Twitter has recently gained popularity in emergency medicine (EM). Opinion leaders on Twitter have significant influence on the conversation and content, yet little is known about these opinion leaders. We aimed to describe a methodology to identify the most influential emergency physicians (EP) on Twitter and present a current list.We analyzed 2,234 English-language EPs on Twitter from a previously published list of Twitter accounts generated by a snowball sampling technique. Using NodeXL software, we performed a network analysis of these EPs and ranked them on three measures of influence: in-degree centrality, eigenvector centrality, and betweenness centrality. We analyzed the top 100 users in each of these three measures of influence and compiled a list of users found in the top 100 in all three measures.Of the 300 total users identified by one of the measures of influence, there were 142 unique users. Of the 142 unique users, 61 users were in the top 100 on all three measures of influence. We identify these 61 users as the most influential EM Twitter users.We both describe a method for identifying the most influential users and provide a list of the 61 most influential EPs on Twitter as of January 1, 2016. This application of network science to the EM Twitter community can guide future research to better understand the networked global community of EM. Twitter is an online social media platform that allows individuals to communicate through tweets. A tweet is an electronic message of 140 characters or less that is accessible to the public. By following other users, you can view their tweets in your personal timeline. Twitter is used by 23% of online adults, making it one of the most popular social media platforms globally. 8Dissemination of information on Twitter can be rapid and viral, and is heavily influenced by important opinion leaders. 12Despite its popularity and potential pitfalls, there is a paucity of data examining influence among Twitter users in EM. Furthermore, existing measures of influence in social media are not directly applicable to Twitter. The influential group of opinion leaders in the EM Twitter community has not been defined. Defining this group is an important step toward understanding the spread of information among EPs on a social media platform.network science, a new type of applied graph theory that incorporates several disciplines. We aimed to both describe a method to identify the most influential EPs on Twitter and present a current list. To perform this task we used This study was granted institutional review board exemption by the University of Washington Human Subjects Division.www.Twitter.com), FollowerWonk (https://moz.com/followerwonk) and Twiangulate (http://twiangulate.com/search/). A snowball sampling technique was used to expand the list by exploring followers’ biographies and the Twitter accounts of organizations and journals related to EM. Twitter lists are a common tool to group users into categories by various criteria. The first curated list of English-language EPs on Twitter was published in 2009. From its January 2016 update, we gathered data about each member using NodeXL computer software . Variables including number of followers and tweets were recorded for each user.Network science helps identify influential people based on several different metrics of influence. This is conceptually important because an individual may have social influence within a community for many different reasons. For example, an EP on Twitter may be influential because he or she has a large number of followers, has followers who are influential themselves, or has a unique group of followers to help disperse information. As such, sociologists have developed contemporary methods to identify influential members in a network and rank them according to different definitions of importance. These measures of importance are called centralities. 16within a network. In the case of our study, it is not the total number of followers a certain user has on Twitter. Instead, it is a measure of how many EPs are following a given user. In this measurement, each follower has equal weight.Degree is a measure of connections based on the number of followers a user has Users with high in-degree centrality are considered to have prominence, prestige, and importance. Messages can spread broadly if retweeted, or passed along, by a few influential users. As such, being followed by one popular Twitter user bestows more influence than being followed by many brand-new Twitter users with few followers. Eigenvector centrality accounts for this by going beyond the number of followers a user has. It measures the collective influence of each follower. Being recognized by someone seen as powerful contributes heavily to one’s perceived influence. Eigenvector centrality elevates those users followed by a smaller, but more influential, number of followers. 