Intravascular fasciitis (IVF) is a rare benign condition characterised by reactive myofibroblastic proliferation arising from the superficial or deep fascia and involving arteries and/or veins. It is a distinct variant of the more common condition of nodular fasciitis, which possesses similar clinical and histological features to IVF, but lacks vascular invasion. A thorough review of the literature revealed 26 reported cases of IVF.We report a case of IVF in a 16-week pregnant lady affecting the hypothenar eminence of the hand associated with the ulnar artery.The characteristic involvement of muscular arteries and veins by reactive myofibroblastic proliferation in IVF suggests a malignant component and often leads to an inappropriate diagnosis for this benign condition. We propose that hormone-related changes associated with pregnancy may play an important role in the aetiopathogenesis of this myofibroblastic lesion. Intravascular fasciitis (IVF) is a term originally described by Patchefsky and Enzinger to describe this distinctive variant of nodular fasciitis . It is ahousewife. There was no history of trauma, insect bites or drainage from the area. There was no significant past medical or surgical history. She was not on any prescribed medication. There was no family history of tumours. She denied smoking, alcohol and drug abuse. On examination, a non-tender, firm to palpation, well-demarcated and tethered to the subcutaneous tissues mass, surrounded by mild erythema, and measuring approximately 2.5 cm × 3.0 cm was revealed, in the absence of palpable lymph nodes at the ipsilateral elbow or axilla. There were no other abnormal signs.In August 2003, a 20-year-old 16-week pregnant mother of one, presented to the orthopaedic outpatient clinic with a 2-month history of a slowly growing painless mass located in the right hypothenar eminence. She was right-hand dominant and a Haematological and biochemical investigations were within the normal range. A plain X-ray of the right hand and forearm was unremarkable. Magnetic resonance imaging showed the mass to have infiltrated all skin layers and to have encircled the ulnar artery without involvement of the underlying bones Figure . The absThree weeks after initial presentation, the lesion was excised preserving the ulnar nerve and artery. Histology showed a multi-nodular, well-circumscribed uniform spindle cell lesion with varying cellularity and fibrosis. In one section, central cyst formation was observed. Occasional mitoses of normal morphology were seen. Necrosis was not a feature. In several areas, the lesion appeared to be intravascular, the vessel was lined by an apparent endothelium Figure . ScatterThe patient had an uneventful recovery and required no further therapy. She continues to be evaluated for local recurrence every 6 months, with no sign of recurrence at the 2-year follow-up.IVF was first described as a subset of cases closely resembling the clinical and histological picture of nodular fasciitis (NF) . RecogniPre-existing trauma, viral infection, and venous thrombosis resulting in myofibroblastic transformation of the vessel wall have all been implicated in the aetiopathogenesis of IVF, but there was no evidence for either of these in our case affecting the hypothenar eminence. Pregnancy was the only significant co-existing medical condition.We hypothesised that hormone-related changes seen during pregnancy may have contributed to the development of IVF in our patient. Oestrogen in particular, has for long been known to stimulate fibroblast and smooth muscle cell types and has been implicated in fibroproliferative diseases such as carpal tunnel syndrome, Dupuytren's contracture, and dermoid tumours. A weak presence of oestrogen receptors has also previously been demonstrated in nodular fasciitis . OestrogIVF is a benign myofibroblastic proliferative lesion that involves arteries and/or veins, a feature distinguishing it from other pseudosarcomatous proliferative lesions. Nevertheless, it may be easily mistaken for a malignant sarcomatous invasion of the vasculature. Based on our case presentation, we propose that pregnancy and oestrogen-related changes may be another risk factor for IVF and play an important role in the aetiopathogenesis by influencing proliferative changes in fascia myofibroblasts, however, more research and further cases need to be studied prior to establishing such hypothesis.The author(s) declare that they have no competing interests.1 reviewed the literature and prepared the manuscript. SRC5 provided the case and revised the manuscript. AN3 obtained patient consent and helped in preparation of the manuscript. ET6 and EMT2 conception and identification of case hypothesis, critical revision of the manuscript and coordination of the study. AA4 provided the histology images and histology information. All authors read and approved the final manuscript.AA


Subsequent clinical follow-up and CT examination of the patient has not revealed recurrent disease at 14 months.Carcinoma within a long-standing fistula-in-ano is rare and may be defined by specific neoplastic involvement of the fistulous track in the absence of rectal mucosal carcinoma. The presence of a carcinoma of mucinous histology occurring synchronously in the perianal region and the colon is exceptionally rare. We present a case with a review of the literature concerning its aetiopathogenesis and treatment. A 72-year-old man with a 2 months history of dark red rectal bleeding and mucus per rectum with alternating constipation and diarrhoea, was observed. Clinical examination and a barium enema showed a perianal fistula and an annular stenosing lesion of the rectosigmoid. Preoperative CT scan confirmed the colonic lesion. Colonic resection and wide fistula excision were performed. Histology showed an adenocarcinoma with a clear resection margins. The fistula also showed a similar histology. Chemoradiation (5-Fluorouracil (425 mg/m Carcinoma within a long-standing fistula-in-ano is rare and may A 72-year-old Afro-Caribbean male presented to our University Hospital with a 2 months history of dark red rectal bleeding and mucus per rectum with alternating constipation and diarrhoea as well as 20 lb. weight loss. There was no family history of colorectal neoplasia. There was an attendant history of perianal discharge for the previous 2 years and the patient volunteered that he had been seen in the United Kingdom and told that he had a perianal fistula in 1999. Clinical examination was unremarkable apart from the perianal area where a perianal fistula was evident which appeared to be low trans-sphincteric in nature. It was firm and indurated. A barium enema which had been performed outside the hospital showed an annular stenosing lesion of the rectosigmoid. Preoperative CT scan confirmed the colonic lesion but was otherwise unremarkable. In view of the presentation he was taken to the operating theatre after full bowel preparation for elective sigmoid colectomy and wide fistula excision.2) and leucovorin (20 mg/m2) before and after 4500 cGy external beam radiotherapy (EBRT) to the perineum and inguinal portals. Clinical and CT examination of the patient has not revealed any signs of recurrent disease at 14 months.Histology of the colon showed a moderate to well differentiated adenocarcinoma with extensive mucinous components and none out of 10 lymph nodes resected was involved with tumour. (Stage IIA American Joint Committee Cancer) The resection margins of the tumour were clear. The fistula also showed a similar histology Figure  The postSynchronous adenocarcinoma of the rectosigmoid and the perianum is exceptionally rare and we could only find 6 previously reported cases -10. VeryThe presence of perianal malignancy of mucinous histology may have several aetiologies. One possibility is malignant degeneration within a long-standing anal fistula and this, although uncommon, has been well reported. The histology of such cases tends to be either squamous or mucinous, with Rosser first describing in 1931 the 3 basic criteria for definitional purposes which determine that a fistula has undergone malignant transformation. For thiet al.[Viable tumour cells which represent clones capable of transplantation and proliferation have been retrieved from the lumen of the large intestine distal to established rectal tumours. In thiset al. where tuet al. where thThe management of this patient is unclear since too few cases have been reported to provide any evidence base for therapy. In our case, we performed a sphincter saving procedure as opposed to that of Hyman and Kida who recommended an extended abdominoperineal resection The rôleNS: Assisted in the photography of the case and in the literature searchPP: Assessed the histology and the photomicrographsAC: Assisted in the format and design of the paperAPZ: Assisted in the literature search and the writing of the articleAll authors read and approved the final manuscript.


This article reports measurements of household levels of gamma and cosmic rays at the addresses of children with cancer at the time of diagnosis and six months before, and of similar data at the addresses of control children. There is no indication of increased risk with increasing dose rates either in matched or unmatched analyses, with or without adjustment for deprivation. Sub-division by diagnostic group did not reveal any association with any specific types of malignancy. Studies of the relationship between household gamma rays and radon concentration show no evidence of any interactions.British Journal of Cancer (2002) 86, 1727–1731. doi:10.1038/sj.bjc.6600277www.bjcancer.com© 2002 Cancer Research UK To address this question separate household measurements were made of radon gas concentration and of penetrating external radiation . Whilst there was special interest in the biological effects of high linear energy transfer (LET) irradiation from radon and its short term decay products  variation in dose rate . The UK The UKCCS was designed as a population-based matched case–control study covering the whole of Great Britain. Ten regional centres  Family Health Services Authority  or Scottish Health Board of residence. Children were eligible if they were born in Great Britain, had no prior malignancy and were not in local authority care. Subjects were ineligible if they themselves or their parents had lived outside Great Britain for the three months leading up to diagnosis. Eligible controls who declined to participate were replaced until two controls were enrolled .A face-to-face interview was conducted with each child's parents, covering social, occupational and medical histories of the child and parents. A full residential history for the child was also collected and all addresses lived in by the child for 6 months or more were targeted for measurement. A written request to participate was then made to each residence. Following agreement, two radon and two thermoluminescent dosimeters (TLD) or ‘gamma’ detectors were sent to each household with instructions to place one of each in the main bedroom and in the main living area. After 6 months had elapsed a letter was sent recalling the detectors, which were returned to the NRPB for processing and measurement of the cumulative exposure. This study reports on the results of the address of the case at diagnosis, that is, those who had lived at that address for at least the previous 6 months.The system used for passive environmental photon monitoring consisted of a modification of a standard body TLD used for personal photon monitoring and the assessment of dose is based on two 30% LiF;Mg;Ti detector elements. These detectors respond to all types of penetrating ionising radiation including terrestrial gamma rays and secondary particles resulting from cosmic rays.The results are expressed as absorbed dose to air. Annual estimates are in micro gray (μGy) per year and hourly dose rates in nano gray (nGy) per hour. The results given in this study include the dose from cosmic rays; this varies with height above sea level, but differences within the UK are small. The mean has been estimated to be 32 nGy per hour  or 280 μThe main problems encountered with the detectors were occasional damaged detectors, faulty readouts, and differences between the recorded doses on the two detectors of greater than 20%. In all these cases the result was excluded from the analysis. It is estimated that this occurred for between 5 and 10% of measurements. Where the readings of the two detectors differed by less than 20%, the mean value was used. Doses accumulated away from the household of measurement were allowed for by subtracting 2.3 μGy for each day in storage, and 0.9 μGy for each day in transit.Detectors were intended to be in place for 6 months but the period over which measurements were actually made varied. The analyses were restricted to the measurements of the doses that had accumulated with the detectors in place within a household for both living-room and bedroom for between 5 and 7 months. Over 99% of all detectors met this requirement.An area-based index of deprivation for the child's residence at the time of diagnosis, and 6 months before, based on an index used in a previous study of childhood leukaemia . The indAll statistical analyses were performed using Stata version 6 . All estThe parents of 3838 children with cancer and 7629 children without cancer were interviewed, representing 87% of eligible cases and 64% of eligible controls. Following interview, measurements were obtained from the home at diagnosis of 2165 cases and 5086 controls. Nearly all (97%) estimates were based on readings obtained from both the bedroom and the living room.