of viruses, presently containing 24 recognized species [The  species  and seve species ,12,13,14Among the OWAVs, both Lassa virus (LASV) and the recently identified Lujo virus (LUJV) are capable of causing hemorrhagic fever (HF) ,15. WhilIn the case of the NWAV serocomplex, viruses can be further divided into three clades  based on phylogenetic relationships , althougIn total arenaviruses cause at least seven distinct hemorrhagic fevers, making them the largest family of HF-causing viruses currently known. In addition, both the NWAVs and OWAVs contain a number of human apathogenic strains, which are in some cases quite closely related to the agents of HF disease. The basis for these marked differences in virulence among genetically closely related viruses remains unknown. Similarly, despite their significance for public health, many details of the arenavirus lifecycle, including the pathways used for virus morphogenesis and budding, remain poorly understood. However, in recent years significant progress has been made in this area and has revealed interesting commonalities in the mechanisms used by the various arenaviruses but also striking differences that remain to be fully explained from a mechanistic stand-point. Here we attempt to summarize the current state of our knowledge, including recent findings, in the field of arenavirus morphogenesis and budding.Arenavirus particles are enveloped and highly pleomorphic, having a diameter of 50-300 nm . They coTogether with NP and L the two ambisense genome segments are assembled into ribonucleoprotein complexes (RNPs), which serve as the templates for transcription and replication by the RNA-dependent RNA polymerase, L. Indeed, studies have shown that together NP and L represent the minimal protein requirements for transcription and replication of both NWAVs and OWAVs ,28,29,30Despite being the smallest of the arenavirus proteins, with a size of only 89 to 103 amino acids, Z has been demonstrated to take part in a number of processes and interactions central to the viral lifecycle . ConsistThe host cell membrane represents a significant barrier to the egress of newly formed or forming viral particles. In order to overcome this physical barrier many viruses have evolved to take advantage of host cell sorting pathways, in particular those directing vesicle formation into multi-vesicular bodies (MVBs). Thus for many enveloped viruses, including arenaviruses, it has been shown that they recruit the endosomal sorting complex required for transport (ESCRT) machinery of the host cell to facilitate their release ,56. HoweIn the uninfected host cell the ESCRT-system is involved in MVB vesicle formation and cytokinesis. It consists of six complexes  that are recruited sequentially to the sites of membrane remodelling and fission. While ESCRT-0 binds to and accumulates ubiquitinated cargo for delivery into the MVBs, the ESCRT-I and ESCRT-II complexes are recruited and co-assemble on the membrane to drive bud formation. ESCRT-III, which is responsible for final membrane scission, is recruited by binding to ESCRT-I and/or Alix/AIP1, which connects these complexes. Finally the AAA-type ATPase Vps4 mediates disassembly of the complex and its subsequent recycling . During For many enveloped viruses it has been shown that the self-budding activity of their matrix protein is functionally dependent on the presence of late domain motifs within their sequences . Late-domain motifs promote viral budding by mediating the interaction of viral proteins with components of the cellular ESCRT-machinery or ESCRT-associated ubiquitin ligases, an observation that appears to hold true for a variety of virus families with which these studies have been conducted. The viral PT/SAP motif has been shown to recruit the ubiquitin-binding ESCRT-I-component tumor susceptibility gene 101 (Tsg101) to initiate viral budding ,68,69,71The Z protein has been shown for several arenaviruses to serve as the viral matrix protein providing the principle driving force for the budding of virus particles, and as such it is capable of forming virus like particles (VLPs) when expressed alone . During Sequence analysis of OWAVs shows that they generally contain either a PPxY motif alone or both a PPxY and a PT/SAP motif at their C-terminus. In addition, several of the OWAVs contain an additional YxxL motif in the RING domain. The exceptions to this are Merino Walk virus, which based on the available sequencing data does not appear to have any known late-domain motifs, and LUJV, which displays an unusual late domain motif arrangement more closely related to that of the NWAVs. This may reflect the fact that while LUJV is classified as belonging to the OWAVs, it is only distantly related to all other known OWAVs , which mInterestingly, despite the presence of multiple late domain motifs in some arenaviruses, the extent to which virus budding is dependent on each of these motifs seems to vary between viruses . For OWAFor the NWAV JUNV, the release of chimeric VLP was shown to be heavily dependent on the PTAP motif , but theNP has been described as an accessory factor for the budding of members of several virus families, including the paramyxoviruses ,82, retrAll evidence to date has clearly demonstrated that Z is the key determinant without which arenavirus budding cannot proceed. However, for TCRV Z, budding is considerably enhanced by the presence of NP , an effeInteraction of NP with Alix/AIP1, via its Bro-1 domain, has also been described for MOPV where, rather than enhancing budding, it was shown to mediate the incorporation of NP into Z-induced VLPs , suggestArenaviruses are serious public health concerns in many parts of the world and thus the development of vaccines and anti-viral therapies remains an important priority. An understanding of the exact mechanisms used for virus morphogenesis and release will help to offer new possibilities to combat viral infection by targeting these pathways. Such targets could include both the interactions among viral proteins (i.e. Z-NP interactions) and interactions between viral and host cell components. Emphasizing this potential, tetherin has recently been identified as a novel antiviral factor that is able to inhibit Z-mediated release of arenaviruses ,87. TethIn addition to the proline-rich and YxxL-type late-domains found within the matrix protein, N-terminal myristoylation also plays a decisive role in viral budding. This post-translational modification takes place at a glycine residue at position 2 (G2) in the Z protein sequence and is universally conserved among all arenaviruses . The myrFinally, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in a variety of cellular processes , has alsThe formation of progeny virus particles and their release is an essential step in the lifecycle of a virus. In recent years significant progress has been made in the field of arenavirus morphogenesis and budding. The findings have revealed many commonalities in the budding mechanisms used by different arenaviruses, but also striking differences.The matrix protein Z of arenaviruses has been shown to provide the driving force for budding, an activity that depends on various distinct motifs that have been identified within the protein. Among these are an N-terminal myristoylation at glycine 2 (G2) of Z, which mediates binding of Z to the host cell plasma membrane, and various late domains . These late domains are responsible for the recruitment of different components of the cellular ESCRT-machinery, normally involved in vesicle formation, to the budding-site where they initiate the release of membrane enveloped virions from the infected host cell . While tIn summary, the current data reveal a budding mechanism for arenaviruses that is ESCRT-dependent, but involves a variety of different players depending on the virus species examined. Clearly there are also a number of unknowns and issues that need to be further investigated, especially given the significance of arenaviruses for public health, as a better understanding of the mechanisms and pathways used for virus morphogenesis might offer new possibilities for the development of antiviral therapies to combat arenavirus infection.