18Betweenness is a measure of information gatekeeping. Users with a high betweenness centrality provide the shortest paths between other users within the network. Because of their position within the network, they have considerable control over information diffusion. They are important in passing along information through a network. Users with high betweenness are frequently viewed as leaders. 19There is no single measure of importance that is paramount in understanding a social network. Rather, these centralities must be taken together to provide a robust measure of a user’s influence. Of the 300 users in the top 100 of each measure of centrality see , there wFifty-three of the 61 (87%) provide their full name in their profile. Of those whose gender was easily discernable from their profile, 9 of 59 (15%) are women. Seventy-one percent of TIs are located in the United States, with others in Europe (13%), Australia (9%), Canada (5%), and Costa Rica (2%). The earliest users joined Twitter in 2007, while the most recent influencer joined in 2014.The strengths of this study lie in a robust network analysis of over 2,200 EPs using three different measures of influence grounded in network science. We provide a network analysis method for determining the most influential EPs on Twitter. We also present a current list of those TIs, or Twitter influencers. This list helps quantify the qualitative concept of social influence and demonstrates a contemporary methodology for defining influence.among emergency physicians, and not broader influence among other healthcare networks or the lay public. For example, there are EPs with influence outside the EM community, like television star Travis Stork, MD, (@TravisStorkMD) who has 159,000 Twitter followers. He does not, however, influence the conversation or content among EPs because he is not followed by them and does not lie between them in the EP Twitter network.It is important to note that this analysis represents influence only Women make up a small percentage of the TIs. This gross disproportionality is consistent with other studies examining influential EPs. A recent study found that only 11% of academic chairs in EM are women. This work builds on Lulic and Kovic’s 2013 derivation of the EM users on Twitter list. Our derived cohort had some overlap with the only other existing measure of social influence, the SMi. Of the 61 Twitter users affiliated with the top SMi blogs and podcasts, 41 (67%) were in our list of TIs. By applying several different, robust measures of influence, this curated list adds to our knowledge of the influential EPs on Twitter.We believe this list of 61 TIs can be used as a valid foundation for future research around Twitter in EM. Rigorous analyses of the 61 TIs will move forward our understanding of the way Twitter is used for content, conversation, and professional development. For example, in-depth content analysis of the tweets of the 61 TIs would give insight into the EM subjects with the most weight on Twitter. A recent analysis of free open-access educational resources found imbalanced and incomplete coverage of EM core content. In response to the concern that social media was gaining too much influence and that we are losing sight of key metrics of scientific value, such as citation indices, the satirical Kardashian Index was described in 2014. This study is limited to English-language speaking EPs. We did not contact the users to verify that they were EPs, though most of the 61 TIs are known to the authors as EPs. While our network analysis examined the number of followers for each user within the network of EPs, it did not analyze recent account activity for these users. It is possible that there are other influential EP users with high eigenvector, in-degree centrality and betweenness centrality who were excluded from our analysis because they have not been identified as EPs on the existing EPs Twitter list. This list is also limited to physicians and does not include those emergency medical services personnel, social workers, nurses, and pharmacists who are influential in the EM Twitter community.In summary, there is a growing network of EPs on Twitter, impacted by a small group of opinion leaders. To understand this network, we both describe a method for identifying the most influential users and provide a list of the 61 most influential EPs on Twitter as of January 1, 2016. This application of network science to the EM Twitter community can guide future research to better understand the networked global community of EM.