−1) to a maximum of 2027 μGy y−1.The case household participation for TLD ‘gamma’ detection was virtually identical to that for radon measurements. Thus all the issues that arose from the differential response rates of the case and control families and from the overall response rates are similar to those discussed in the accompanying radon paper . Table 1Table 1Table 2land See . This isP=0.15)Studies of dose rate by measurement year and months by region showed little variation over the period of the study (results not shown). Table 3−3 is shown in Table 5The relationships between TLD-derived doses divided into thirds  and radon concentrations split into the five predefined levels  of 0–24, 25–49, 50–99, 100–199 and 200+ BqmTo our knowledge this is the first case–control study directly measuring domestic gamma ray and cosmic ray levels and relating this to the risk of all childhood cancers. The results relate to the households of the affected children at the time of diagnosis, and are limited to children who had lived at the address for a minimum period of 6 months, the control households having the same limitations. The study is limited by accuracy of the TLD measurements, including inherent detector variability, storage, transport, householder compliance and the assumption of constant cosmic ray dose rate irrespective of altitude of the household. Some indication of combined uncertainties can be obtained from Cosmic ray dose rates increase systematically with increasing altitude. However, most of the population live at relatively low altitudes (see below), so it is reasonable to assume the variation in dose rates seen in the analysis is mostly due to terrestrial gamma rays.The geographical trends in gamma-ray dose rate results by Region that might be expected from knowledge of geological variations are obscured by variation in population density. For example: studies of Terrestrial gamma rays have been estimated to contribute about 30% of the total natural annual low-LET dose to bone marrow, most of the remainder being due to cosmic rays (∼40%) and natural internal radio nuclides with the body (∼30%) , then ab−1 is very similar to that found in the NRPB material  positive trend. That one such trend should be observed when six groups are examined is hardly surprising. When the estimated mean cosmic ray dose rate is taken into account, the mean household terrestrial gamma-ray dose rate of 62.1 nGy hThe increase in measured household exposure with increase in the deprivation index necessitated adjustment for socio-economic factors, but this makes no significant difference to the results. With the exception of the highest measured gamma-ray dose rate and radon concentration there was no evidence of any association between the gamma-ray and the results of the radon analysis. The number of households for which the highest levels of both were recorded (8) was, however, very small.In conclusion, in line with standard risk estimates, the findings from this study are broadly reassuring.RA Cartwright, G Law, E Roman, E Gilman, OB Eden, M Mott , K Muir, D Goodhead, G Kendall.KK Cheng, Central Region; N Day, East Anglia Region; RA Cartwright, A Craft, North East Region; JM Birch, OB Eden, North West Region; PA McKinney, Scotland; J Peto, South East Region; V Beral, E Roman, South Midlands Region; P Elwood, South Wales Region; FE Alexander, South West Region; CED Chilvers, Trent Region; R Doll, Epidemiological Studies Unit, University of Oxford; GM Taylor, Immunogenetics Laboratory, University of Manchester, Manchester; M Greaves, Leukaemia Research Fund Centre, Institute of Cancer Research; DT Goodhead, Medical Research Council, Radiation and Genome Stability Unit, Harwell; FA Fry, National Radiological Protection Board; G Adams, UK Co-ordinating Committee for Cancer Research.KK Cheng, E Gilman, Central Region; N Day, J Skinner, D Williams, East Anglia Region; RA Cartwright, A Craft, North East Region; JM Birch, OB Eden, North West Region; PA McKinney, Scotland; J Deacon, J Peto, South East Region; V Beral, E Roman, South Midlands Region; P Elwood, South Wales Region; FE Alexander, M Mott, South West Region; CED Chilvers, K Muir, Trent Region.RA Cartwright, G Law, J Simpson, E Roman.Br J Cancer (2000) 82: 1073–1102A complete list of investigators is given in: The United Kingdom Childhood Cancer Study: objectives, materials, and methods.


Biomedical research in the post-genome era is intensely data-driven and increasingly more integrative as new technologies are introduced, such as next- or third-generation sequencing, mass spectrometry, and imaging to identify novel biological insights. The volume and complexity of biomedical data is increasing exponentially as faster high-throughput machines are introduced. As a result, many research institutes, biotech companies, pharmaceutical companies, and computational labs are considering cloud computing as a cost-effective alternative to process and store this vast amount of data. Efforts in next-generation sequencing (NGS) There are multiple cloud providers, both commercial and open source, including Amazon Web Services (AWS), Rackspace, GoGrid, Nimbus, and Eucalyptus, each contributing to the popularity and globalization of cloud computing. For the purposes of this guide, we focus on the use of AWS as the cloud computing platform and adopt the definition of Vaquero, who states that the cloud is “a large pool of easily usable and accessible virtualized resources . These resources can be dynamically re-configured to adjust to variable load , allowing for optimum resource utilization” http://docs.amazonwebservices.com/AWSEC2/2009-11-30/GettingStartedGuide/): Elastic Compute Cloud (EC2), Elastic Block Storage (EBS), and Simple Storage Service (S3). For additional AWS products beyond the scope of this overview, we refer the reader to the AWS Web site (http://aws.amazon.com/). EC2 contains a variety of user selectable instance types that range in computing power and cost (http://aws.amazon.com/publicdatasets/). Access to all of AWS's services can be done using either a Web-based console, for beginners, or through the command line using an AWS-specific application programming interface (API), for advanced users. AWS costs are generally based either on an hourly rate or amount of data transferred or stored or other services used , networking, and operating system, and is an example of “infrastructure as a service” (IaaS). IaaS is popular with computational biologists because it offers more flexibility for designing projects ad hoc. The majority of computational projects will make use of three AWS products (see “Get Started with EC2” at and cost . An instces used .http://media.amazonwebservices.com/pdf/AWS_Security_Whitepaper.pdf).Before beginning to use the cloud, it is important to understand the basic best practices for cost control and data security  or Secure Copy (scp). Both the AWS Web console and command line tools provide a simple interface to generate key pairs when launching an instance. The public key is automatically installed onto an instance, and the private key can then be used on a computer that will ssh into that instance.Recently, AWS introduced Identity and Access Management (IAM) to offer greater control and management of multiple users. Each user has their own set of security credentials to access cloud resources, eliminating the need to share login information and keys for the master AWS account owner. This is important because the master account contains the personal billing information, which, for obvious reasons, should not be accessible to all users. IAM can restrict services based on specific users or group policies. For example, it is possible to restrict a user to a specific S3 bucket between 9 A.M. and 5 P.M. from a specific IP address.A security group defines a set of rules that govern how traffic (data or communication) reaches the AWS instance. By default, the security group restricts all inbound traffic, allows all outbound traffic, and allows other instances within the group to communicate. These rules can be completely customized; for example, it is possible to restrict access to a specific IP address on a specific port address and not allow that instance to communicate with other instances within the AWS account.http://aws.amazon.com/vpc/), encrypted file systems, and encrypted data volumes that may be used by those who have security needs beyond these basic best practices.All security keys should be replaced with new ones every 30–90 days. Installing regular software updates is essential to protect the operating system and third-party software from vulnerabilities. There are many additional security features such as private clouds  and also per gigabyte for persistent storage . It is ehttp://web.mit.edu/stardev/cluster/), Boto (http://code.google.com/p/boto/), Condor (http://www.cs.wisc.edu/condor/), and Hadoop (http://hadoop.apache.org/mapreduce/) are making cluster creation, termination, and job queuing more automated and accessible to bioinformatics specialists with perhaps only a limited understanding of systems administration and architecture. Within AWS, there is also the option to use Elastic Map Reduce (AWS's implementation of Hadoop) or high performance computing instances (http://aws.amazon.com/ec2/hpc-applications/) for an additional cost per instance. Note that an important consideration for a large cluster is to shut down instances when the number of CPUs is greater than the number of jobs. This will reduce the amount of money spent on idle CPU time, which can be substantial for hundreds or thousands of CPUs A large-scale computing environment that scales up or down in response to computational demand is the most commonly perceived use of cloud computing because it takes full advantage of rapid replication and linear scaling of cheap commodity compute cycles. However, it is important to remember that the cloud does not “magically” enable programs to run more efficiently or in parallel . Instead, it requires an understanding of how to connect multiple instances together to form a cluster and knowledge of how to divide a computational task into sub-components that can run simultaneously. Until recently, cluster creation was onerous, requiring substantial amounts of customized solutions handled best by an expert in systems administration and computer science. Fortunately, new advances in open source cluster management software such as StarCluster  and a parallel computing technique called MapReduce To put the previous concepts into practice, we will walk through the analysis of a large amount of NGS data. Specifically, we detail the creation of a pipeline to process an entire human genome's worth of NGS reads using a short read mapping algorithm. We use the ∼4 billion paired 35-base reads sequenced from a Yoruba African male http://www.gnu.org/software/wget/). Next, we format and mount the EBS volume and create three directories for testing the mapping data—small , medium , and all (entire genome). Then, we upload the African genome and the smaller testing files into the appropriate directories. Following the MAQ instructions (http://maq.sourceforge.net/maq-man.shtml) and executing the mapping and assembly commands, we learn that it takes 2 hours to analyze one pair of read files from the “small” directory on the EBS volume. However, only one of the possible eight CPUs on the extra-large instance is in use because we only issued one MAQ map command. While we could manually launch eight MAQ commands, a better approach would be to use cluster management software to automatically take advantage of all eight CPUs and include additional instances.The NGS mapping example begins by prototyping and testing the whole-genome mapping pipeline . At thishttp://gridscheduler.sourceforge.net/index.html) to manage batch queuing across distributed systems, along with OpenMPI to manage job distribution and instance