Persistent left superior vena cava (PLSVC) represents the most common congenital venous anomaly of the thoracic systemic venous return, occurring in 0.3% to 0.5% of individuals in the general population, and in up to 12% of individuals with other documented congential heart abnormalities. In this regard, there is very little in the literature that specifically addresses the potential importance of the incidental finding of PLSVC to surgeons, interventional radiologists, and other physicians actively involved in central venous access device placement in cancer patients. In the current review, we have attempted to comprehensively evaluate the available literature regarding PLSVC. Additionally, we have discussed the clinical implications and relevance of such congenital aberrancies, as well as of treatment-induced or disease-induced alterations in the anatomy of the thoracic central venous system, as they pertain to the general principles of successful placement of central venous access devices in cancer patients. Specifically regarding PLSVC, it is critical to recognize its presence during attempted central venous access device placement and to fully characterize the pattern of cardiac venous return  in any patient suspected of PLSVC prior to initiation of use of their central venous access device. Central venous access device placement is a commonplace practice for many physicians, including surgeons, interventional radiologists, and other physicians, who are involved in the management of cancer patients . Yet, suThe aim of the current report is to review the available literature as it pertains to the specific congential venous anomaly of the thoracic systemic venous return, persistent left superior vena cava (PLSVC), and to discuss the clinical implications and relevance of congenital aberrancies, as well as of treatment-induced or disease-induced alterations in the anatomy of the thoracic central venous system, as they pertain to the general principles of central venous access device placement and venography. A thorough understanding of such principles is of upmost importance to surgeons, interventional radiologists, and other physicians whom are actively involved in central venous access device placement in cancer patients.The patient was a 53 year old Caucasian woman, without any previous major medical problems, who was recently diagnosed with synchronous bilateral breast cancers and who underwent a right modified radical mastectomy and a left total mastectomy and left axillary sentinel lymph node biopsy for a pT2, pN1, estrogen receptor positive, progesterone receptor positive, HER-2/neu negative invasive lobular carcinoma of the right breast and a pT1b, pN0, estrogen receptor positive, progesterone receptor positive, HER-2/neu negative invasive ductal carcinoma of the left breast, respectively. The patient was subsequently recommended for placement of a subcutaneous implanted port for administration of postoperative adjuvant systemic chemotherapy.Therefore, the patient was taken to the operating room by the surgeon for subcutaneous port placement. At the request of the patient, this procedure was done under general anesthesia. The left side was selected, as it represented the side of her earlier-stage breast cancer. A left cephalic vein cutdown approach was undertaken in the left lateral infraclavicular region, by the methodology as previously described by Povoski . Upon crSame-day consultation with the interventional radiologist revealed a similar opinion. However, based upon the intrinsic limitations of the non-digital subtraction intraoperative venography procedure performed, an accurate assessment of the point of insertion of the PLSVC into the venous return of the heart and the anatomy of the contralateral right-sided central venous system could not be adequately determined. Therefore, subsequent standard digital subtraction venography was recommended by the interventional radiologist.The subcutaneous port placement procedure was uneventfully completed by the surgeon by placing the tip of the 9.6 French single lumen silicone catheter to approximately the 15 cm mark within the recognized PLSVC and attaching the 9.6 French single lumen silicone catheter to an implantable port  and closing the port insertion surgical skin incision site that was located in the left lateral infraclavicular region.A subsequent posterioranterior and lateral chest x-ray Figure  was perfIn the subsequent weeks after left-sided subcutaneous port placement, digital subtraction venography of the left-sided central venous system (by way of the left-sided subcutaneous port) Figure  and digiAt approximately two weeks after left-sided subcutaneous port placement, the patient underwent digital subtraction venography of the left-sided central venous system by way of the left-sided subcutaneous port Figure , in ordeApproximately four weeks later, the patient underwent digital subtraction venography of the right upper extremity veins and the right-sided central venous system by way of a peripheral vein in the dorsum of the right hand Figure , in ordeThereafter, the patient was allowed to use her left-sided subcutaneous port for continued administration of postoperative adjuvant systemic chemotherapy, blood draws, and all necessary subsequent contrast-based imaging. The patient had no detectable problems during the utilization of her left-sided subcutaneous port and had no resultant complications. The patient's left-sided subcutaneous port was eventually removed after she completed her postoperative adjuvant systemic chemotherapy, some seven months after its original placement.th century to 18th century [The exact origin of the first description of PLSVC remain a matter of much great debate within the historical scientific literature, although it appears to have likely occurred at some time during the 17 century ,7. Some  century -9, the E century ,7,10,11, century ,12,13, t century ,7,14, th century ,7,15, an century ,7,16,17.Since that time, a plethora of, and too numerous to cite, papers have been published on various aspects of PLSVC, including characterization of central venous anatomy and central venous anomalies, embryologic development of the central venous system, identification of PLSVC during implantable pacemaker and cardioverter defibrillator placement, identification of PLSVC during various forms of central venous access device placement, impact of PLSVC on various cardiac surgery procedures, and indications for surgical correction of PLSVC. To date, in PubMed.gov , a searcThose papers in the literature that have specifically addressed the incidental finding of PLSVC at the time of placement of some sort of central venous access device or some sort of central venous monitoring device ,44-123 hPLSVC represents the most common congenital venous anomaly of the thoracic systemic venous return ,43. It iPLSVC can occur in several anatomic variations. Most commonly, PLSVC coexists with a right SVC in up to 80% to 90% of cases . While iIf it is suspected that a patient has a PLSVC at the time of attempted central venous access device placement, then it is essential for that patient to undergo subsequent appropriate investigations to fully characterize their central venous anatomy. This is important in order to confirm the presence of PLSVC, to characterize the central venous anatomy of the contralateral right side, to characterize the pattern of cardiac venous return to the right atrium or to the left atrium, and to evaluate the patient for other potential coexisting congential heart abnormalities. Multiple venous imaging modalities can be utilized, as well as used in concert with one another, to accomplish complete characterization of the central venous anatomy. These venous imaging modalities include conventional contrast venography, transthoracic echocardiography, transesophageal echocardiography, multidetector computed tomography venography, and magnetic resonance venography ,127-134.As previously mentioned, the incidental finding of a PLSVC during central venous access device placement is of great potential importance to surgeons, interventional radiologists, and other physicians who are actively involved in central venous access device placement in cancer patients. With PLSVC occurring in only 0.3% to 0.5% of individuals in the general population and since there is theoretically only a 50% chance of encountering a PLSVC in an individual who has a PLSVC , then it is very plausible that most physicians who place central venous access devices in their clinical practice may possible never, or only once, come across this congenital venous anomaly during their careers. In this regard, a resultant patient outcome in this rarely encountered scenario could potentially be devastating if the possibility of PLSVC was not thought of and/or not recognized by the physician at the time of a "difficult" central venous access device placement procedure.It is important to discuss the implications of PLSVC as it applies to the pattern of cardiac venous return  in any given patient suspected of PLSVC at central venous access device placement. As previously discussed, the venous return from the PLSVC drains into the left atrium in approximately 10% to 20% of cases ,44,111. From the surgeon's perspective, intraoperative venography during attempted central venous access device placement can be a very useful tool for immediate characterization of central venous anatomy, including for recognition of congenital venous anomalies such as PLSVC, as well as for recognition of treatment-induced or disease-induced alterations in thoracic central venous anatomy. The use of intraoperative venography techniques during attempted central venous access device placement has been previously discussed in detail, from the surgeon's perspective, by one of the present authors for the venous cutdown approach and for the percutaneous venipuncture approach in cancer patients ,3, as weFrom the surgeon's perspective, an important point of discussion regarding the performance of intraoperative venography during attempted central venous access device placement relates to the method of venous access  and to the stepwise timing of intraoperative venography during attempted central venous access device placement ,3. In thPerformance of intraoperative venography by the surgeon via a venous cutdown approach  at the time of attempted central venous access device placement represents a very safe, straightforward, and highly useful means for obtaining detailed intraoperative characterization of the central venous anatomy ,3. By inOn the other hand, performance of intraoperative venography by the surgeon via a percutaneous venipuncture approach  at the time of attempted central venous access device placement has some intrinsic limitations ,3. AlthoFrom the interventional radiologist's perspective, the approach to central venous access device placement and to venography is somewhat different than the surgeon's approach, as it relates to the method of venous access, the vein selection site, and the method of venography. In this regard, whereas surgeons primarily utilize the left subclavian vein or right subclavian vein percutaneous venipuncture approach (more commonly without ultrasound guidance), interventional radiologists almost exclusively utilize an ultrasound-guided right internal jugular vein percutaneous venipuncture approach, and alternatively an ultrasound-guided left internal jugular vein percutaneous venipuncture approach when there is a contraindication to central venous access device placement on the right side. After initial successful placement of the venipuncture needle by the ultrasound-guided right internal jugular vein or left internal jugular vein percutaneous venipuncture approach, the interventional radiologist passes the guidewire and watch the guidewire pass down through the thorax region under fluoroscopy and use the course of the guidewire and its behavior within the central veins as reasonable validation of standard/normal central venous anatomy. If there is any suspicious behavior by the guidewire , then the venipuncture needle is generally removed over the guidewire, a 5-French dilator is passed over the guidewire, the guidewire is then removed, and a venogram is performed. Injection of contrast into the 5-French dilator at this very peripheral initial point of entry into the right internal jugular vein or left internal jugular vein by the interventional radiologist will again allow for a relatively detailed venous roadmap of the ipsilateral internal jugular vein, subclavian vein, innominate vein, and SVC using digital subtraction intraoperative fluoroscopy equipment that is routinely available in the interventional radiology suite. Such an approach by the interventional radiologists has been developed out of necessity and in response to the increasing number of patients that are encountered with treatment-induced or disease-induced alterations in thoracic central venous anatomy.It is well-established within the radiology , anestheA thorough understanding of venous anatomy, including the recognition of congenital venous anomalies (such as PLSVC) and the recognition of treatment-induced or disease-induced alterations in thoracic central venous anatomy, as well as having a good working knowledge of alternative and supplemental strategies for placement central venous access devices, are all critical factors to maximizing the success of central venous access device placement and to minimizing the risk of potential complications. A thorough understanding of these principles is of upmost importance to surgeons, interventional radiologists, and other physicians whom are actively involved in central venous access device placement in cancer patients.Specifically regarding PLSVC, it is critical to recognize its presence during attempted central venous access device placement and to fully characterize the pattern of cardiac venous return  in any patient suspected of PLSVC prior to initiation of use of their central venous access device.PLSVC: persistent left superior vena cava; SVC: superior vena cavaWritten informed consent was obtained from the patient for publication of this review paper and accompanying images. A signed copy of the written consent form from the patient is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.SPP was the surgeon who performed the central venous access device placement procedure and the intraoperative venogram procedure. HK was the interventional radiologist who performed the postoperative venogram procedures. Both of the authors were involved in writing and editing this manuscript. Both of the authors have read and approved the final version of this manuscript.