The AFRIM study by Obonyo et al.  presentsdecrease in stroke volume index, with a decrease of 48% in one case! It would interest us what the authors believe the reason for this effect is.The purpose of a fluid bolus is to increase stroke volume and hence cardiac output. Although these patients were dehydrated, only 5 out of 11 in group 1 and 5 out of 9 in group 2 demonstrated a 10% increase in stroke volume following the fluid boli. Two patients in group 1 demonstrated a 10% We believe that the administration of fluid, even in hypovolaemic patients, is more complex than often appreciated, with an interaction of many factors related to vascular biology, and further investigation is required to enable an optimisation of fluid delivery and to avoid the detrimental effects of fluid overload. We are encouraged by the interest in fluid resuscitation in children with severe acute malnutrition and hypovolaemic shock, and have read with keen interest the contribution by Dr. Ellis Muggleton highlighting the limited utility of IVCCI in spontaneously breathing patients.In the manuscript, while presenting these data , the autReduction in stroke volume index following fluid bolus administration is indeed an unexpected finding. Nonetheless, our findings stimulate a new direction of inquiry investigating the mechanisms underlying the pathophysiology of hypovolaemia with severe malnutrition as well as the interpretation of the patients’ relative position on the Frank-Starling curve with respect to FR. This is particularly important in the context of little understanding on the effect that fluid administration has on the complex patho-biological interaction of intravascular hypovolaemia and severe malnutrition. Previous research has shown that hypovolaemia and dehydration are associated with higher mortality in severe malnutrition . The AFR


A drug delivery system targeting the tumour microenvironment produces outstanding therapeutic efficacy on triple-negative mammary and pancreatic models. The development of novel therapeutic strategies allowing the destruction of tumour cells while sparing healthy tissues is one of the main challenges of cancer chemotherapy. Here, we report on the design and antitumour activity of a low-molecular-weight drug delivery system programmed for the selective release of the potent monomethylauristatin E in the tumour microenvironment of solid tumours. After intravenous administration, this compound binds covalently to plasmatic albumin through Michael addition, thereby enabling its passive accumulation in tumours where extracellular β-glucuronidase initiates the selective release of the drug. This targeting device produces outstanding therapeutic efficacy on orthotopic triple-negative mammary and pancreatic tumours in mice (50% and 33% of mice with the respective tumours cured), leading to impressive reduction or even disappearance of tumours without inducing side effects. However, the scope of such targeting devices is restricted to only the treatment of tumours expressing a high level of the targeted cell surface marker. Here we show that the non-internalizing drug delivery system 1, which targets the tumour microenvironment, induces remarkable anticancer activity on different animal models and is independent of the cancer cell’s surface hallmarks 10 and a maleimide-bearing side chain in a single entity . The latter reacted with commercially available O-(2-aminoethyl)-O′-(2-azidoethyl)-nonaethylene glycol in the presence of Cu(CH3CN)4PF6 to form the triazole 6 (77%). The full deprotection of the glucuronide moiety was carried out using LiOH and the crude product was engaged in the next step without purification. Finally, reaction with the hydroxysuccinimide ester 7 afforded the drug delivery system 1 with a 33% yield after purification by preparative HPLC (purity > 95%).The drug delivery system terature .22 First1 was incubated with human serum albumin (HSA) at 37 °C in order to form the macromolecular assembly 2. Under these conditions, more than 90% of 1 was converted in two hours as a result of its rapid binding with the protein , this tumour model is highly responsive to folate–drug conjugates.2 Thus, it was possible to compare the efficacy of the β-glucuronidase-catalysed drug delivery process in the tumour microenvironment with a well-established internalising approach using the drug delivery system 823  were well tolerated without any body weight loss  transplanted in mice. The animals received i.v. injections of glucuronide 1 at 4 mg kg–1  weekly for five weeks. Following this protocol, an impressive reduction of tumour volume was observed in all treated animals  is a clinically aggressive disease for which there is no targeted chemotherapy available. To explore the potential of prodrug  animals . Moreove imaging , without imaging . In cont1 for the therapy of pancreatic cancer, which is the