communication. The cluster is composed of a master instance, which is responsible for managing a larger set of worker instances. In this example, each worker instance is able to process eight jobs concurrently and will contain the necessary software to run the analysis. In order to get StarCluster running, we need to work through a few steps that involve configuring the StarCluster instance type, setting the proper security, and installing StarCluster on your local computer to remotely create and terminate a cluster.Next, we will introduce the use of StarCluster to create and manage a small test cluster of two instances. StarCluster is customized for use on AWS and uses the open source version of Sun Grid Engine (http://docs.amazonwebservices.com/AWSEC2/2011-02-28/UserGuide/), we record the AMI ID—we will use this later in the StarCluster configuration file. We take a snapshot of the EBS volume to back it up in S3. We will use the snapshot ID later in the StarCluster configuration file to allow each instance access to the data.First, we will configure the StarCluster base instance type or Amazon Machine Image (AMI) with our required software. An AMI packages the operating system, installed programs, and user settings into a binary file that can be launched to exactly replicate an environment. Amazon creates a unique private ID (default) or public ID for each AMI to launch identical instances. We locate the StarCluster AMI through the AWS console under Community AMIs , launch it, and attach the previously created EBS volume containing the NGS data to the running instance. Then we install MAQ and any additional processing scripts as before. Next, we need to bundle the instance into an AMI in order to allow StarCluster to launch multiple identical instances. After bundling the AMI . The installation package includes the necessary scripts and configuration files to manage a cluster. The configuration file contains the various parameters to specify the cluster creation such as AMI ID, number of instances to launch, AWS account credentials, instance type, key pair, EBS snapshot ID containing the NGS data, and security group.Third, we install StarCluster on our local computer following their documentation . When the jobs are finished, we can save the results on the EBS volume and shut down the cluster.At this point in our case study, we are interested in testing the scalability of the NGS mapping pipeline by creating a small cluster and confirming that the environment is functioning as expected . Using SWe now expand our case study to the next level of usage, one that best exemplifies the most common conception of cloud computing: a virtually unlimited computational environment, which an analysis task will harness for rapid completion. However, getting to this point requires successful prototyping of an application, namely the prior two stages outlined above, on the cloud and ensuring that your application and pipeline can run on two or more inter-communicating instances.Returning to our case study, we want to create the environment to process the entire human genome . The prehttp://aws.amazon.com/importexport/). More information about cloud computing, detailed cost analysis, and security can be found in references In this overview to biomedical computing in the cloud, we discussed two primary ways to use the cloud (a single instance or cluster), provided a detailed example using NGS mapping, and highlighted the associated costs. While many users new to the cloud may assume that entry is as straightforward as uploading an application and selecting an instance type and storage options, we illustrated that there is substantial up-front effort required before an application can make full use of the cloud's vast resources. Our intention was to provide a set of best practices and to illustrate how those apply to a typical application pipeline for biomedical informatics, but also general enough for extrapolation to other types of computational problems. Our mapping example was intended to illustrate how to develop a scalable project and not to compare and contrast alignment algorithms for read mapping and genome assembly. Indeed, with a newer aligner such as Bowtie


BCAM promoter and repressed transcription. Thus, these data indicate that BCAM is a suppressive oncoprotein, and that FBI1/Akirin2 is involved in tumorigenicity and metastasis of hepatoma through the downregulation of suppressive oncogenes.Basal cell adhesion molecule (BCAM), known to be a splicing variant of Lutheran glycoprotein (LU), is an immunoglobulin superfamily membrane protein that acts as a laminin α5 receptor. The high affinity of BCAM/LU for laminin α5 is thought to contribute to the pathogenesis of sickle red blood cells and to various developmental processes. However, the function of BCAM in carcinogenesis is poorly understood. Based on microarray expression analysis, we found that BCAM was one of the target genes of the oncogenic 14-3-3β-FBI1/Akirin2 complex, which acts as a transcriptional repressor and suppresses MAPK phosphatase-1 gene expression. To elucidate the detailed function of BCAM in malignant tumors, we established BCAM-expressing hepatoma K2 cells. These cells lost the malignant characteristics of parental cells, such as anchorage-independent growth, migration, invasion, and tumorigenicity. Moreover, luciferase reporter assays and chromatin immunoprecipitation analysis revealed that the 14-3-3β-FBI1/Akirin2 complex bound to the  Furthermore, overexpression of 14-3-3β in NIH3T3 cells confers tumorigenicity in nude mice via activation of the mitogen-activated protein kinase (MAPK) cascade. Mutations in ras family oncogenes and suppressive oncogenes such as p53 and Rb are not detected in K2 cells 14-3-3 proteins regulate many cellular processes, including the cell cycle, metabolism, signal transduction, malignant transformation, and apoptosis. We previously reported that 14-3-3β is implicated in the positive regulation of cell cycle progression and tumorigenesis mitogen-activated protein kinase phosphatase 1 (MKP-1) transcription, resulting in the promotion of tumorigenicity and metastasis basal cell adhesion molecule (BCAM) gene as one of the genes downregulated by FBI1/Akirin2.To further analyze the oncogenic function of 14-3-3β, we screened for 14-3-3β binding partners by the yeast two-hybrid system using 14-3-3β as a bait BCAM, known to be a splicing variant of Lutheran glycoprotein (LU), is an immunoglobin superfamily membrane protein and acts as a laminin α5 receptor 1-induced rat hepatoma were cultivated with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air (2).K2 cells established from AFB32P-labelled probes as previously described NotI-BamHI fragment of full-length BCAM, and the 0.7-kb NotI-XhoI fragment of FBI1/Akirin2.Total RNAs were isolated from various cell lines by the acidic guanidine thiocyanate/phenol/chloroform method Total RNAs were prepared from various cell lines as described above. Reverse transcription (RT) reactions were performed using total RNAs and Moloney murine leukemia virus reverse transcriptase, according to the manufacturer's instructions (Invitrogen). The RT reaction mixtures were subjected to PCR amplification using specific primers as follows: mmp9-forward, 5′-TGG CTC TAG GCT ACA GCT TTG CTG C-3′, mmp9-reverse, 5′-CGA AGG AGT CAT CGA TCA CGT GTC G-3′; cyclinD1-forward, 5′-CTC CAT GTT CCA AAA CCA TTC C-3′, cyclinD1-reverse, 5′-GGG CAA CCT TCC CAA TAA ATA C-3′; FBI1-forward, 5′-TGG ATT TCG ACC CAC TGC TTA GC-3′, FBI1-reverse, 5′-GAT CAT CCC AAC CTG CCT TAG AG-3′; mkp1-forward, 5′-CCA TGG TGA TGG AGG TGG GCA TCC T-3′, mkp1-reverse, 5′-CCT TCA GCA GCT CGG AGA GGT TGT G-3′.BCAM expression vector was constructed by the insertion of a BCAM open reading fragment into the pcDNA3 expression vector. K2 cells were transfected with BCAM expression vector or empty vector using Lipofectamine regent (Invitrogen) according to the manufacturer's instructions. After 2 weeks of selection with 1 mg/ml G418, resistant clones were expanded and analyzed for the expression level of BCAM by western blotting.Western blotting was performed as described previously 4) were plated in 24-well plates containing DMEM supplemented with 5% FCS and cultivated for various times. The cell number was counted using a hemocytometer. For soft agar assays, cells (1×103) were suspended in 0.3% agar medium containing 5% FCS and layered on a 0.5% agar-coated 35-mm dish and cultivated for 2 weeks. The colonies formed were stained with 0.25% 1-p-iodophenyl-p-nitrophenyl-5-phenyltetrazolium chloride  for 12 h, and the number of colonies (>0.2 mm in diameter) was counted Cells . Cells were allowed to migrate for 12 and 24 h at 37°C, and the same fields were photographed again. Scratched areas were measured with ImageJ software , and recovered surface areas over 6, 12, and 24 h were calculated compared to control cells. Photographs of the wounded areas were taken at different times after wounding the monolayer.4 cells were plated on the top chamber with a Matrigel-coated membrane   in medium with 0.1% FCS, and medium supplemented with 10% FCS was used as a chemoattractant in the lower chamber. After 48 h, cells on the lower surface of the membrane were stained with crystal violet and counted.For the invasion assay, 6×104/200 µl of phosphate-buffered saline/flank) were inoculated subcutaneously into 6-week-old SCID mice . Tumor volume was calculated according to the formula V = a×b2×0.52, where a is the largest diameter and b the smallest diameter of the tumor. The average volumes of the tumors were represented by the mean tumor value ± SE (n = 5).Cells .5′-CGT CCT AAA ACT CAA CAA TAG CCA AAG-3′, and 3′-primer for -1942Luc, 5′-ATT CCC TGC AGT GGC GGC AG-3′. For each transfection, 1×104 cells/96-well plate were transfected with 30 ng of each pGL4.10 luciferase reporter vector, 3 ng of Renilla luciferase expression vector pGL4.74, and a total of 10 ng of pcDNA3 empty vector and pcDNA3-FBI1 in various combinations. Twenty-four hours after transfections, cells were lysed with the lysis buffer of the Dual-Luciferase Reporter Assay System (Promega), and luciferase activities were determined according to the manufacturer's instructions using an ARVO Light (Perkin Elmer). Reporter gene activities were normalized using Renilla luciferase activity as an internal control.Luciferase reporter gene plasmids driven by the rat BCAM promoter were constructed as follows. Rat BCAM promoters were amplified by PCR using K2 cell genomic DNA and introduced into pGL4.10 vector (Promega) and designated as -1942Luc. The plasmids -1281Luc, -705Luc, -208Luc, -61Luc, and -4Luc were generated by cloning the region of -1942Luc extending from −1281, −705, −208, −61, or −4 to +10. The primer sequences used were as follows: 5′-primer for -1942Luc,  GCC AGC AGG ACT GCG AGC AAC AG -3′).Cells were cross-linked with 1% formaldehyde for 10 min. The nuclear fraction was isolated and sonicated to shear genomic chromatin. Chromatin extracts were precleaned with protein G-Sepharose/salmon sperm DNA beads at 4°C for 3 h and then incubated with anti-FBI1 and anti-14-3-3β antibodies at 4°C overnight. Protein G-Sepharose/salmon sperm DNA beads were then added to the mixture for 3 h, and immunoprecipitated DNA-protein complexes were isolated from beads after several washing steps. Reversal of the cross-linking of chromatin was performed at 65°C for 6 h, and proteinase K digestion was allowed to proceed for 1 h at 55°C. DNA was extracted by the phenol/chloroform method. PCR was carried out on purified DNA using primers corresponding to -309 to +49 of the BCAM promoter region  degrade extracellular matrix, and the expression level of MMPs is correlated with the metastatic ability of cancer cells observed . Also, cobserved .3, whereas BCAMS1 and BCAMS2 cells formed smaller tumors with mean volumes of 2.41 and 0.87 cm3, respectively , casein kinase II (CKII), and PKA MKP-1 transcription via binding with the GC box in the promoter region BCAM gene expression, K2 cells were transfected with various amounts of Flag-FBI1 expression vector, and after 24 h expression levels of BCAM protein were analyzed. BCAM protein expression was dose-dependently downregulated by the ectopic expression of FBI1, up to a maximum of 0.2 µg of FBI1, at which point an approximately 50% reduction was observed Click here for additional data file.Figure S2Expression levels of FBI1/Akirin2 mRNA in various cell lines. Total RNAs were extracted from various cell lines, including rat hepatocarcinoma cells , rat glioblastoma cells (C6), embryonic carcinoma cells (P19), human hepatocarcinoma cells (HepG2), cervical carcinoma cells (HeLa), and normal prostate tissue analyzed by northern blotting using 32P-labeled BCAM cDNA as a probe.(TIF)Click here for additional data file.