The microenvironment at the site of tumor metastasis plays a key role in determining the fate of the metastasizing tumor cells. This ultimately has a direct impact on the progression of cancer. Bone is the preferred site of metastasis of breast cancer. Painful, debilitating osteolytic lesions are formed as a result of crosstalk between breast cancer cells and cells in the bone, predominantly the osteoblasts and osteoclasts. In this paper, we have discussed the temporal and spatial role of hedgehog (Hh) signaling in influencing the fate of metastatic breast cancer cells in bone. By virtue of its secreted ligands, the Hh pathway is capable of homotypic and heterotypic signaling and consequently altering the microenvironment in the bone. We also have put into perspective the therapeutic implications of using Hh inhibitors to prevent and/or treat bone metastases of breast cancer. The overwhelming numbers of cancer patients (≥90%) that die due to the dissemination of cancer cells rather than the primary tumor throw the process of metastasis to the centre stage of clinical management of cancer . HoweverEvolving literature supports that metastasis is a second disease imposed on the primary tumor. The outcome of metastasis is determined by the interplay between the subpopulation of metastatic cells and host homeostatic factors in the specific organ microenvironment . The metThe metastasizing tumor cells hijack many of the pathways that play major roles during normal development. Many of the embryonic developmental signaling pathways, such as the Wnt, Hedgehog (Hh), and Notch pathways, affect the survival of tumor stem cells and orchestrate a complex microenvironment that promotes tumor survival and progression. In this review, we will highlight the significance of the Hh pathway in developmental biology and our present understanding of its role in regulating breast cancer metastasis to bone. We will elaborate how a pathway that is so critical in normal development of the embryo is usurped by the breast cancer cells to serve their own purpose of invading the tissue of its origin, extravasation, survival during translocation, and adaptation at the distant site to bring about proliferation and colonization.The Hh pathway plays a central role in embryonic development and maintenance of stem or progenitor cells in many adult tissues . The Hh The involvement of the Hh pathway, in particular the ligand SHH, with the skeletal system begins with embryonic development, where SHH is expressed in the notochord, the floorplate of the neural tube, the brain, the zone of polarizing activity in the developing limbs, and the gut , 12. SHHin vitro and for proliferation and survival of human melanoma in vivo [The Hh pathway is required for normal proliferation of human melanocytes  in vivo , 20. In  in vivo , while i in vivo . Li et a in vivo . This is in vivo . SHH is  in vivo . As such in vivo , 25–48  , 54. Tumates OPN . OPN is ates OPN , and infates OPN , 59. OPNates OPN , 61 and ates OPN . It is hates OPN , 63, 64.For the purpose of this review, we have focused the remainder of the article on discussing the role of Hh signaling in impacting breast cancer metastasis to the bone. This complication of breast cancer continues to present a challenge to oncologists and reduces the chances of survival for breast cancer patients. Among breast cancers that become aggressive, metastasis to bone marrow is common. Detection of bone metastasis often signals the onset of the life-threatening phase of breast cancer. The 5-year survival rate is 98% for breast cancer when detected early; this precipitously drops to 83% for patients initially diagnosed with regional spread and to 26% for those with distant metastases. In the following sections, we will discuss the role of Hh signaling in mediating a crosstalk between breast cancer cells and cells in the bone and the overall impact on the ability of breast cancer cells to sculpt the bone microenvironment and cause osteolysis Figures  and 2.The bone microenvironment comprises osteoblasts, osteoclasts, mineralized bone matrix, and other cell types, such as the osteocytes embedded within bone. Of these, the most important ones (from the perspective of this article) are the bone-resorbing osteoclasts and bone-forming osteoblasts.β, and so forth are known to take part in the differentiation of preosteoblasts into mature osteoblasts. Eventually, mature, mineralizing osteoblasts become embedded in the newly secreted bone matrix and undergo terminal differentiation to form osteocytes. Although the osteocytes have much reduced activity as compared to osteoblasts, their long processes allow them to connect the entire matrix via a series of canaliculi. It is understood that the osteocytes ensure communication between sites deep in the bone and the extraosseous world; they create an enormous increase in mineral surface exposed to extracellular fluid and cellular activity and function as mechanosensory cells of bone, involved in the transduction of mechanical loads into biochemical signals [Osteoblasts are derived from mesenchymal stem cells, which can also give rise to chondrocytes, fibroblasts, myocytes, or adipocytes . Formati signals .κB (RANK) on the surface, while the ligand RANKL is expressed by the bone marrow stromal cells and osteoblasts. Binding of the ligand to the receptor commits the precursor cells to the osteoclast lineage. The same interaction is also critical for osteoclast formation and can also promote osteoclast activity, since RANK is also present on the surface of terminally differentiated osteoclasts. The fusion of osteoclast precursor cells results in the formation of large multinucleated active osteoclasts.Osteoclasts, on the other hand, are large multinucleated terminally differentiated cells with a unique ability for bone resorption . They arOsteoprotegerin (OPG) is a soluble decoy receptor and a competitor of RANKL in its binding with RANK and thus can inhibit osteoclastogenesis. Therefore, the balance of RANKL and OPG is critical for osteoclast formation and activity. Osteoclasts attach to the bone surface via actin-rich podosomes enabling them to form sealed zones with ruffled borders. Proteolytic enzymes such as CTSK (Cathepsin K) and MMPs are secreted into this isolated environment, resulting in degradation of the bone matrix, dissolution of the bone mineral, and resorption of the bone . Evidentβ and insulin-like growth factors activate tumor expression of VEGF, PDGF, and endothelin (ET-1) [β, and IL-11 produced by breast cancer cells favor osteoclast maturation and osteolysis, leading to the release of growth factors that stimulate malignant tumor growth [vicious cycle of events  . Factorsr growth . In factr growth . Thus, sf events .Hh-signaling-activated GLI2 transcription mediates osteoblast differentiation . This isOPN is one of the abundant noncollagenous proteins in bone. It promotes osteoclast function and is consistently overexpressed in highly metastatic cells. OPN accumulates at cement lines in remodeling bone  and is lThe Hh ligands also mediate a direct dialogue between breast cancer cells and preosteoclasts and induce changes in preosteoclasts that influence the production of OPN and essential bone-resorbing proteases, CTSK, and MMP9 by osteoclasts . Thus, HThus, the vicious cycle of bone metastasis involves a complex crosstalk between the metastasizing breast tumor cells and the bone microenvironment through multiple extracellular factors and signaling pathways with the Hh pathway playing an essential role. Based on our findings, we would like to propose that the newly arrived breast tumor cells induce initial osteoblast differentiation which stimulates osteoclast differentiation. Soon, the situation is overwhelmed by osteoclast differentiation followed by intense bone resorption leading to the local release of generous amounts of growth factors that not only encourage their growth but also alter their phenotype, making them (cancer cells) resistant to standard cytotoxic antitumor treatments see the appendix , 86.The bone microenvironment with ongoing bone resorption almost resembles sites of wound healing. The bone stroma is almost guaranteed to provide hospitable sites for disseminating colonization-competent breast cancer cells . This en


Fucus vesiculosus and increasing reproduction area of perch Perca fluviatilis, but decreasing reproduction areas for pikeperch Sander lucioperca following successful implementation of the Baltic Sea Action Plan. Despite variability in estimated non-market benefits among countries, such changes were highly valued by citizens in the three Baltic countries investigated. We conclude that predictive models are powerful and useful tools for science-based management of the Baltic Sea.We evaluated performance of species distribution models for predictive mapping, and how models can be used to integrate human pressures into ecological and economic assessments. A selection of 77 biological variables  across the Baltic Sea were modeled. Differences among methods, areas, predictor, and response variables were evaluated. Several methods successfully predicted abundance and occurrence of vegetation, invertebrates, fish, and functional aspects of biodiversity. Depth and substrate were among the most important predictors. Models incorporating water clarity were used to predict increasing cover of the brown alga bladderwrack  Marine and coastal ecosystems contribute substantially to the global pool of biodiversity and production of goods and services , Marine Strategy Framework Directive (MSFD), and a recently proposed directive for maritime spatial planning and integrated coastal management (MSP). All of these initiatives emphasize actions, adaptivity, and the need for integrating ecological and socio-economic systems. It is also evident that these frameworks pose several new methodological challenges, for example, how to make status assessments, how to detect and evaluate human impacts, and how to achieve integrated planning. The project PREHAB  was designed to address one particular challenge relevant in many management contexts: the need for comprehensive and spatially explicit information about structural and functional aspects of biodiversity in the marine environment.Maps and other information of areal extent are essential for assessing the status according to the MSFD and for implementing future MSP directives. A major difficulty is that data on vegetation, invertebrates, and fish in the marine environment are very scattered and sparse. This is because they are usually collected using dives, corers, videos, or nets. To develop comprehensive distribution maps, methods that predict the state in unsampled sites by integrating existing data are needed , which predictors are generally powerful and which methods generally perform well. This would be a foundation for evidence-based recommendations on biodiversity mapping to authorities in the Baltic Sea region. Furthermore, the aim was to explore how the use of SDMs could be extended into evaluation of ecological and economic effects of management actions. The rationale and results of these efforts are briefly summarized below.The scientific activities of PREHAB were structured into three main parts: (1) evaluating the potential for predictive modeling in the Baltic Sea, (2) modeling responses to management scenarios, and (3) monetary valuation of management scenarios.First, the scientific core of PREHAB was a systematic assessment of the general applicability of predictive, empirical SDMs in the Baltic Sea , and root mean square error (RMSE) were used for abundance models. RMSE values were normalized to the range of each response (NRMSE). In addition to these analyses, we also developed a strategy for assessing (5) the importance of spatial resolution in one of the case-study areas.This resulted in approximately 200 quantitative and 300 occurrence models of vegetation, benthic invertebrates, fish, and indices of biodiversity from a wide range of environmental conditions representing different parts of the Baltic Sea. These models were used to assess (1) overall predictability of benthic species and habitats, (2) differences in performance among modeling approaches , (3) differences in predictability among types of organisms, and (4) differences in predictive power among different types of predictors. Each model was assessed against a test dataset , which was left out at the training stage. Area under the receiver operating characteristic curve (AUC) values were used to validate models of presence versus absence, whilst the coefficient of determination  or ranges of salinity, wave exposure, annual ice cover, and coastal morphology.The performance of models was generally quite good across all taxa, study areas, and modeling methods Fig. . The majR2, and NRMSE was relatively similar among the methods . It has been suggested that mobile, widespread, and generalistic species are difficult to model, as they are ubiquitous and show a very weak response to changes in the environment . Environmental predictors important in quantitative models were also important in qualitative models. Bottom topography (primarily depth) and bottom substrate were generally the most powerful and important predictors, with strong effects on abundance and occurrence patterns of invertebrates and vegetation. For fish, geographic location and hydrographic variables , tended to be more powerful predictors than depth and wave exposure, both regarding abundance and occurrence. Overall, the most striking conclusion is the vital role of detailed information on water depth and bottom substrate. Accordingly, access to high-resolution data on depth and substrate can greatly improve modeling and mapping, and is more or less a requirement for fulfilling the potential of predictive modeling of species distribution in benthic environments. An important message to any commissioning authority or other user is