gastrointestinal malignancy with the worst prognosis and a 5 year survival rate of less than 5%. To date, chemotherapy is ineffective against this disease, prompting the need for new therapeutic approaches. Within this framework, human MIA PaCa2 cells exhibiting the four most common mutations for pancreatic adenocarcinoma27 were injected into the pancreas of nude mice and tumour growth was examined by 3D echography. Mice bearing intra-pancreatic tumours were treated with two 4 mg kg–1 doses of the glucuronide prodrug 1. As soon as the first dose was given, tumours started to regress in all animals that received 1, whereas treatment with MMAE (3 × 0.5 mg kg–1) only resulted in retardation of tumour growth (1 and 33% of mice were tumour free at day 70 post-implantation, as assessed both by echography and bioluminescence imaging (We next considered prodrug r growth . Tumour  imaging .3. As determined by photoacoustic imaging these tumours displayed strong hypoxia, therefore making this model more predictive (see the ESI1 at 4 mg kg–1 administered weekly for nine weeks. This therapy induced remarkable antitumour activity with a dramatic reduction of the initial tumour volumes (versus untreated mice, for which tumour growth led rapidly to death (1 results in an outstanding size reduction of tumours.29Since pancreatic cancers in humans are usually detected at a late stage and characterized by severe tumour hypoxia, rendering malignant cells resistant to chemotherapy, we conducted a new trial on mice bearing MIA PaCa2 orthotopic xenografts with sizes ranging from 2.5 to 3.5 cm volumes . Survivato death . In compin vivo for the treatment of various solid tumours.36 However, owing to their rapid renal clearance and/or the limited potency of the targeted drug, these prodrugs have to be administered at very high doses to achieve significant anticancer activity, hampering their transfer to the clinic. Therefore, the therapeutic efficacy demonstrated by 1 represents a real breakthrough that could have significant impact in the field of drug delivery for cancer chemotherapy.Some glucuronide prodrugs have already been evaluated 1 is a selective, efficient and potentially versatile therapeutic strategy. We believe that this approach could be employed in combination with internalizing drug delivery systems in targeted poly-chemotherapy. The efficacy observed in pre-clinical models offers a new hope for the chemotherapy of solid tumours, especially for the treatment of pancreatic ductal adenocarcinoma, for which there is an urgent need for novel therapeutic strategies.Overall, this study demonstrates that the targeting of the tumour microenvironment by means of the β-glucuronidase-responsive albumin-binding prodrug in vivo studies and analysed the data. T. L. and J. A. developed early versions of the glucuronide prodrugs. I. T. O. prepared the folate–drug conjugate. B. E. undertook trypsin-digestion experiments. P. P. conducted HPLC/HRMS experiments and analysed the data. S. L. and A. L. P. designed and supervised in vivo experiments. O. K. and S. K. conducted pharmacokinetic studies. E. P. carried out in vitro biological experiments. J. C. supervised in vitro biological experiments and analysed the data. S. P. designed the study and wrote the manuscript.B. R. synthesised, purified and characterised the glucuronide prodrugs and carried out enzymatic reactions. F. R. designed and conducted


Wilms’ tumour (nephroblastoma) is the most common renal tumour in children. Wilms’ tumour in adults is extremely rare and has a poorer prognosis than paediatric Wilms’ tumour. It is difficult to differentiate adult Wilms’ tumour from renal cell carcinoma based on radiological findings alone. The diagnosis in adults is often serendipitous following nephrectomy for presumed renal cell carcinoma. Because of the paucity of literature, there are no standard protocols for the management of adult Wilms’ tumour, and therefore, it is managed as per paediatric Wilms’ tumour. Herein, we report the case of adult Wilms’ tumour in a 43-year-old man, which was diagnosed unexpectedly following nephrectomy for presumed renal cell carcinoma. The patient was then referred to an urologist for further management.A 43-year-old Caucasian man, with a 4-week history of right upper quadrant and right flank pain radiating to his right testicle, presented to his general practitioner. There was no preceding trauma, haematuria or any other systemic symptoms. The past medical history included osteoid osteoma in the left upper femur, which was removed in 2007. He also had L4/5 microdiscectomy. There was no positive family history of medical problems or cancer. He was a non-smoker, had no allergies and was not taking any regular medications. The general practitioner organized an ultrasound, which revealed a large subcapsular haematoma measuring 7 × 5 × 7 cm in the right kidney compressing