The temperature dependence of ionic conductivity from 303 K to 353 K exhibits Arrhenius plot behaviour. The thermal stability of the polymer electrolyte system is studied by using thermogravimetric analysis (TGA) while the structural and morphological properties of the polymer electrolyte is studied by using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction analysis (XRD), respectively.Gel polymer electrolytes (GPEs) are developed using poly(1-vinylpyrrolidone-co-vinyl acetate) [P(VP-co-VAc)] as the host polymer, lithium bis(trifluoromethane) sulfonimide [LiTFSI] as the lithium salt and ionic liquid, and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide [EMImTFSI] by using solution casting technique. The effect of ionic liquid on ionic conductivity is studied and the optimum ionic conductivity at room temperature is found to be 2.14 × 10 Polymer electrolytes are gaining great interest due to their high ionic conductivity and wide electrochemical windows. They also possessed interesting properties such as thin-film forming ability, flexibility, and transparency –3. There3SO3−) and BF4−. The obtained films are freestanding and flexible with ionic conductivities ranging from 1.1 to 5.8 mS cm−1 at room temperature /lithium bis(trifluoromethane) sulfonimide [LiTFSI] polymer electrolytes system is investigated using ac impedance spectroscopy at different temperatures. In addition, the thermal stability, structural and morphological properties of the polymer electrolyte system is analysed using thermogravimetric analysis (TGA), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction analysis (XRD), respectively.4 g mol−1 (Sigma-Aldrich), lithium salt LiTFSI with 97% purity (Sigma-Aldrich), and ionic liquid EMImTFSI with 98% purity (Basionics) which were used without further purification in this study. The gel polymer electrolytes were prepared by using solution casting technique using distilled water as a solvent.The materials used in this research work are P(VP-co-VAc) with a molecular weight of ~5.0 × 10The casted thin films were then characterized on its electrical, thermal, structural, and morphological properties.Electrical Properties: Ac Impedance Spectroscopy. HIOKI 3532-50 LCR HiTESTER was used to measure the impedance of the samples. The thickness of the samples was measured using a micrometer screw gauge. The impedance measurement for each sample was done in the frequency range from 50 Hz to 5 MHz at room temperature to 80°C. The samples were placed on the sample holder in between two stainless steel electrodes with diameter of 4.9087 cm2. Ionic conductivity, σ, was determined from the following equation:L is the sample thickness (cm), A is the cross-sectional area of electrode and sample contact (cm2), and Rb is the bulk resistance (Ω) of the sample.Thermal Properties: Thermogravimetric Analysis (TGA). TA TGA Q500 was used to study the thermal stability of the samples. The samples were cut and weighed ~2.0 mg. Then, the samples were put into the sample holder and heated under nitrogen atmosphere from 25°C to 505°C at a heating rate of 10°C min−1.Structural Properties: Fourier Transform Infrared (FTIR) Spectroscopy. The FTIR studies were carried out by using Thermo Scientific Nicolet iS10 spectrophotometer at room temperature in the wave region between 4000 cm−1 and 600 cm−1 with a resolution of 4 cm−1. Morphological Properties: X-Ray Diffraction Analysis (XRD). The XRD study was conducted by using D5000 diffractometer to determine the polymer electrolytes films which are either crystalline or amorphous in nature based on the characteristic pattern obtained from X-ray diffractogram. Coherent length study is done to justify that the crystallinity of P(VP-co-PVAc)—LiTFSI—EMImTFSI system is reduced. The coherent length, C, is calculated from the Scherrer equation:λ is the X-ray wavelength; θb is the glancing angle; and Δ2θb is the full width at half maximum (FWHM).−10 S cm−1. The maximum ionic conductivity 2.14 × 10−6 S cm−1 is achieved with addition of 25 wt% of EMImTFSI to the polymer electrolyte system.σ) against 1000/T (in Kelvin) are shown in Ea, for each system can be determined from the slope of its Arrhenius plot. Ea for the polymer electrolytes.Plots of linear variation of log ionic conductivity . The addition of LiTFSI into the polymer system shows that the diffraction peaks corresponding to the salt are disappearing, suggesting that LiTFSI is fully complexed with P(VP-co-VAc).The diffractograms of P(VP-co-VAc)-LiTFSI with different wt% of EMImTFSI are shown in Figures + and TFSI− ions in the ionic liquid are mobile, thus increasing the number of charge carriers available for conduction. Besides, with the low viscosity of EMImTFSI, the crystallinity of the polymer matrix can be reduced, hence increasing the mobility of the charge carriers [Incorporation of ionic liquid, EMImTFSI, to the polymer electrolyte gives plasticizing effect which increases the flexibility of the polymer backbone and enhances the amorphous phase of the system. In addition, both Imcarriers . The inccarriers .The increase in ionic conductivity as a function of temperature is due to the decrease in viscosity of the polymer systems that increases the chain flexibility . This phσo is the preexponential factor; Ea is the activation energy; k is the Boltzmann constant; and T is the absolute temperature.From Ea, can be defined as the energy needed to overcome the reorganization and reformation of Li+ to relocate to neighbouring sites. From + to move faster inside the polymer network. Upon addition of EMImTFSI, the interaction between the polymer system and lithium ions is disturbed causing less energy which is needed for the Li+ hopping process [Activation energy,  process .There are three degradation stages which can be observed from Figures For the second degradation stage, it is shown that the pure polymer is starting to degrade at 295°C with 24% weight loss. In EMIm 0, the second degradation stage occurred at 275°C with 30% weight loss while in EMIm 10 and EMIm 25 it occurred at temperature 265°C with weight loss of 30% and 275°C with 25% weight loss, respectively. This stage corresponds to the degradation of poly(vinyl acetate) (PVAc) due to the total loss of acetic acid as shown in 3 [ cis-trans isomerisation, aromatization, and cross-linking [The third stage of degradation happened due to the degradation of vinylpyrrolidone  and viny3 . The pol-linking . These r-linking . For theAs EMImTFSI is nonflammable and nonvolatile, these properties contribute to the increase in heat resistivity of the samples resulting in EMIm 25 to have less weight loss compared to pure P(VP-co-VAc) with residual mass of 12.44% and 9.66%, respectively. Therefore, it can be concluded that samples with addition of EMImTFSI are more thermally stable than pure P(VP-co-VAc).−1 and 2954 cm−1 which is assigned to O–H stretching and C–H stretching of P(VP-co-VAc), respectively, is observed to be shifted to 3405 cm−1 and 2958 cm−1, respectively, in EMIm 0. It is also observed that the peaks of C=O stretching of both PVAc and PVP at 1731 cm−1 and 1655 cm−1 have shifted to 1732 cm−1 and 1651 cm−1, respectively, in the PE system. These shifting might be due to the interaction between free Li+ with the ester C=O in PVAc and amide C=O in PVP. Besides, the peak shift of C=O in PVP (Δf = 4 cm−1) is more intensive as compared with PVAc (Δf = 1 cm−1); therefore it is claimed that interaction of Li+ with C=O of PVP is more favourable [vourable .−1 ), 781 cm−1  + vibrational (S–N)), 1193 cm−1 s), and 1356 cm−1 a) are shifted to 739 cm−1, 788 cm−1, 1182 cm−1, and 1350 cm−1 in the EMIm 0, respectively. These shifting can be attributed by the presence of free TFSI− in the EMIm 0. Two characteristic peaks of LiTFSI δCH3 at 2979 cm−1 and 2876 cm−1 have disappeared after interacting with P(VP-co-VAc) and C–H stretching peaks of P(VP-co-VAc) at 2954 cm−1 shifted to 2958 cm−1 in EMIm 0. Besides, the characteristic peak at 1423 cm−1 in P(VP-co-VAc) has shifted to 1441 cm−1 in EMIm 0. Characteristic peak at 1021 cm−1 in P(VP-co-VAc) has also shifted to wavenumber 1022 cm−1 and becomes the shoulder peak to LiTFSI characteristic peak that shifted from wavenumber 1065 cm−1 in LiTFSI to 1055 cm−1 in EMIm 0.From the figure, we can also observe the shifting in the LiTFSI characteristic peaks. The peaks of LiTFSI at 740 cm−1 in the EMIm 0 is observed to be shifted to 3390 cm−1 and 3403 cm−1 in EMIm 10 and EMIm 25 systems, respectively. The C=O stretching of PVAc and PVP in the EMIm 0 system is also shifted from 1732 cm−1 and 1651 cm−1, respectively, to 1731 cm−1 and 1652 cm−1 in EMIm 10 and to 1731 cm−1 and 1654 cm−1 in EMIm 25.−1 which shifted to a shoulder peak in EMIm 10 and disappears in EMIm 25.Upon adding EMImTFSI into the polymer electrolyte system, there are no significant changes occurring to LiTFSI characteristic peaks except for the peak of wavenumber 1022 cm−1 and 1573 cm−1 have disappeared after being added to EMIm 0 system as shown in Figures −1 and 1168 cm−1 in −1 in −1 in It can also be observed from As shown in Based on Figures In general, coherent length defines the crystallite size. As the diffraction peak width is longer, the crystallite size is shorter and thus brings higher amorphous phase and lower crystallinity of the polymer system . Based o−6 S cm−1, which suggested that addition of ionic liquid in the polymer electrolytes systems increases the ionic conductivity values. The temperature dependence ionic conductivity study shows that all samples exhibit Arrhenius type which is favourable for Li+ ion hopping mechanism at higher temperature as proved by the increasing in ionic conductivity values with temperature. In addition, the addition of EMImTFSI also enhanced the thermal stability of the polymer electrolytes systems. The FTIR and XRD studies showed the complexation of the materials inside the polymer electrolytes systems which revealed that with the incorporation of EMImTFSI the amorphous fraction is increased, resulting in the increasing of the ionic conductivity values.In this work, GPEs are developed by incorporating different amount of EMImTFSI into the polymer electrolytes system. Among all the polymer electrolytes systems, EMIm 25 has the highest ionic conductivity, 2.14 × 10