therefore that efforts to provide accurate high-resolution data on water depth and bottom substrate are needed to improve the quality of biodiversity maps.The predictive performance of empirical models is affected by uncertainty in the estimation of the response and predictor variables as well as uncertainties associated with the structure and formulation of a model. All of these sources of uncertainty are dependent on the spatial scale, i.e., extent and resolution. This means that the choice of sampling resolution can affect the accuracy of models and maps. In a specific study from the Swedish west coast using data collected in a hierarchical design, we assessed effects of spatial resolution on the predictive power of models of benthic flora and fauna  and precision (RMSE) that would be expected based on uncertainty of estimates in the biological variables of interest. The precision and predictive power of these simulations were compared to the observed performance of a simple linear model (LM) and of a more flexible method (RF) , pikeperch (Sander lucioperca), eelgrass (Zostera marina), and bladderwrack (Fucus vesiculosus) in a 40 000 km2 archipelago area of the northern Baltic Sea.In the study on eutrophication, potential ecological effects of eutrophication mitigation in accordance with the targets of the politically adopted BSAP were explored  were compared to estimate effects of changes in water clarity on species distributions under a set of scenarios based on the BSAP. The three methods gave qualitatively similar results, although quantitative responses differed between them Fig. . The anawww.prehab.gu.se). Maps of shoreline constructions in the form of jetties and marinas, which may affect habitats directly through building and dredging and indirectly through increased boating  the general performance documented in our analyses across the Baltic Sea (Sundblad et al. First, the Baltic-wide synthesis of models suggest that models using species–environment relationships, derived from a range of statistical methods, can be used to predict both abundance and occurrence of vegetation, benthic invertebrates, and fish. Some differences in predictability can be explained by data quality and differences among taxonomic groups, but variability among species was generally unpredictable. Whether the performance of the models is sufficiently accurate for practical management depends on the demands of the management situation (e.g., Fielding Second, by combining information on human pressures and predictive models and by linking a study of economic valuation to different scenarios, we assessed ecological and economic benefits of a range of management scenarios (Kosenius and Ollikainen www.prehab.gu.se. Nevertheless, the work toward sustainability is a continuing and adaptive process requiring cross-fertilization between policy and research. Some of the more prolific areas of interaction are those of marine spatial planning, linking structural and functional aspects of biodiversity, and integrated assessment of ecological and socio-economic systems (e.g., Crowder and Norse Third, the increasing pressures and impacts on the Baltic Sea environment require new tools for implementing more sustainable use and management. PREHAB was developed to contribute in some of these areas, and the results of these efforts have been formulated specifically for authorities and policy-makers in a user-friendly web-resource at


Planning and license applications concerning groundwater diversion in areas containing water-dependent or water-favored habitats must take into account both hydrological effects and associated ecological consequences. There is at present no established methodology to assess such ecohydrological responses. Thus, this paper describes a new stepwise methodology to assess ecohydrological responses to groundwater diversion from, e.g., water-drained pits, shafts, tunnels, and caverns in rock below the groundwater table. The methodology is illustrated using the planned deep-rock repository for spent nuclear fuel at Forsmark in central Sweden as a case study, offering access to a unique hydrological and ecological dataset. The case study demonstrates that results of ecohydrological assessments can provide useful inputs to planning of monitoring programs and mitigation measures in infrastructure projects. As a result of the assessment, artificial water supply to wetlands is planned in order to preserve biological diversity, nature values, and vulnerable species.The online version of this article (doi:10.1007/s13280-013-0404-8) contains supplementary material, which is available to authorized users. Environmental impact assessments (EIA) are commonly performed and reported as part of planning and license applications for infrastructure projects. These EIA must identify and assess risks, associated with various types of disturbances during construction and operation, to humans and the surrounding environment. Examples of typical disturbances in infrastructure projects  are landscape encroachments, noise, vibrations, and dusting due to rock blasting and crushing, as well as emissions of water and air pollutants from the use of contract machines.If the cavity is located below the groundwater table, inflowing groundwater must be diverted in order to keep the cavity drained. In addition to the disturbances mentioned above, in such cases the EIA must also include an assessment of the resulting groundwater-table drawdown and other hydrological effects in the surroundings of the cavity. Moreover, depending on the characteristics of the area in which the cavity is located, it is required that the EIA consider consequences for house foundations and infrastructure, water-supply wells, agriculture, forestry, and other ecological conditions. In particular, planning and license applications for construction and operation of water-drained cavities in areas that contain natural habitats for water-dependent or water-favored flora and fauna need to take into account ecohydrological responses to the diversion of groundwater, i.e., hydrological effects and associated ecological consequences.Compared with other environmental disturbances, such as noise and vibrations, the hydrological effects of groundwater diversion from cavities in rock and associated ecological consequences are relatively difficult to predict, and any such predictions are normally subject to uncertainty. For instance, detailed predictions of hydrological effects may require extensive data on geometries and hydraulic properties of fracture zones in the rock as well as the hydraulic properties of the interface between regolith and rock  is relatively new and it has been given increasing attention during recent years among the ecological and hydrologic scientific communities, including debates regarding the subject’s scope and definition  methodology that links diversion of groundwater from the rock to hydrological effects and ecological consequences in surface systems. Forsmark has been selected as the site for the planned Swedish deep-rock repository for spent nuclear fuel, and it offers access to a unique hydrological and ecological dataset  consists of a spiral-shaped access ramp and vertical shafts, a central area with a number of rock caverns, and a repository area with tunnels for deposition of canisters  and fen orchid (Liparis loeselii). These two species are also legally protected according to the EU’s Habitats Directive  contains a well-connected network of geological structures, consisting of so-called sheet joints, i.e., geological structures that may have high horizontal permeability, and steep fracture zones that locally are in contact with the regolith (Follin The regolith (unconsolidated deposits overlying the rock) in Forsmark is dominated by relatively permeable till. The groundwater table is shallow and generally follows the ground-surface topography, which causes small-scale groundwater flow systems near the ground surface to overlie larger-scale flow systems in the rock. The upper 100–150 m of the granitic rock  fens were investigated using the national method for rich-fen inventory in Sweden , the specific objectives of the predictions, and the severity of potential ecological consequences of groundwater diversion from the rock. In the Forsmark case study, the numerical water-flow modeling tool MIKE SHE, which is fully integrated with the channel-flow code MIKE 11  for the assessment should be based on a site-specific conceptual hydrological model . Hence, this simulation case provides safety margins for the ecohydrological assessment and is likely to overestimate the groundwater-table drawdown that would occur in practice. The MIKE SHE calculation results were temporally and spatially distributed, and could hence be used to assess hydrological effects for individual nature objects. The classes shown in Table In addition to the hydrometeorological parameters mentioned above, MIKE SHE simulation cases were also executed to investigate the influence of the hydrogeological properties of regolith and rock, the number of open, water-drained tunnels in the repository, the hydraulic conductivity of grouted rock around subsurface cavities, as well as the locations of model-domain boundaries and the spatial discretization of the model domain. In order to capture influences of both temporal hydrometeorological variability and the listed parameter and model uncertainties, the simulation case chosen as input to the integrated ecohydrological assessment represents a purely hypothetical situation with a fully open, water-drained repository and a relatively permeable grouted zone , MIKE SHE calculations show that diversion of groundwater during construction and operation of the deep-rock repository has potential to yield groundwater-level drawdown in the rock across large areas , the purely hypothetical MIKE SHE simulation case that represents a fully open, water-drained repository and a permeable grouted zone yields an influence area for the groundwater-table drawdown, i.e., annual average drawdown of 0.1 m or larger, which is 2.5 kmCombining the outcomes of the previously described steps, the results of the integrated ecohydrological assessment for the Forsmark case study Fig.  show thaForest habitats and forest species are generally less water-dependent or water-favored compared with wetland habitats and wetland species, and the former are therefore less sensitive to changes in the level of the groundwater table. Accordingly, the results of the Forsmark assessment show that the groundwater diversion will cause ecological consequences that are noticeable (consequence class 3) or of less severity for the delineated forest objects, and it is predicted that there will be noticeable consequences for red-listed forest fungi. The assessment shows that the groundwater diversion will lead to insignificant or small ecological consequences for aquatic ecosystems, due to their relatively limited nature values and the predicted small hydrological effects on such systems. There is an assigned Natura 2000 area in the easternmost part of the investigation area. The assessment shows that the protected habitats and species in this area will not be harmed or subject to any disturbance that significantly renders difficult preservation of protected species.This paper demonstrates that ecohydrological concepts and methods are important parts of planning and permit applications for construction and operation of water-drained cavities in areas with water-dependent or water-favored nature objects. Specifically, such assessments provide useful guidelines for focusing monitoring and mitigation measures on a limited number of subareas.Underlying classification and modeling tools are inevitably associated with different types of assumptions and uncertainties, in particular assumptions and parameter and model uncertainties related to water-flow models that collectively propagate to prediction uncertainties in the integrated ecohydrological assessment. Geometries and hydraulic properties of fracture zones in crystalline rock, and hydraulic properties of the interface between the regolith and the rock, are important factors that always should be accounted for in assessments of hydrological effects. In the Forsmark case study, temporal variability of hydrometeorological parameters (including conceivable ranges of climate and sea-level changes) and uncertainties related to, e.g., the geometries and the hydraulic properties of fracture zones in the rock have been handled by comprehensive, transient model calibrations, and sensitivity analyses. The simulation case used in the Forsmark assessment captures conceivable ranges of temporal hydrometeorological variability, various parameter and model uncertainties, and it predicts the theoretical upper limit of the drawdown.As part of preparations for repository construction and operation in Forsmark, the assessments presented in this paper are used as one of the inputs to further develop the hydrological and ecological monitoring programs, including monitoring of reference objects at other, similar sites. The EIA document that accompanies the deep-repository permit application according to the Swedish Environmental Code proposes artificial water supply to substantially reduce potential negative consequences for wetlands, i.e., habitats for rare and protected species such as the pool frog and fen orchid.It is concluded that further research is needed, based on observed ecological responses in water-dependent or water-favored habitats to observed changes in hydrological conditions. This research requires input from systematic ecological and hydrological monitoring as part of infrastructure projects, in areas containing such habitats, which include construction and operation of water-drained cavities in rock below the groundwater table.Supplementary material 1 (PDF 199 kb)Below is the link to the electronic supplementary material.