Multiple independent genomic profiling efforts have recently identified clinically and molecularly distinct subgroups of ependymoma arising from all three anatomic compartments of the central nervous system . These advances motivated a consensus meeting to discuss: (1) the utility of current histologic grading criteria, (2) the integration of molecular-based stratification schemes in future clinical trials for patients with ependymoma and (3) current therapy in the context of molecular subgroups. Discussion at the meeting generated a series of consensus statements and recommendations from the attendees, which comment on the prognostic evaluation and treatment decisions of patients with intracranial ependymoma (WHO Grade II/III) based on the knowledge of its molecular subgroups. The major consensus among attendees was reached that treatment decisions for ependymoma  should not be based on grading (II vs III). Supratentorial and posterior fossa ependymomas are distinct diseases, although the impact on therapy is still evolving. Molecular subgrouping should be part of all clinical trials henceforth. Ependymoma is a histologically defined intrinsic tumor that involves the three major anatomic compartments  of the central nervous system and affects both children and adults. The current standard of care therapy for patients with intracranial ependymoma remains surgical resection combined with radiotherapy. The survival benefit of chemotherapy for ependymoma and the prognostic ability of histopathological grading criteria to risk-stratify patients are still both inconclusive and contentious. No molecular or tumor-specific immunohistochemical markers are in routine current clinical use for ependymoma. Recent advances in the biological characterization of ependymal tumors have demonstrated the existence of nine clinically, demographically, and molecularly distinct entities, with three occurring in each anatomic compartment. These findings offer new opportunities to create a precise, reliable, and objective platform for stratification of ependymoma patients, and the potential for altering therapeutic decisions based on molecular features. Herein, we discuss the current consensus on the molecular subgroups of intracranial ependymoma (WHO Grade II/III) in children and adults, as well as recommendations for integration into future clinical trial designs. These discussions and recommendations were made by a collection of neuro-oncologists, neurosurgeons, neuro-pathologists, radiation oncologists, and basic scientists, meeting at the global ependymoma consensus conference   would dramatically benefit from a matched control to correct for aberrations inherent to the germline. As such, an agreement among most attendees was established that submission of blood samples should also be mandatory for enrollment in a clinical trial. It should be recognized that arguments were made against the mandate of fresh-frozen tissue, owing to the logistical issues of collection, storage, and submission, particularly in small community centers. Additionally, there were ethical concerns regarding the mandated submission of blood. Attendees recognized that efforts would need to be established to create standard operating procedures in smaller centers to enable reliable collection and submission of frozen tissue. Many of those agreeing on a mandate of frozen tissue and blood argued that given the rapid developments in the field of molecular genetics, with the emergence of increasingly powerful analytical devices and computational tools, the time is now to collect tissue specimens in combination with high-quality clinical data. This would enable the use of such advances to improve the care of future ependymoma patients.Clinical management of intracranial ependymomas (WHO Grade II/III) is challenging and the optimal treatment strategy is contentious. Intracranial ependymoma, particularly before administration of any therapy, demonstrates predominantly locally invasive growth patterns and has only very low metastatic potential. Surgery plays a primary role for local tumor control and the extent of neurosurgical resection has been the most consistent independent prognostic factor reported in the last decades , 6, 34. In addition to surgery, post-operative field radiotherapy dosed at 54–59.4 Gy is considered the standard of care for patients with non-disseminated ependymoma to lower the risk of local recurrence . Radiatin = 820 cases) found that patients with either PF-EPN-A or PF-EPN-B tumors benefit from gross total resection, with the survival rates being particularly poor for sub-totally resected PF-EPN-A, even in the setting of radiation therapy [It should be emphasized that all prior studies that evaluated the therapeutic value of neurosurgical interventions and external beam radiation in posterior fossa ependymoma have not accounted for molecular subgroup affiliation and might therefore be confounded by clinical differences in response to therapy between these subgroups. Data from a current retrospective study on four independent non-overlapping cohorts of posterior fossa ependymomas  and were associated with a poor prognosis with 5-year progression-free and overall survival of 29 and 75%, respectively. Interestingly, the level of resection did not significantly affect the outcome within the ST-EPN-RELA-positive subgroup in this retrospective analysis in patient samples collected over a long period of time (>20 years). The two remaining supratentorial subgroups, ST-SE and ST-EPN-YAP1, were restricted only to adults  and predominantly to children , respectively, with both of these variants showing an excellent prognosis. As the cited studies and other available collections of single cases markedly differ regarding age distribution, therapy modalities and availability of molecular data, variations in outcome cannot be reliably linked to specific treatment approaches or molecular subgroups. It was, therefore, concluded that there was not enough evidence yet to recommend distinct treatment approaches for ST-EPN-RELA ependymoma. Molecular analyses of supratentorial ependymomas from clinically well-annotated international trial cohorts as well as from large retrospective cohorts with long-term follow-up have now been initiated. The authors expect that this approach will help to clarify questions about the clinical outcome of the molecular variants of supratentorial ependymoma and result in explicit therapy recommendations.Observation for gross totally resected supratentorial ependymomas has also been advocated based on retrospective series that were not molecularly characterized. For example, a retrospective, multicenter study comprising 92 patients  with gross totally resected and non-anaplastic supratentorial ependymal tumors did not find evidence of decreased progression-free or overall survival with the omission of external beam radiation . The 5–1In contrast to surgery and radiotherapy, the role of chemotherapy in the management of ependymoma remains unproven despite extensive investigation. Cohorts of pediatric or adult patients in which the role of chemotherapy was retrospectively analyzed either failed to demonstrate a survival advantage or showed substantial variation between individual patients , 13, 28.EPHB2)-driven ST ependymoma models—also highly expressed in ST-EPN-RELA tumors—have pinpointed 5-fluorouracil treatment as a potential cytotoxic therapy with efficacy in murine models and is currently being evaluated in early phase ependymoma clinical trials [Because of the recognition that ependymal tumors comprise molecularly distinct subtypes, with potentially distinct clinical management, the generation of subgroup-specific pre-clinical models for the development and assessment of novel therapies is required. The identification of candidate cells of origin for ependymoma has permitted the generation of novel mouse models that can be leveraged for novel therapeutic discovery and evaluation , 27, 30.l trials , 16, 38.l trials , 39. In We now recognize that ependymal tumors from different compartments of the central nervous system are biologically distinct and there are phenotypically divergent subgroups within each anatomic compartment. Future clinical trials, the development of pre-clinical model systems, and the identification and testing of subtype-specific therapeutics must accompany molecular classification to be useful to ependymoma patients and to the neuro-oncology community. The differentiation between histologically defined grade II versus grade III/anaplastic ependymomas is problematic and of limited utility for clinical decision-making, and therefore should be used with great caution outside the setting of a clinical trial. For patients with PF-EPN-A ependymoma over the age of 12 months of age, the recommended standard of care is maximal safe micro-neurosurgical removal followed by local radiotherapy, but probably does not include the routine use of chemotherapy outside the setting of a clinical trial. A subset of PF-EPN-B ependymoma patients who undergo gross total micro-neurosurgical resection are likely cured in the absence of radiotherapy, and a clinical trial to test the possibility to avoid radiotherapy in the context of complete resection for PF-EPN-B patients is indicated. The characteristics and heterogeneity between molecular subgroups of supratentorial ependymoma require additional study before specific treatment recommendations can be made. The division of an already uncommon entity (“ependymoma”) into nine new entities will necessitate great co-operation and international collaboration with the pediatric and adult neuro-oncology community if clinical trials are to be properly and expeditiously completed.


The ability to preoperatively predict postoperative complication risks is valuable for individual counseling and (post)operative planning, e.g. to select low-risk patients eligible for short stay surgery or those with higher risks requiring special attention. These risks however, are not well established in pituitary surgery.We conducted a systematic review of associations between preoperative characteristics and postoperative complications of endoscopic transsphenoidal surgery according to the PRISMA guidelines. Risk of bias was assessed through the QUIPS tool.In total 23 articles were included, containing 5491 patients (96% pituitary adenoma). There was a wide variety regarding the nature and number of risk factors, definitions, measurement and statistics employed, and overall quality of mainly retrospective studies was low. Consistent significant associations were older age for complications in general, and intraventricular extension for cerebrospinal fluid (CSF) leaks. Associations identified in some but not all studies were younger age, increased BMI, female gender, and learning curve for CSF leaks; increased tumor size for complications in general; and Rathke’s cleft cysts for diabetes insipidus. Mortality (incidence rate 1%) was not addressed as a risk factor.Based on current literature, of low to medium quality, it is not possible to comprehensively quantify risk factors for complications. Nevertheless, older age and intraventricular extension were associated with increased postoperative complications. Future research should aim at prospective data collection, reporting of outcomes, and uniformity of definitions. Only then a proper risk analysis can be performed for endoscopic pituitary surgery.The online version of this article (doi:10.1007/s11102-017-0839-1) contains supplementary material, which is available to authorized users. Over the past two and a half decades, pituitary surgery has undergone major technical developments, the introduction of the endoscope perhaps being the most important one. Several systematic reviews show relatively better results in terms of gross total resection, with reduced complication rates for endoscopic surgery compared to microscopic surgery –10. ThesA systematic review was conducted according to a predefined protocol, which was based on the PRISMA criteria for systematic reviews  and regiA literature search was conducted on May 15 2017, with the guidance of a trained clinical librarian (J.S.). The following databases were searched: PubMed, Embase, Web of Science, Cochrane, CINAHL, Academic Search Premier and ScienceDirect. Terms included were ‘pituitary adenoma’, ‘non-functioning adenoma’, ‘acromegaly’, ‘Cushing’s disease’, ‘prolactinoma’, ‘Craniopharyngioma’, ‘Rathke’s cleft cyst’, ‘complications’, ‘risk factors’ and ‘prognosis’, and derivatives or synonyms of these words. The complete search strategy can be found in online supplement 1. Reference checking of included studies was performed to screen for additional studies.Inclusion criteria were: (1) articles reporting on outcomes of ETS for pituitary tumors; (2) describing an association between ≥1 preoperative characteristics and ≥1 postoperative complications; (3) published in English; (4) peer-reviewed; (5) containing original clinical data; and (6) including >10 adult patients (>18 years). Excluded studies were: (a) microscopic, endoscopic-assisted surgery, or combined microscopic and endoscopic approaches without a separate description of endoscopic results, (b) articles without a described association, and (c) articles including >10% other pathologies than pituitary adenoma.A meta-analysis appeared to be infeasible because of heterogeneity in (the definition of) risk factors and outcomes. In addition, the number of studies assessing the same association for a complication was too small. This review focuses on complications that directly intricate the postoperative course. Perioperative CSF leaks can be managed adequately during surgery and were therefore not included. Other reviews have addressed specific complications occurring during surgery; e.g. internal carotid artery (ICA) injuries  or laterThe selection consisted of two phases: (1) title and abstract screening for potentially eligible articles, and (2) full text screening of these articles. During both phases, the same inclusion and exclusion criteria were used. During phase 1, in case of doubt, the full text paper was retrieved. Since a variety of risk factors can be investigated within the same cohort, a decision was made not to omit overlapping cohorts.Extracted study characteristics included: institution, study period, study design, number of patients, number of procedures, percentage females, tumor type, approach, length of stay, and duration of follow-up. Preoperative factors were categorized into groups  and all potential associations were categorized into complications in general, neurosurgical and endocrine complications. Risk factors were considered consistent when they were reported as significant in ≥2 independent studies. Inconsistent when ≥2 positive or negative and ≥1 neutral (non-significant) associations were reported and conflicting when ≥1 positive and ≥1 negative associations were reported.Assessment of risk of bias was done by means of the Quality in Prognostic Studies (QUIPS) tool . The QUIThe search resulted in 2596 unique titles and abstracts. The screening of titles and abstracts resulted in the selection of 472 full-text articles retrieved for the second phase of the selection process. Finally, 23 articles were included in the present systematic review Fig. .The study characteristics are summarized in Table The results of the scoring of the methodological quality of the studies are shown in Table Overall, the methodological quality was low: only one study had a low risk of bias (4.3%), two a moderate risk (8.7%) and the remaining twenty studies had a high risk of bias (87.0%). A high risk of bias was found twelve times for study confounding .The incidence rates of complications described in the included studies are described in Table Eight studies investigated the potential risk factors for complications in general , 31, 35.Age was assessed in two studies. Both studies found an increased risk for older age . Age was defined as a categorical parameter: (1) age ≥70 versus <60 years   and (2) 3 versus <10 cm3 OR 6.3 (1.6–25.0) [Tumor size and volume were significantly associated with increased complications in general in two out of three studies. Definitions used were (1) macroadenoma versus microadenoma OR 3.98  , (2) tum.6–25.0) , (3) tum.6–25.0) , and (4).6–25.0) .Four out of six investigated risk factors showed increased risks for complications in general. Tumor extension was defined in five different ways: (1) intraventricular extension, (2) Knosp grade, (3) supra-/parasellar extension, (4) extension into the anterior cranial fossa (ACF), and (5) cavernous sinus invasion. Intraventricular extension OR 7.85 (2.88–21.43) , supra-/Tumor type was investigated in one study; however, no significant effect was detected .Previous radiation was associated with an increased risk in one study  . The surFourteen studies investigated the potential risk factors for postoperative CSF leaks , 36, 37.Younger age was inconsistently associated with a higher risk of CSF leaks. Three studies, including two with overlapping cohorts , 34, fouIn two of five studies, female gender was associated , 33, 34 As expected, three out of four studies found a significant increase in postoperative CSF leaks in patients with a higher BMI , 25, 34.One study evaluated various comorbidities in relation to CSF leaks Table  and founOnly one out of five studies looking at tumor size or volume found a significant association with CSF leaks , 33, 36.Tumor extension was analyzed in seven studies , 36, 37,Four studies looked at the relationship between various forms of pathology and CSF leaks , 25, 34.Previous surgery was not reported as a risk factor for CSF leaks , 33, 34.Even though the frequency of surgical resection after radiotherapy is low, one study found an increased risk for patients with prior radiotherapy; 4/14 patients had a postoperative CSF leak . The othOne out of four studies considering the surgeon’s learning curve found a significant increase in postoperative CSF leaks , 23, 32.Two out of three studies reporting associations found a significant association for intracranial infections , 27, 38.Only two studies looked at risk factors for bleedings: (1) ICA injury , and (2)Eight studies looked at risk factors for DI , 32, 33;Age (2 studies) , 33, gen3 (no significant effect) [One study reported an association with permanent or overall DI , 29, 33. effect) , 33, and effect) .Knosp grade was not associated with a significant increase of DI .In overlapping cohorts, RCC was significantly associated with an increased risk of DI compared to other tumor types: (1) 47.6 versus 20.2% (p < 0.05) , and (2)Previous surgery was defined as (1) prior non-endoscopic surgery , (2) priLearning curve was assessed in three studies, but not associated with an increased risk of DI , 23, 32.One out of three studies looking at adrenal insufficiency addressed potential associations . IncidenThree complications were only analyzed once: cranial nerve injury, vision loss and sinusitis. One study found a significant relationship between patients with a history of an extrasellar tumor and cranial nerve injury  . One stuThis systematic review on preoperative risk factors for postoperative complications in ETS identified only two consistent risk factors: older age for complications in general and intraventricular extension for CSF leakage. Clear and uniform definitions of postoperative complications were mostly missing and almost all studies were retrospective. This resulted in a lack of standard reporting of complications, causing a large variation between studies regarding reported risk factors and incidence rates of complications.The most frequently studied complication, CSF leaks, was consistently associated with intraventricular extension. Other risk factors were not consistent, but did not report conflicting results. At this stage, we conclude that intraventricular extension increases the risk of CSF leaks and lower age, female gender, and high BMI potentially increase the risk Table . The secA distinction can be made between amendable and non-amendable risk factors. Even though often difficult to change, these should be taken into consideration in cases with increased risks. The learning curve is perhaps one of the easiest to amend. Experience (learning curve), for instance, appears to be an important risk factor for a lower risk of CSF leaks. This was confirmed in some but not all studies, while learning curve was not associated with any other complication. Obviously, learning curve statistics can be biased since a more experienced surgeon will operate on a more complex case mix with an innate higher risk of CSF leaks, which cannot be extracted from the currently available series. In a national survey, Ciric found that for most complications, surgical learning curve is an important factor . This waIncreased BMI and younger age were risk factors for postoperative CSF leaks. This might be explained by the increased intra-abdominal pressure . PerhapsSince generally only large tumors have suprasellar, intraventricular extension or extension into the ACF, these risk factors can be considered correlated and classified under tumor size. When taking this into account, large or giant pituitary tumors are associated with complications in general and postoperative CSF leaks. Literature on endoscopic resection of giant adenoma is scarce, however increased risk of complications can be found . In partPathology, also a non-amendable risk factor, might also be an important risk factor for postoperative complications. In particular, several associations were described for RCCs and craniopharyngiomas; however, only those for DI have been reported more than once (in overlapping cohorts). These two tumors have an increased risk of DI, possibly also for postoperative CSF leaks. The relationship with pathology can likely be explained by the tumor etiology. Whereas RCCs are typically located between the anterior and posterior lobe, compression/manipulation of the posterior lobe is likely to occur prior to or during surgery. Craniopharyngiomas commonly arise in the pituitary stalk, which is vulnerable to surgical manipulation; therefore, surgery is more likely to cause DI. Another risk factor found for RCCs was postoperative CSF leaks.Despite being addressed only in one study, previous radiation showed an association between complications in general and carotid artery injury. Even though radiotherapy prior to surgery is not common, some adenomas are very therapy resistant and need additional surgery. Boling et al. presented data from nine patients who had received radiotherapy prior to surgery, showing a complication rate of 33% . This miOne of the most reviewed subjects in pituitary surgery literature is the comparison between endoscopic and microscopic surgery. We found several reviews assessing the topic. Because the influence of surgical technique was the primary interest of comparison, patient-related risk factors were not investigated in these reviews. Typically, gross-total resection and complications have been compared between microscopy and endoscopy, most showing equal or superior results in favor of endoscopy; however, patient-related risk factors have not been further determined –10. In tThe main purpose of the study was to improve preoperative patient counseling and to identify high- and low-risk patients. The low quality of the studies precludes firm conclusions based on this review. Overall, most studies were retrospective and too small to allow multivariable analyses. Also, no meta-analysis could be performed because of the heterogeneity and low number of associations. Complications were often defined differently and mostly gave limited descriptions. This complicates generalizability, and future researchers should aim at clearly defining (presented) complications in an effort to improve the clinical impact of future research on daily practice. Furthermore, reporting of outcomes, not only by centers of excellence, and prospective registration will lead to further evolvement in the field. Many examples, like the Value Based Healthcare concept , have shWe realize that only a subset of the total number of studies reporting on complication rates in ETS could be included in this study since the vast majority did not perform risk factor analysis. Furthermore, studies that presented only pooled data between microscopic and endoscopic surgery did not give a utilizable overview of potential risk factors for complications specific for patients treated through an endoscopic transsphenoidal approach, as in many daily practices nowadays.Although many studies assessed individual risk factors of different types of postoperative complications, there were no prognostic models found in the current literature. Prognostic models in other fields have shown added value in individualized decision-making and patient counseling. Such a model could have different types of outcomes, based on the aim of the model: complications in general, prediction of potential candidates for short stay. Before implementation of such a model, it should be thoroughly internally and externally validated.Although on average the reported mortality rate is around 0.6%, unfortunately no associations were found in the current literature. Even though incidence rates are low, they are not negligible. Suggested improvements for definitions and registration of complications might give us a better understanding of the etiology of these complications.We present an overview of preoperative risk factors for postoperative complications. Only two risk factors were consistently associated with increased risks: older age for complications in general and intraventricular extension for CSF leakage. This does not mean that there are no other important risk factors, and further emphasizes the need for uniform definitions, reporting of outcomes and prospective registration. The low methodological quality of included studies, inconsistent results, and lack of uniform definitions make firm conclusions difficult. Nevertheless, we believe that awareness of presented risks may benefit patient counseling and surgical case selection.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 15 KB)Supplementary material 2 (DOCX 19 KB)Supplementary material 3 (DOCX 33 KB)


Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3+ cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes.LEN, at the concentration range reached In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity. The development of these drugs represented a paradigm shift in the treatment of multiple myeloma (MM) [The Immunomodulatory drugs (IMiDsoma (MM) , 2. LEN-oma (MM) –5. Moreooma (MM) .® have been recently elucidated highlighting the role of Cereblon (CRBN) and its target factors [Several mechanisms of action have been described, , 8 inclu factors , 11. LEN factors . Through factors . Recentl factors  and in M factors .Currently, few data are reported on the possible effects of LEN on dendritic cells (DCs) populations. , 16–18 IDifferent studies reported an increased incidence of acute Graft versus host disease (aGvHD), with a possible enhancement of the graft versus MM effect, in patients treated with LEN after allo-transplantation –23. SincBased on these evidences, in this study we investigated whether LEN may affect maturation, phenotype and functionality of DCs as antigen presenting cells (APCs), either directly or through the modulation of human mesenchymal stromal cell (hMSC) effect on DCs.in vivo in MM patients, [vs LEN 0.1 μM, 45.82 ± 4.55 vs 59.45 ± 8.21, p = 0.029; DMSO vs LEN 1 μM, 45.82 ± 4.55 vs 73.52 ± 7.71, p = 0.001), CD86  and CD209    differentiated from BM aspirates and PB of MM patients. Despite a reduction of both number and % of mature DCs, LEN, at the concentration range reached atients,  signific) Figure , comparevs LEN 0.1 μM, 147.49 ± 45.08 vs 200.44 ± 44.22, p = 0.002; DMSO vs LEN 1 μM, 147.49 ± 45.08 vs 249.61 ± 42.10, p = 0.016) and CD209   expression was found in DCs differentiated from PB CD14+ cells  (Friedman test)  and the percentage of DCs obtained in vitro    at 10vs LEN 0.1 μM vs LEN 1 μM: 1076 vs 1755 vs 2193 pg/ml, p < 0.05), CC chemokine ligand (CCL)2 , CCL5  and TNF-α  and slightly decreased the production of IL-6  , by mo-DCs differentiated from MM patients, compared to vehicle  assay in the presence of LEN or vehicle.+ cell proliferation was significantly higher in co-culture with LEN-treated DCs, compared to DMSO-treated DCs      Figure . However) Figure .+ cells of MM patients were differentiated into DCs in the presence of LEN or DMSO treated human telomerase reverse transcriptase transduced hMSC (hTERT-hMSC) conditioned medium (CM).To investigate a possible MSC-mediated indirect effect of LEN on DCs, BM CD14in vitro system  and CD86    Figure .PTGS2) gene expression levels at all tested concentrations  , IL6, CCL5 and transforming growth factor beta 1 (TGFB1).Thereafter, we examined whether LEN treatment affected the expression of immunosuppressive factors in hTERT-hMSCs, by Real-time PCR (RT-PCR). Interestingly, we found that LEN significantly down-regulated prostaglandine 2  Figure  but not To investigate the molecular mechanism involved in the effect of LEN on MSCs, firstly we checked the expression profile of Cereblon and its target proteins in hTERT-hMSCs showing that they expressed Cereblon Figure  but not in vitro DC differentiation from BM CD14+ cells of 2 MM patients. Interestingly, we found that the effect of hTERT-hMSCs on DC maturation markers were reverted by the down-regulation of CK1-α   .in vivo LEN treatment on DC maturation markers, we compared the expression profile of DCs differentiated from PB CD14+ cells of 9 MM relapsed patients, purified at the baseline (DAY 0) and after 7 days of LEN treatment, just before the start of the weekly treatment with Dex. All the patients were responsive to LEN treatment.Finally, to evaluate the effect of in vivo LEN treatment significantly increased the expression of HLA-DR  and CD209 , and CD86 without reaching statistical significance   . Flow-cytometry histograms from one representative MM patient were reported in Interestingly, we found that DCs of MM patients are known to be functionally defective, with a decreased expression of maturation markers and antigen presentation ability . The proin vivo in MM patients treated with this drug 5–25 mg daily, [−8 M, consistent with several studies that reported Dex inhibitory effects on DC maturation and functions [vs LEN alone [Studies performed on murine models, showed that both LEN and POM treatment increased the expression of DC maturation markers, enhanced DC endocytotic activity, increased the production of TNF-α and CCL2, and the DC-dependent T-cell expansion , 17. Howg daily, , 34 signg daily, –37 both unctions , 39. Howunctions  on high-EN alone . ConversEN alone , 42.in vitro DC differentiation was associated with an increased DC functional activity to stimulate T cell proliferation that was abrogated by the combination with Dex.Consistent with the effect of LEN on DC maturation markers, we found that LEN treatment increased DC production of IL-8, CCL2, CCL5, and TNF-α, in line with data observed on murine models , 17. TheSubsequently, we investigated the molecular mechanism beyond LEN effects on DCs. It is widely demonstrated that LEN exerts its anti-MM activity through the modulation of Ikaros and Aiolos , 13. MorPTGS2, known to inhibit the transitional processes of differentiation of monocytes into DCs [Along with a direct effect of LEN on DC maturation, our data suggested a potential indirect effect, through the modulation of MSC immunomodulatory properties such as the production of cytokines and chemokines. –26, 45 Winto DCs , 47. Theinto DCs , 48 receinto DCs , 48. Intinto DCs .in vitro evidences were expanded and confirmed by ex vivo DC cultures in relapsed MM patients treated with LEN 25 mg/day, as mono-therapy for one week, just before the start of the weekly treatment with Dex. After 7 days of treatment we found an increased PB DC differentiation. Of note, all analyzed patients were responsive to LEN treatment. This early effect was in agreement with recent data reporting the in vivo increase of T and NK cells, with a rapid decline of Ikaros, after 7 days of POM treatment without Dex in MM patients [Lastly, our patients .in vitro and in ex vivo cultures, enhancing DC ability to stimulate T cell proliferation and to release chemokines involved in the immune response. LEN treatment also reduces the immunosuppressive properties of hMSCs, suggesting new possible effects of IMiDs® on the allo-reactivity against MM cells.In conclusion, our data indicate that LEN increases the expression of mature DC markers both BM and/or PB were obtained from 30 consecutive patients with active MM , including both newly diagnosed and relapsed MM, admitted to our hematological Unit. Patient samples were obtained after informed consent, according to the Declaration of Helsinki. The study was approved by the Institutional Ethical Review Board of our Hospital.Moreover, PB were obtained from 9 patients with relapsed MM  I: 4, International Staging System (ISS) II: 3, International Staging System (ISS) III: 2), at the baseline and after 7 days of treatment with LEN 25 mg/day (days 1–21), just before the start of the weekly treatment with Dex.in vitro studies.Mononuclear cells (MNCs) were isolated from BM and PB samples after Ficoll gradient separation and used for further −6M). All cell lines were authenticated and tested for mycoplasma contamination.The human myeloma cell line (HMCL) JJN3, purchased by DSMZ  and the human monocytic cell line THP-1, obtained from the American Type Culture Collection , were maintained in culture in RPMI 1640 medium with 10% FBS; hTERT-hMSCs were kindly gifted from Dr Giuseppe Gaipa  and maintained in culture with RPMI 10% FBS with hydrocortisone . CD3+ cells were isolated following the same protocol, using anti-CD3 mAb from PB of MM patients. The presence of potential contaminating cells in each fraction was evaluated by flow cytometry analysis, using the fluorescence-activated flow cytometer BD FACS Canto II with Diva software ; Franklin Lakes, NJ). Purity of cell samples was > 92%.BM and PB CD14+ cells, cultured in vitro at 1 × 106 cells/ml in RPMI 10% FBS, with recombinant human (rh) granulocyte macrophage colony-stimulating factor (GM-CSF) (50 ng/ml) and IL-4 (50 ng/ml) , for 8 days , in the presence of LEN  or vehicle (DMSO), at concentration 0.1 and 1 μM. TNF-α at 10 ng/ml  was added to the culture medium for the last 24 h, in order to induce DC terminal maturation. At the end of culture period, both cells and CM were collected for further analysis. In some experiment, the combination of LEN and Dex  was tested on DC differentiation. Briefly, DCs were differentiated from BM CD14+ cells of MM patients, in the presence of LEN (0.1 and 1 μM) or vehicle, as reported above. At the end of culture period, cells were collected and reseeded (5 × 104/ml) in fresh medium with Dex (10−8M) or vehicle (EtOH) for 48 h. After Dex treatment, cells were collected and analyzed for DC maturation markers. For the ex vivo studies, PB CD14+ cells were isolated from MM patients at day 0 and after one week (day 7) of LEN (25 mg/day) treatment, just before the start of the weekly treatment with Dex. Cells were then differentiated into DCs, following the above protocol, without LEN in vitro treatment.DCs were differentiated from purified CD14DCs were also differentiated from THP-1 cell line, by adding rhIL-4 (200 ng/ml), rhGM-CSF (100 ng/ml), ionomycin (200 ng/ml)  and rhTNF-α (20 ng/ml) for 72 h to the culture medium ; then LEN or DMSO were added for the last 24 h of culture period. THP-1-derived DCs (THP1-DCs) were detached with EDTA 2 mM on ice for 2 h and cell pellets collected for further analysis.+ cells of MM patients, in the presence or absence of the CM  of hTERT-hMSCs treated with LEN or DMSO. Briefly, 1 × 104 hTERT-hMSCs were seeded in T75 flasks and cultured in RPMI 10% FBS, in presence of LEN (0.1 and 1 μM) or DMSO, for 5 days. At the end of culture period, the medium was replaced with RPMI 10% FBS in order to discard LEN, and after 48 h, the CM was collected and used during DC differentiation, as previously reported. In some experiments, after 5 days of LEN treatment, hTERT-hMSC pellets were collected for immunoblotting and RT-PCR analysis.In some experiments DCs were differentiated from BM CD143 cells/w) in round-bottomed 96well-plates, in RPMI 15% AB human serum. DCs were co-cultured with autologous PB CD3+ cells (1 × 104) for 6 days. At the end of culture period, an MTT assay  was performed in order to measure T cell proliferation.DCs were differentiated, as previously reported, in presence of LEN or vehicle , from BM of 6 MM patients, for 8 days. Then, treated cells were collected, analyzed by flow-cytometry and partly re-seeded : 1) anti-CD14-FITC/anti-CD83-PE/isotype control-PE-Cy5/isotype control-APC, 2) anti-CD14-FITC/anti-CD83-PE/anti-CD86-PE-Cy5/anti-HLA-DR-APC, 3) anti-CD14-FITC/anti-CD83-PE/anti-CD80-PE-Cy5/anti-CD209-APC. Four color, six-parameter acquisition and analysis were perfomed on a two-laser FACSCalibur instrument (BD Biosciences) using CellQuest software (BD Biosciences). Mature DCs were identified as CD14−CD83+ cells and the MFI of the maturation markers was compared between cells treated with LEN and/or Dex vs the relative control, for each experiment.After in vitro DC differentiation, by using multiplex bead-based sandwich immunoassay kits , following the manufacturer's instructions. Measurement were performed by a reader . For TNF-α level evaluation, the obtained results were normalized for TNF-α concentration measured in the control medium .The concentration of Interferon (IFN)-γ, IL-6, IL-8, IL-10, IL-12, IFN-γ induced protein (IP)-10, CCL2, CCL5 and TNF-α was evaluated on DC CM, collected after Nuclear and cytosolic extracts were obtained using a commercial kit  following the manufacturer's protocol from THP1-DCs and hTERT-hMSCs, treated with LEN or DMSO. Immunoblotting was performed as previously reported  using th4 cells were infected with a multiplicity of infection (MOI) of 4, in the presence of 8 μg/ml polybrene . 