This review refers to the origin and current state of the assessment of the SpondyloArthritis International Society (ASAS) criteria for the classification of axial and peripheral spondyloarthritis (SpA) and the possible implications in the pediatric population. The ASAS criteria evolved from the idea that the earlier the recognition of patients with ankylosing spondylitis, the better the efficacy of tumor necrosis factor blockers. Strategies included the development of new concepts, definitions, and techniques for the study of clinical signs and symptoms. Of relevance, the new definition of inflammatory back pain (IBP) and the introduction of sacroiliitis by magnetic resonance imaging represented the most important advance in the early identification of AS in the “pre-radiographic stage” of the disease. AS is considered in this paper as a disease continuum with symptoms depending on age at onset. The application of those specific strategies in children and adolescents with SpA seems limited because the most important manifestation in the early stage of disease is not IBP, but peripheral arthritis and enthesitis. In this instance, the logical approach to juvenile onset SpA according to ASAS criteria should not be through the axial criteria but rather the peripheral set of criteria. Ankylosing spondylitis (AS) is the prototype of a group of arthritis conditions collectively called SpA1, which includes undifferentiated SpA (u-SpA), reactive arthritis (ReA), and subsets of psoriatic arthritis (PsA), Crohn’s disease, and ulcerative colitis. The characteristics of these conditions include axial and peripheral enthesitis and arthritis, certain extrarticular manifestations, family aggregation, and HLA-B27 association. The spectrum of SpA spans from early, undifferentiated forms to well-defined diseases such as AS. Despite the fact that u-SpA might be the initial stage of any definite form of SpA, mainly AS, a number of patients may have only a short and mild course of symptoms followed by complete remission of inflammation and no permanent consequences.In contrast to this mild, self-limited disease, AS is the result of a long-standing process that combines chronic inflammation and new bone formation, mainly at the tendon and ligament attachments to bones. AS is usually characterized by inflammatory back pain (IBP) and morning stiffness, progressive reduction of the spinal mobility, lower-limb joint and entheses involvement, anterior uveitis, and non-specific inflammatory bowel disease (IBD) . The preThe diagnosis of AS is usually made eight to ten years after the onset of complaints (mostly IBP) and depends on the presence of axial signs and symptoms and radiographic changes of the sacroiliac joints Table  3]. The. The3]. The term “ankylosing spondylitis” means “stiff vertebrae” (from the Greek ankylos and spondylos). Alternative names, most in disuse, include seronegative polyarthritis, seronegative spondarthritis, seronegative spondyloarthritis, seronegative spondylarthropathies, spondyloarthritides. The stereotypic AS patient has a long-standing and severe disease characterized by spinal deformity and vertebral ankylosis for which no effective therapy has been available. Yet, it is clear that not all patients with AS fit into that stereotype. Moreover, the introduction of tumor necrosis factor (TNF) blockers has resulted in the control of signs and symptoms related to inflammation and the improvement of most outcome measures, including HRQoL, particularly in patients with a short disease duration.Within the last ten years, clinicians have tried to recognize and diagnose AS in the early inflammatory stage of the process that ultimately leads to bone proliferation ,10. The In this paper, the characteristics and development of the ASAS criteria for the classification of axial and peripheral SpA are reviewed. These classification criteria and their possible role in the classification of children and adolescents with SpA are examined by defining and comparing the juvenile and adult criteria as they apply to pre-radiographic AS, AS, u-SpA, and ASAS axial and peripheral SpA.Although the most common age at onset of AS is around 25 years, a variable percentage of patients start complaining in childhood or adolescence. Despite the fact that juvenile and adult onset forms differ in their mode of presentation, both forms represent a disease continuum in which age-related factors might play a role in clinical expression. Compared with juvenile onset AS, patients with adult onset disease have a higher prevalence of axial symptoms and, in contrast, a much lower prevalence of peripheral enthesitis and arthritis in the initial years of the disease -22. IncoThe consequences of AS seem more severe in juvenile patients -17,19-22In keeping with the traditional idea of Moll and Wright about the SpA group , we concThe rationale for the development of the ASAS axial and peripheral classification criteria was to facilitate the recognition and detection of patients with back pain at risk of AS. It is widely known that most patients with early AS present with undifferentiated manifestations, most characteristically with IBP, less frequently with peripheral arthritis and enthesitis, and rarely with extrarticular manifestations. Thus, the initial stage of AS corresponds to that of u-SpA, a group that accounts for an important proportion of SpA in the community ,31, specPeripheral u-SpA has a childhood counterpart, which is traceable to clinical descriptions embraced under terms such as “probable Still’s disease” , type IIThere are important variations in the proportion of adult-onset and juvenile-onset patients with u-SpA fulfilling AS criteria that occur throughout the disease courses in various studies Figure . The reaIn adults with u-SpA, progression to AS has been inconsistently associated with several factors. These include uveitis ,46,48, HIn contrast, in children with JIA or JRA, radiographic sacroiliitis and AS associate in univariate or multivariate analyses with a somewhat different set of factors at different intervals such as HLA-B27 ,61, DRB1Prospective data of patients with axial SpA according to ASAS criteria or IBP differ in some important aspects from retrospective studies. These differences are seen in the prospective German Spondyloarthritis Inception Cohort (GESPIC) , the MaaGESPIC  consisteThe combination of severe sacroiliitis on MRI and HLA-B27 was highly predictive for AS. The DESIR  study inThe importance of such prospective studies is that up to one-third of patients with AS fulfill the modified New York criteria for AS within two years of symptoms and 50% by five years. While cohort studies include patients with IBP, those studies in retrospect include a significant number of patients with peripheral symptoms. Despite a trend for male sex, HLA-B27, and uveitis in patients with AS, most features of this peripheral AS disease do not differ from those of patients with axial SpA. Ultimately, these prospective risk factors include some factors already identified in retrospective studies and help define and illustrate the relationships between u-SpA, pre-radiographic AS, and axial and peripheral SpA.Although Zeidler, et al.  proposedRudwaleit, et al.  first caBased on real-world clinical findings, Heuft-Dorenbosch, et al.  proposedThere is no such analyses yet in juvenile-onset SpA, but the association of some variables with the development of AS and radiographic sacroiliitis suggest a role for genetic, demographic, and clinical features for the progression of u-SpA to AS Table . EssentiToday, the concept of axial SpA has moved from the non-radiographic sacroiliitis stage of AS to the wider spectrum of SpA, including the axial and peripheral categories ,12 Tabl. The ASANevertheless, there are more issues in axial  and peri1) The existence of two sets of criteria has academic and research implications, yet their validation in various populations and clinical scenarios are needed before they would be widely used in clinical practice;2) The cost of MRI and HLA-B27 testing may limit the applicability of ASAS criteria in countries with budget restrictions or in segments of the population not covered by any health security system;3) Despite the fact that the definition of some variables listed in both axial and peripheral SpA criteria differ from each other to avoid classification overlaps, the existence of different definitions may be confusing Table ;4) Except for radiographic sacroiliitis, signs and symptoms in the ASAS criteria for SpA refer to active inflammatory and not structural damage; and5) Regarding nomenclature, the term “peripheral SpA” may appear to be contradictory in itself and confusingASAS criteria for axial and particularly peripheral SpA may have important implications for the recognition of children and adolescents with SpA and the understanding of the relationship between juvenile and adult SpA. Children and adults would be classified under the same criteria, the long-term follow-ups of children with SpA would be more easily carried out, and the results of clinical trials and management would be interchangeable to some extent.Before quickly accepting these ASAS criteria for children and adolescents, however, it is important to answer two specific questions:1) Is there a rationale for alternative criteria for children with ERA, PsA, and undifferentiated arthritis according to ILAR criteria? ERA and PsA (and perhaps some cases of undifferentiated arthritis) are the subgroups representing juvenile-onset SpA in the ILAR JIA classification criteria for JIA . The con2) If needed, which set of ASAS criteria is more appropriate for children, axial or peripheral? It seems clear that axial and peripheral SpA classifications have different purposes. While the former  is intenThe dilemma of how to apply adult AS and related SpA criteria to children has challenged pediatric rheumatologists for decades. The ILAR JIA criteria for PsA and ERA do not corresponds well with adult AS criteria. The new ASAS criteria offer rheumatologists a chance to reexamine how children with SpA, ERA, PsA, and undifferentiated arthritis can fit and not fit into these new criteria. Adult and pediatric rheumatologists want to diagnose these patients described by the ASAS criteria early and be able to offer these patients aggressive TNF inhibitor therapy, when possible and affordable, to possibly prevent joint and bone damage.The application of those specific strategies in children and adolescents with SpA is challenging as the most important manifestation in the early stage of disease is not inflammatory back pain as it is in adults, but peripheral arthritis and enthesitis. In this instance, the best approach to juvenile onset SpA according to ASAS criteria may be not to use the axial criteria but rather to use the peripheral set of criteria. The question of whether pediatric rheumatology needs new separate criteria for SpA, ERA, PsA, and undifferentiated arthritis remains controversial and goes against our need to encompass such similar adult and pediatric diseases under the umbrella of one set of criteria.The author declares that he has no competing interests.RB-V is the author and corresponding author and has designed and written the article. As author read and approved the final manuscript.