24 h later, the infected medium was replaced with fresh growing medium. Puromycin selection (0.5 μg/ml) was initiated 2 days after transduction. Once a cellular clone was established, to induce CK1α silencing, cells were incubated with 500 μM IPTG  every 2–3 days for a total of one week. Then, fresh medium without IPTG and puromycin was added for further 48 h. At the end of culture period, cell pellets were collected and analyzed by western blotting to check CK1-α down-regulation and select the more efficient clone. CM was also collected and used for in vitro DC differentiation.RNAi was performed through the generation of inducible shRNA stable cell lines. hTERT-hMSC were transduced with the IPTG inducible lentiviral particles carrying CSNK1A1-specific shRNA . Two independent shRNAs  sequences were chosen. 3 × 10IDO1 (Assay ID:103804), IL6 (Assay ID: 144013), CCL5 (Assay ID: 113395), PTGS2 (Assay ID: 102471), TGB1 (Assay ID: 101210), and GAPDH (Assay ID: 102052). The expression of selected genes was checked by Real Time PCR by Light Cycler 480 . To normalize the differences in RNA quality and reverse transcription efficiency, we applied the comparative Ct method using the endogenous reference gene GAPDH.Total RNA was extracted from hTERT-hMSCs, after all different experimental conditions, using the RNeasy total RNA isolation kit . RNA (1 μg) was reverse-transcribed with 400 U Moloney murine leukemia reverse transcriptase  in accordance with the manufacturer's protocol. Real Time PCR was performed by adding complementary DNA to a universal Light Cycler 480 Probes Master and RealTime ready Catalog Assay  for the following genes: t-test was used to analyze flow cytometry data of in vitro DC differentiation from BM and PB of MM patients and for the ex vivo studies. Non-parametric Friedman test, Wilcoxon test and Mann-Whitney test were used for the other experiments with a lower number of samples. Results were considered significant at p < 0.05. GraphPad Prism 6.1™  was used for all the statistical analyses.Data were expressed as mean ± SEM or median values. Paired Student's


This Special Issue relates to the 18th biannual International Gap Junction Conference (IGJC2017), held at the Crowne Plaza Hotel, Glasgow, U.K., from the 29 July–2 August 2017. The special issue, entitled: Interplay of Connexins and Pannexins in Tissue Function and Disease focused on six key state of the art reviews written by chairs of the sessions highlighting the assembly and functional interactions of connexins and pannexins in diverse tissues and disease states, with translational outputs emerging. A further 14 articles detail specific contributions that were presented as oral communications . The meeConnexins and pannexins are tetramembrane spanning channel proteins with a shared topology and related functional properties. They oligomerise to form channels in the plasma membrane with two extracellular loops that project into the extracellular space and an intracellular carboxyl tail is subject to post-translational modification. Within the connexin family (21 members in man), these extracellular loops interdigitate and dock with loops from neighbouring cells to form dodecameric intercellular gap junction channels (GJCs) that link the cytoplasm’s of neighbouring cells. These GJCs enable the regulated exchange of over 300,000 metabolites of less than 1000 Da in size, in so doing co-ordinating specific cell and tissue homeostasis. Connexin compatibility is highlighted by phylogenetic classification into three–four specific subgroups, with only specific heteromeric channel combinations possible and in so doing facilitating the segregation of tissue compartments.Over the last 15 years, it has emerged that connexin hemichannels can be triggered to open under conditions of cell stress releasing signalling molecules such as ATP, glutamate and Nicotinamide adenine dinucleotide (NAD) into the extracellular environment and subsequently elicit localised extracellular signalling cascades via purinergic receptors. Pannexins, proteins forming hemichannel-like structures, identified about 15 years ago, share a common topology but no sequence homology with connexins, are thought to be evolutionary related to the innexins, gap junction forming proteins in invertebrates. Unlike connexins (and innexins) pannexins are highly glycosylated proteins and act to release ATP, engaging with downstream signalling pathways. In particular, pannexin signalling has been closely linked with inflammatory mediated events, where caspase1 cleavage of the carboxyl tail renders these channels constitutively open and triggers cell death ‘find me’ signals.Due to the diversity of connexin and pannexin expression in tissue networks, their importance cannot be underestimated and they are now firmly established as key proteins in diverse tissue networks including the cardiovascular system, the skin, the nervous system, the liver, the ocular, respiratory and immune system. Further, changes in connexin expression and function occur in disease states associated with all of these tissues: Tumorigenesis, diabetic retinopathy, cardiac arrhythmia, atherosclerosis, stroke, Alzheimer’s disease, chronic non–healing skin wounds, and inflammation of epithelial tissue, to name a few. A range of mutations in connexins are associated with clinical disease, where mutations in Cx26 are among the most common in recessively inherited hearing impairment, Cx32 with the demyelinating disorder Charcot Marie Tooth-Linked disease, Cx43 with oculodentodigital dysplasia, and Cx46 and Cx50 with familial cataract formation. All these diseases impact on the quality of life, healthcare resources, ageing populations and many with conditions that can be managed with current therapies, but not cured.Thus, connexins and pannexins have emerged as prime therapeutic targets for a diverse range of disease states. These channels are amenable to targeting therapeutically and a range of antisense and peptidomimetic strategies have emerged as key regulators of channel function. Such regulators were first identified over 25 years ago when Evans and Warner synthesised the first mimetic peptides and antibodies that mimicked amino acid sequences on the extracellular loops. These tools are now widely used by the research community to define the role of connexins and pannexins in tissue function and are proving successful in translational research where the success of clinical trials of a connexin targeted therapy, from bench to bedside, for improving wound healing events is reported in this special issue.The Editors thank all contributors to this special issue, delegates and in the conference support team for the successful running of IGJC2017. The follow-up meeting will be held in Victoria, Vancouver Island, July 2019 (#IGJC2019).ReviewsInt. J. Mol. Sci.2018, 19(5). [Aasen, T.; Johnstone, S.; Vidal-Brime, L.; Lynn, K.S.; Koval, M. Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease. Int. J. Mol. Sci.2018, 19(5). [Sorgen, P.L.; Trease, A.J.; Spagnol, G.; Delmar, M.; Nielsen, M.S. Protein–Protein Interactions with Connexin 43: Regulation and Function. Participant ContributionsInt. J. Mol. Sci.2018, 19(6). [Ek-Vitorin, J.F.; Pontifex, T.K.; Burt, J.M. Cx43 Channel Gating and Permeation: Multiple Phosphorylation-Dependent Roles of the Carboxyl Terminus. Int. J. Mol. Sci.2018, 19(6). [Spagnol, G.; Trease, A.J.; Zheng, L.; Gutierrez, M.; Bazu, I.; Sarmiento, C.; Moore, G.; Cervantes, M.; Sorgen, P.L. Connexin43 Carboxyl-Terminal Domain Directly Interacts with β-Catenin. Int. J. Mol. Sci.2018, 19(7). [Sanchez-Pupo, R.E.; Johnston, D.; Penuela, S. N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1. , 19(7). Int. J. Mol. Sci.2018, 19(9). [Batissoco, A.C.; Salazar-Silva, R.; Oiticica, J.; Bento, R.F.; Mingroni-Netto, R.C.; Haddad, L.A. A Cell Junctional Protein Network Associated with Connexin-26. 2018, 19(9). [Schadzek, P.; Helmes, D.; Stahl, Y.; Dilger, N.; Ngezahayo, A. Concatenation of human connexin26 (hCx26) and human connexin46 (hCx46) for the analysis of heteromeric gap junction hemichannels and heterotypic gap junction channels. , 19(9). ReviewInt. J. Mol. Sci.2018, 19(6). [Molica, F.; Figueroa, X.F.; Kwak, B.R.; Isakson, B.E.; Gibbins, J.M. Connexins and Pannexins in Vascular Function and Disease. Participant ContributionsInt. J. Mol. Sci.2018, 19(1). [Carballo, S.; Pfenniger, A.; Carballo, D.; Garin, N.; James, R.W.; Mach, F.; Shah, D.; Kwak, B.R. Differential Association of Cx37 and Cx40 Genetic Variants in Atrial Fibrillation with and without Underlying Structural Heart Disease. Int. J. Mol. Sci.2018, 19(4). [Noureldin, M.; Chen H, Bai D. Functional Characterization of Novel Atrial Fibrillation-Linked GJA5 (Cx40) Mutants. , 19(4). Int. J. Mol. Sci.2018, 19(4). [Viczenczova, C.; Kura, B.; Egan Benova, T.; Yin, C.; Kukreja, R.C.; Slezak, J.; Tribulova, N.; Szeiffova Bacova, B. Irradiation-Induced Cardiac Connexin-43 and miR-21 Responses Are Hampered by Treatment with Atorvastatin and Aspirin. , 19(4). Int. J. Mol. Sci.2018, 19(6) [Boucher, J.; Simonneau, C.; Denet, G.; Clarhaut, J.; Balandre, A.C.; Mesnil, M.; Cronier, L; Monvoisin, A. Pannexin-1 in Human Lymphatic Endothelial Cells Regulates Lymphangiogenesis. 8, 19(6) Int. J. Mol. Sci.2018, 19(7). [Htet M, Nally, J.E.; Shaw, A.; Foote, B.E.; Martin, P.E.; Dempsie, Y. Connexin 43 Plays a Role in Pulmonary Vascular Reactivity in Mice. , 19(7). ReviewInt. J. Mol. Sci.2018, 19(6). [Graham, S.V.; Jiang, J.X.; Mesnil, M. Connexins and Pannexins: Important Players in Tumorigenesis, Metastasis and Potential Therapeutics. , 19(6). Participant ContributionsInt. J. Mol. Sci.2018, 19(3). [Busby, M.; Hallett, M.T.; Plante, I. The Complex Subtype-Dependent Role of Connexin 43 (GJA1) in Breast Cancer. , 19(3). Int. J. Mol. Sci.2018, 19(7). [Iikawa, N.; Yamamoto, Y.; Kawasaki, Y.; Nishijima-Matsunobu, A.; Suzuki, M.; Yamada, T.; Omori, Y. Intrinsic Oncogenic Function of Intracellular Connexin26 Protein in Head and Neck Squamous Cell Carcinoma Cells. , 19(7). ReviewsInt. J. Mol. Sci.2018, 19(5). [Chanson, M.; Watanabe, M.; O’Shaughnessy, E.M.; Zoso, A.; Martin, P.E. Connexin Communication Compartments and Wound Repair in Epithelial Tissue. , 19(5). Participant ContributionsInt. J. Mol. Sci.2018, 19(2). [Faniku, C.; O’Shaughnessy, E.; Lorraine, C.; Johnstone, S.R.; Graham, A.; Greenhough, S.; Martin, P.E. The Connexin Mimetic Peptide Gap27 and Cx43-Knockdown Reveal Differential Roles for Connexin43 in Wound Closure Events in Skin Model Systems. Int. J. Mol. Sci.2018, 19(5). [Chen, J.; Liang, C.; Zong, L.; Zhu, Y.; Zhao, H.B. Knockout of Pannexin-1 Induces Hearing Loss. ReviewInt. J. Mol. Sci.2018, 19(6). [Montgomery, J.; Ghatnekar, G.S.; Grek, C.L.; Moyer, K.E.; Gourdie, R.G. Connexin 43-Based Therapeutics for Dermal Wound Healing.