Although musculoskeletal ultrasound (MSUS) has emerged as an indispensible tool among physicians involved in musculoskeletal medicine in the last two decades, only recently has it become more attractive to pediatric rheumatologists. Thereafter, the use of MSUS in pediatric rheumatology has started to increase. Yet, an ever-growing body of literature shows parity and even superiority of MSUS when compared to physical examination and other imaging modalities.MSUS is suitable for examination of children of all ages and it has certain advantages over other imaging modalities; as it is cheaper, mobile, instantly accessible bedside, easy to combine with clinical assessment (interactivity) and non-invasive. It does not require sedation, which facilitates repetitive examinations. Assessment of multiple locations is possible during the same session. Agitation is rarely a problem and small children can be seated in their parents' lap or they can even play while being examined. Although musculoskeletal ultrasound (MSUS) has emerged as an indispensable tool among physicians involved in musculoskeletal medicine in the last two decades, only recently has it become more attractive to pediatric rheumatologists. Thereafter, the use of MSUS in pediatric rheumatology has started to increase. Yet, an ever-growing body of literature shows parity and even superiority of MSUS when compared to physical examination and other imaging modalities .Several noninvasive techniques have been proposed to assess articular involvement of the pediatric rheumatoid diseases; however, magnetic resonance imaging (MRI) and MSUS have come to the forefront. MSUS has a tremendous advantage over MRI in that the examination can be performed quite rapidly. Rheumatoid patients, especially the younger ones, are easily bored and cannot tolerate lying motionless on hard table for the time required for an MRI examination. In addition, during sonographic examination, the patient may move other extremities relatively freely, and the procedure does not require sedation. Cost and availability factors also strongly favor MSUS. The real time imaging capability of US allows dynamic assessment of joint and tendon movements, which can often aid the detection of structural abnormalities . On the MSUS is most commonly used in the assessment of soft tissue disease or detection of fluid collection. It can also be used to visualize musculoskeletal structures, such as muscle, fascia, tendon, para-tenon, ligament, synovium/capsule, hyaline and costal cartilage, fibrocartilage, nerve and bone surfaces . HoweverMost musculoskeletal work is performed using gray scale, which means images are produced in black-white format; each white dot in the image representing a reflected sound wave. Sound waves travel in a similar way to light waves and hence the denser a material is (e.g. bone cortex) the more reflective it becomes and accordingly, the whiter it appears on the screen. On the other hand, water is the least reflective tissue and therefore it appears as black while the sound waves travel straight through it. Two factors influence reflectivity: The acoustic impedance of materials and the angle of incidence of the sound beam. Acoustic impedance is the product of a material density and the speed of sound within that substance. According to the intensity of the echo, images are categorized in three forms. Anechoic: A structure that does not produce any internal echoes. Hypoechoic: A term used to describe an area that has decreased brightness of its echoes relative to an adjacent structure. Hyperechoic: A term used to describe a structure which has increased brightness of its echoes relative to an adjacent structure.The transducer is an essential part of the US equipment and is responsible for the generation of the US beam and the detection of returning echoes. A variety of linear-array transducers, including large (> 40 mm), medium-sized (< 40 mm) and small-field of view (hockey-stick-shaped) probes, are currently available in the frequency range used for musculoskeletal examinations. Selection of the most appropriate transducer primarily depends on the frequency  whereby high-frequency probes (e.g. 10-18 MHz) are used to visualize superficial structures and low-frequency ones (e.g. 5-10 MHz) for deeper tissues.The improvement in fast digital computer processing and memory storage capacity have recently improved the possibility of applying 3-Dimensional technology to US. 3D acquisition can be achieved with US using either 2D conventional transducers equipped with a small electromagnetic positional sensor or dedicated "3D-volume transducers" which are larger than standard probes. Although it is difficult to handle those probes, they provide better assessment of each scanning plane.Newer US techniques, including color and power Doppler imaging, provide color maps of tissues. The amount of color is related to the degree of blood flow, which may be of use in the assessment of vascular tissues as in soft tissue inflammation .In this review, we will focus on major topics whereby MSUS has improved our diagnostic, interventional and follow-up abilities in pediatric rheumatology.6 children per year [Juvenile idiopathic arthritis (JIA) is the most common chronic inflammatory arthropathy in childhood, accounting for approximately 6-19 cases per 10per year . It is cper year ,9.Similar to the stituation in adult rheumatoid arthritis, MSUS has proven to be valuable in the early diagnosis of JIA, for evaluation and follow-up of disease activity and for the assessment of treatment response . It is eThe synovial membrane is an important connective tissue lining the inner surface of the joint capsule, tendon sheath, and bursa. Therefore, it is essential to understand the pathogenesis and the pathological changes seen in inflammatory synovium in order to perform a complete scan of the synovial joints. In JIA, as in any other inflammatory arthritis, the synovium undergoes significant changes leading to the formation of a mass of synovial tissue. This is the result of edema, multiple redundant folds, and villae. The presence of joint, bursal or tendon sheath effusion is used as an excellent, indirect correlate of synovial inflammation. Further, its presence (as an anechoic structure) technically enables a better visualization of the synovial thickening, proliferation and villous formation during MSUS imaging .In the absence of an effusion, synovitis is diagnosed by the presence of an abnormally thickened hypoechoic region, usually measured in a standard plane with reference to an established normal range or to the contralateral normal joint. Therefore, MSUS can easily detect significant degrees of synovitis which is not determined by clinical examination ,15 and cWith MSUS, synovial hypertrophy is detected as solid, non-compressible, hypoechoic tissue in connection to joint lines or surrounding tendons . In chilEvaluation has been enhanced on machines with power Doppler setting which depicts the increased vascularity of the hypertrophied synovium by demonstrating microvascular flow. The Doppler signal can distinguish between active and inactive synovitis, correlating to clinical and laboratory data -19, MRI Athough it is nonspecific, joint effusion is a valuable indicator of active joint disease. US has been shown to be one of the best methods for detection of increased intra-articular fluid and synovial proliferation. Graded compression is useful in distinguishing isolated effusion from synovial proliferation. Effusions, as small as 1 mL can be detected with ultrasound and interobserver agreement for ultrasound detection of effusion in hand and foott joints is reported to be 79% .Using MSUS to detect and localize small joint effusions is effective in clinical practice. In patients with inflammed MCP and proximal interphalangeal (PIP) joints, MSUS improved accurate needle placement from 59% by palpation guidance to 96% by MSUS guidance . MSUS haIn children with JIA, MSUS imaging has been shown to be a sensitive modality to detect alterations in the articular cartilage . It alloCartilage edema in early stages of JIA can be detected sonographically as thickening of the articular cartilage. Chronic inflammation of the cartilage results in permanent damage to the articular surface. This is observed sonographically as blurring of the articular surface. Continued destruction of the cartilage due to rheumatoid disease is seen as pitting of the articular surface and measurable thinning of the cartilage. Cartilage loss is better detected than on plain films especially in young children with thick epiphyseal cartilage and at the early stages of the disease .In a cohort of healthy children of different ages, the reliability of the assessment of cartilage thickness with US has been recently demonstrated by Spannow et al . The autThe presence of juxta-articular flow at color Doppler examination in the growing child may either represent normal flow of the well-vascularised cartilage of the epiphysis or synovial hyperemia indicating inflammation. Flow in the cartilage probably indicates normal cartilaginous flow in contrast to flow inside the synovium which probably indicates hyperemia .Studies assessing long-term outcome of JIA have shown that a relevant proportion of patients may develop progressive joint destruction and serious physical disability . The devIn line with previous literature concerning rheumatoid arthritis, recent studies on JIA confirmed that MSUS is equal or superior to plain radiography in detecting cortical erosions in sonographically accessible areas, but that it is less reliable in detecting intramedullary lesions and those within the centres of larger joints, due to the acoustic shadowing from overlying bones ,28,33.Bone is often regarded as a barrier to the use of MSUS in clinical practice. On the other hand, one of the most exciting applications of MSUS is the evaluation of bone erosions in rheumatologic diseases. On MSUS, bone erosions can be seen as interruption of the smooth, continuous hyperechogenic line corresponding to the bony cortex. Marginal erosions are usually seen in rheumatoid diseases and are identified as crater-like defects in the bony contours along the edges of the articular cartilage. Recent studies support the superiority of MSUS over radiography in detection of erosions ,35. ExpeJuvenile spondyloarthropathies which form the second most common form of chronic arthritis in children, are a group of disorders that affect the axial and extra-axial joints ,37. JuveMSUS is useful for detection of synovial effusions/hypertrophy and various forms of enthesopathy, i.e. calcifications, enthesophytes, bony erosions at insertion sites. It may also show loss of the normal fibrillar echogenity of tendons, absence of the homogeneous pattern, blurring of tendon margins, and irregular fusiform thickening . The abiMusculoskeletal involvement of systemic lupus erythematosus includes arthralgia and arthritis/synovitis typically affecting the small joints of the hands, wrists and knees. It is generally not erosive, but can be deforming . MSUS caThere are some innovations regarding ultrasound technology like sono-elastography . It is aMSUS is suitable for examination of children of all ages and it has certain advantages over other imaging modalities ,47; it iMSUS: Musculoskeletal Ultrasound; JIA: Juvenile Idiopathic Arthritis; MRI: Magnetic Resonance ImagingThe authors declare that they have no competing interests.FT, ED and LO have made substantial contributions to conception and design.FT, ED and LO have been involved in drafting the manuscript or revising it critically for important intellectual content.


The vast amount of data published in the primary biomedical literature represents a challenge for the automated extraction and codification of individual data elements. Biological databases that rely solely on manual extraction by expert curators are unable to comprehensively annotate the information dispersed across the entire biomedical literature. The development of efficient tools based on natural language processing (NLP) systems is essential for the selection of relevant publications, identification of data attributes and partially automated annotation. One of the tasks of the Biocreative 2010 Challenge III was devoted to the evaluation of NLP systems developed to identify articles for curation and extraction of protein-protein interaction (PPI) data.The Biocreative 2010 competition addressed three tasks: gene normalization, article classification and interaction method identification. The BioGRID and MINT protein interaction databases both participated in the generation of the test publication set for gene normalization, annotated the development and test sets for article classification, and curated the test set for interaction method classification. These test datasets served as a gold standard for the evaluation of data extraction algorithms.The development of efficient tools for extraction of PPI data is a necessary step to achieve full curation of the biomedical literature. NLP systems can in the first instance facilitate expert curation by refining the list of candidate publications that contain PPI data; more ambitiously, NLP approaches may be able to directly extract relevant information from full-text articles for rapid inspection by expert curators. Close collaboration between biological databases and NLP systems developers will continue to facilitate the long-term objectives of both disciplines. Before the explosion of online data archives such as Medline and PubMed, searches of the scientific literature for specific data content was a tedious practice that relied on dedicated paper-based services such as Current Contents. With the advent of electronic text databases and Internet access, the entire corpus of biomedical literature can be readily queried by author name and free-text keywords, such as gene or disease names. Nevertheless, whilst retrieving the literature of interest is now a relatively trivial task, mining and archiving the individual biological data elements contained within each of the millions of publications is still not possible. De facto there is no well-validated procedure that enables extraction of relevant information from the biomedical literature by automated parsing algorithms. This situation exists for several reasons, not least because information is embedded in non-standard descriptive natural language. The problem is exacerbated by the fact that publication authors typically fail to use unambiguous identifiers to describe bio-molecular entities and by the fact that data, with few exceptions ,2, are nTo date, only the perseverance of expert curators at specialized biological databases enables a fraction of the available data to be accessed for automatic codification and computation. Manual curation, although more accurate and significantly more reliable than automated annotation , is a trHigh-throughput technologies have recently permitted the rapid accumulation of vast collections of genome-scale data for mRNA expression , proteinH. sapiens recorded in PubMed. Despite the cooperative efforts of protein interaction databases through the International Molecular Exchange (IMEx) consortium [To date, however, these interaction database initiatives have relied exclusively on manual parsing and curation of the literature. Although complete coverage of the literature has been achieved for some model systems, notably budding and fission yeast ,19, the nsortium ,18,20-27The Critical Assessment of Information Extraction in Biology (BioCreative) -31 initiBioGRID and MINT annotate only data that is explicitly corroborated by experimental evidence reported in the peer-reviewed literature. MINT primarily annotates protein-protein interactions (PPI), whereas BioGRID annotates both protein and genetic interactions. While both databases are members of the IMEx consortium, MINT as active member and BioGRID as an observer, the two databases adhere to slightly different curation standards. MINT annotates interaction data according to the PSI-MI (Proteomics Standards Initiative–Molecular Interactions) controlled vocabulary developed and maintained by a working group of the Human Proteome Organization Proteomics Standards Initiative (HUPO-PSI) . BioGRIDBioGRID and MINT contributed high quality manually curated datasets as gold standards for three tasks of the Biocreative III Challenge, namely gene normalization (GN), article classification (ACT) and interaction method (IMT). The BioGRID dataset was curated from a pre-selected collection of publications provided by the Biocreative organizers while the MINT dataset was derived in the course of routine curation for the database. The aforementioned datasets formed the competition test set. The assembled data was revealed at the close of the competition.Gene normalization is the process of linking genes or proteins to stable database identifiers and as such is a crucial step in the annotation of biological interactions. Expert curators from both BioGRID and MINT participated with curators from other databases in the annotation of the test set for the gene normalization task. Curation specifications were set by the BioCreative III organizers and, for each gene mentioned in the full-text, required the annotation of taxon and Entrez Gene identifier. If either of these conditions could not be met, the gene was not annotated.From previous Biocreative editions, it has clearly emerged that classification of publication relevance for PPI data requires the analysis of full-text articles rather than abstracts . Indeed,Unfortunately, full-text articles are often not accessible to text mining tools, in contrast to abstracts, which are freely available through PubMed in a common XML format. While open access initiatives have gained momentum, particularly as supported by the NIH, HHMI and Wellcome Trust, full-text articles are typically not freely available from for-profit high impact journals. In addition, XML specifications differ from one journal to another, requiring the development of specific tools to parse articles gathered from various publishers.The BioCreative consortium organized an article classification task in order to assess the capability of available systems to classify pertinence of articles for PPI data based solely on abstracts. Participants were provided with a collection of recent abstracts, where for many of them free full-text articles were available. Systems were then tested for their ability to carry out a binary classification for relevance to PPI data, and were evaluated by comparing to manual curation results.BioGRID (one curator) and MINT (two curators) manually classified a development and a test set of for relevance to PPI. Although the two databases do not share the same evidence codes and annotation vocabularies, both adopt the same rules for articles selection. Articles were considered suitable for curation only if the abstract suggested the presence of at least one experimentally verified protein interaction. As a consequence indirect functional connections or predicted interactions were not considered for positive classification. The resulting datasets were used as a benchmark to evaluate the precision of the dataset generated by the organizers for the training and test phases of the competition.As the datasets annotated by BioGRID and MINT were partly overlapping (200 articles in common), it was possible to assess the inter-annotation agreement between the two databases. The percentage overlap between independent MINT and BioGRID curation was 95%, a remarkably high value given the different strategies and diverse expertise of different curators. Further analysis showed that the residual 5% discrepancy was not due to classification error per se, but rather due to contextual ambiguities in the abstract. For instance, in one of the scrutinized articles (PMID:19628465) a phosphorylation event was cited in the abstract even though no experimental evidence was proved.A crucial aspect in the annotation of PPI data is the determination of the experimental method used to support the interaction. The reliability of any given interaction is correlated with the accumulation of experimental evidence obtained by diverse techniques . The PSIBoth BioGRID and MINT annotated physical interaction and co-localization evidence, BioGRID in accordance with the MIMIx recommendations and MINT in accordance with the IMEx curation guidelines . The divEmbo Journal, EMBO Reports, Developmental Cell, Molecular Biology of the Cell, Molecular Cell, Molecular and Cellular Biology, Proceedings of the National Academy of Sciences, The Journal of Biological Chemistry and The Journal of Molecular Biology. The MINT test set was composed of protein-protein interactions extracted from articles published in EMBO Journal and EMBO Reports. These manual curation efforts resulted in a dataset of 157articles that contained evidence for 954 interactions for BioGRID and 66 articles that contained evidence for 3093 interactions for MINT. The reason for the high number of interactions in the MINT dataset is due to the chance presence of articles that reported almost 2300 interactions derived from medium and high-throughput experiments .The BioGRID test set was composed of protein-protein interactions extracted from articles published in the journals Detailed results of comparisons between these manually annotated interaction datasets and interactions parsed automatically from the same publications are described elsewhere in this issue. Nevertheless, even from casual inspection, it is still possible to observe a substantial discrepancy between manual and automated curation ,38, suggThe advent of genomics has enabled the systematic description of entire genomes . The nexHowever, the comprehensive annotation of interaction maps is far from complete , both beIn the future, this problem may be largely solved by the adoption of rigorously structured scientific abstracts that contain author-annotated data attributes, including standard gene identifiers and interaction evidence codes. Interaction data may then be captured automatically by various databases. Nascent efforts are underway to develop and implement computable abstracts as a new aspect of the scientific literature . In the At this juncture, the performance of current automated information extraction systems is not comparable with manual curation. Text-mining tools are thus still unable to reliably capture the richness of experimental details from full-text articles and associated figures, tables and supplementary data nearly as effectively as human curators. Nevertheless text mining is placed to play an increasingly important role in improving the efficiency of manual curation by assisting the selection of relevant articles and facilitating the information extraction process.On these premises, the 2010 version of the BioCreative Challenge was shaped with the explicit aim of directing the development of text mining systems towards the immediate needs of biocurators. The correct assignment of gene/protein identifiers is a sine qua non of systematic curation and, although gene mention detection methods have high accuracy, automated approaches are still far from effectively achieving correct database identifier assignment. Although it is now clear that the most reliable results are attained by mining the full-text of articles, abstracts are frequently the only freely-available resource. Thus, the aim of the Article Classification Task was the development of tools that would permit curators to obtain a more refined list of articles than from, for example, a simple PubMed query. Moreover, through parsing abstracts, available text mining systems are able to place each retrieved article in rank order of likely relevance, thereby greatly assisting the curator in the selection of articles more likely to contain protein interaction data, or other data types.As the annotation of the experimental method employed to detect the interaction is a crucial aspect of the curation pipeline, the purpose of the Interaction Method Task was to develop tools able to assist curators in assignment of experimental details. Although performance of current systems in this task was far from optimal, these initial efforts represent an important starting point for the delivery of more efficient tools that facilitate this key aspect of biocuration . RefinemThe realization of high-performance user-oriented text mining systems will require ever-closer collaborations between tools developers and biological interaction databases. In particular, the assembly and release of high quality benchmark datasets will be crucial for the refinement of text-mining algorithms. It will be of particular interest to develop rule sets that enable the capture of more subtle textual features that define biological interactions and evidence codes. These rules in turn will help establish the basis for structured scientific abstracts that are implicitly machine-readable. The BioCreative Challenge III competition of 2010 demonstrates that alliances between text mining groups and protein interaction databases, such as BioGRID and MINT, facilitate the research interests of all, to the overal benefit of the biomedical research community. The advancement of information extraction tools should enable the goal of full literature curation of biological interactions to be achieved in a reasonable time frame.For the article classification task curators from BioGRID and MINT, assisted by MyMINER software , classifEmbo Journal (issue 22 from 2008), EMBO Reports (issue 5 from 2009), Developmental Cell , Molecular Biology of the Cell , Molecular Cell , Molecular and Cellular Biology , Proceedings of the National Academy of Sciences , The Journal of Biological Chemistry  and The Journal of Molecular Biology . MINT articles were chosen from issues of EMBO Journal  and EMBO Reports . Both datasets are available for download at http://www.biocreative.org/resources/corpora/biocreative-iii-corpus/.BioGRID and MINT annotated the test set to assist the Interaction Method Task. Both databases curated protein-protein interactions in accordance with the PSI-MI controlled vocabulary, choosing the deepest possible child term of PSI-MI controlled vocabulary root term ‘interaction detection method’. UniProtKB  identifihttp://thebiogrid.org/downloads/archives/Other%20Datasets/Biogrid_Biocreative_2010_IMT.txt.zip.BioGRID dataset is also available at PMID:18398472, PMID:19393081, PMID:20502630, PMID:20502631). For each identified gene, the taxon and EntrezGene identifier were reported. This annotated dataset served as test set for the Gene Normalization task.BioGRID and MINT curators annotated genes from articles provided by the Biocreative organizers (ACT: Article Classification Task; GN: Gene Normalization, HHMI: Howard Hughes Medical Institute; HUPO: Human Proteome Organization; IMEx: International Molecular Exchange Consortium; IMT: Interaction Method Task; MI: Molecular Interaction; MINT: Molecular INTeraction; NIH: National Institute of Health; NLP: Natural Language Processing; PPI: Protein Protein Interaction; PSI: Proteomics Standards Initiative.AC, GC and MT conceptualized the project. AC wrote the manuscript with input from GC and MT. AC, AW, LP, LL, MI, LC, GC participated in curation.The authors declare that they have no competing interests.


Androgen Insensitivity Syndrome (AIS) could be considered as a disease that causes resistance to androgens actions, influencing both the morphogenesis and differentiation of the body structures, and systems in which this hormone exerts its effects. It depends on an X-linked mutations in the Androgen Receptor (AR) gene that express a variety of phenotypes ranging from male infertility to completely normal female external genitalia. The clinical phenotypes of AIS could vary and be classified into three categories, as complete (CAIS), partial (PAIS), and mild (MAIS) forms, according to the severity of androgen resistance. We will describe a case of CAIS in a 16-year-old patient. Androgen Insensitivity Syndrome (AIS) could be considered as a disease that causes resistance to androgens actions, influencing both the morphogenesis and differentiation of the body structures and systems in which this hormone exerts its effects. It depends on an X-linked mutations in the Androgen Receptor (AR) gene that express a variety of phenotypes ranging from male infertility to completely normal female external genitalia. The first who described this syndrome was John Morris , but we The patient was 16 years old, with female habitus and voice, and normal intellectual function. Informed parental consent, patient consent, and approval by the Hospital Ethics Committee were obtained before initiating the report.The patient was sent to our observation by a provincial ambulatory service of gynecology and obstetrics, with diagnosis of hypergonadotropic hypogonadism. Previous exams showed elevated blood gonadotropins levels and infantile internal genitalia. At general physical examination, we found adipose tissue well represented, poor body hair, and hypotrophic breasts. The gynecological examination evidenced infantile external genitalia and viable vagina. Considering the suspected diagnosis of hypergonadotropic hypogonadism, we administered Hormone Replacement Therapy with oral estroprogestinics. At the follow-up after three months, the patient referred that there had been no change in primary amenorrhea. For this reason, we decided to repeat the hormonal and ultrasound examinations, obtaining the following results.The hormonal levels were the following .We performed transabdominal ultrasound that evidenced absence of uterus and of structures related to the ovaries in the area where it is commonly found, and then we determined a “male” 46,XY karyotype. After communicating the results to the parents (who preferred not to inform the patient), we decide to perform laparoscopy (which confirmed the results described by ultrasound) and to take biopsies of testicular-like tissue held in the pelvic cavity. Histological examination of biopsy tissue showed testicular parenchyma, seminiferous tubules of smaller size than the norm, and agenesis of the germinal epithelium. For this reason, considering the high incidence of malignant degeneration of the gonads held in the abdominal cavity, we addressed the patient to a bilateral gonadectomy.The histological examination of the removed gonads enlightened presence of spermatic cord and epididymal-didymus parenchyma. With reference to testicular tissue, we highlighted seminiferous tubules arranged in irregular lobular formations, for a process of interstitial and peritubular fibrosis. The tubular spaces were widely devoid of germ epithelium with the presence of immature Sertoli's cells in vacuolar degeneration, marked vascular congestion of parenchymal and hilar vessels. Moreover, there were associated findings of congestion and interstitial fibrosis of the funiculars sections and of the epididymal intertubular connective tissue.All these findings were consistent with Complete Androgen Insensitivity Syndrome. Therefore, the patient was referred to a hormone replacement therapy. Parents preferred not to disclose the diagnosis to the daughter, to whom it was explained that, due to the absence of the uterus and ovaries, hormone therapy was needed but could not resolve nor amenorrhea nor infertility. This news was so devastating for a young psychologically female, who hoped to solve her problems in other ways.