the renal cortex. Further investigation with computed tomography (CT) scan of the abdomen with intravenous contrast revealed enlarged right kidney with subcapsular haematoma and a mass lesion within the right lower pole measuring up to 7.4 × 7.5 × 9.2 cm with associated paracaval lymphadenopathy producing compression of the right renal vein and inferior vena cava , and the estimated glomerular filtration rate was 66 ml/min. CT staging showed a large right renal tumour and significant paracaval lymphadenopathy producing compression of the right renal vein and inferior vena cava. The size of the tumour was substantially increased in size to 11.5 × 11.6 × 11.5 cm when compared with that in the previous CT scan.Figure 3). There was an extensive haematoma within the lumen of the cyst and possible necrosis. The retrocaval lymph node measured 4 cm. Histologically, the tumour comprised of blastemal, stromal and anaplastic elements. The blastemal elements were present in nodular fashion and had syncytial pattern with no diffuse infiltrative component . The anaplastic elements were focally present in stromal elements . The stromal elements were present with focal anaplastic elements. There were features of necrosis, haemorrhage and cystic degeneration. The morphologic and histopathology features favoured adult Wilms’ tumour with synovial sarcoma as a differential diagnosis. A fluorescence in situ hybridization analysis showed loss of heterogeneity (LOH) for 1p 16q, which is associated with poor outcomes in paediatric Wilms’ tumour. The pathological staging was pT3a, pN1 and cM0 . The patient was subsequently treated with adjuvant combination chemotherapy and radiotherapy.A preliminary diagnosis of renal cell carcinoma was made based on the clinical findings and investigations. The patient underwent open radical right nephrectomy, retroperitoneal lymph node dissection and partial excision, and reconstruction of vena cava. The renal samples were sent for histopathology. The right kidney specimen (with fibro adipose tissue) weighed 1291 g and 175 × 140 × 110 mm in size. The tumour was located in the mid-pole and lower pole of approximately 100 × 95 × 100 mm. On gross inspection, the tumour was cream and pink in colour with mixture of solid and cystic components (Based on the age of presentation, unfavourable histology and chromosomal LOH for 1p16q, the tumour was considered high-risk, and chemotherapy was initiated as per the Children’s Oncology Group UH-1 protocol, including early radiation to the tumour bed, and multiagent chemotherapy including actinomycin D, vincristine and doxorubicin. Radiation and chemotherapy were commenced concurrently within 3 weeks of surgery. The intensity of chemotherapy was maintained as per protocol with granulocyte support. The patient has been followed up at regular intervals with CT scans and routine blood tests. There has been no radiological or clinical recurrence at the end of 12 months of initial diagnosis.Wilms’ tumour is the most common renal tumour accounting for 85% of paediatric renal neoplasms. The tumour is most prevalent in the first 5 years of life with peak incidence between 3 and 4 years of age . The annTreatment of Wilms’ tumour invariably is multimodal involving surgery, chemotherapy and/or radiotherapy. Two accepted management protocols are the NWTS and SIOP. NWTS has always recommended upfront nephrectomy to define the accurate stage of the tumour and the histology, on which further treatment stratification is decided. In contrast, the SIOP investigators pioneered the concept of prenephrectomy chemotherapy in all patients over 6 months of age to reduce the tumour size and prevent intraoperative tumour rupture causing spillage and to increase the proportion of children with a lower tumour stage that required less overall treatment .The NWTS group has reported an overall survival rate of 82% in adults with favourable histology Wilms’ tumour . TerenziIt is well documented that Wilms’ tumours are radio- and chemo-sensitive, and delay in treatment can adversely affect outcomes . There aAdult Wilms’ tumour is a rare entity with mainly case reports-only literature. There are no established protocols for treatment of this group of patients. Being so rare, the diagnosis is generally made on postoperative pathology. Treatment of this tumour is multimodal incorporating surgery and lengthy chemotherapy, which needs to be tailored to the patient. Its prognosis is poorer than that of Wilms’ tumour in the paediatric population. Relapse in the first year after treatment is a predictor of poor outcome. It is recognized that Wilms’ tumour in adults is a rare entity, and the importance of registering such patients to international data bases will assist in the research and development of future management guidelines for this tumour.


Galectin-1  has been described to promote tumour growth by inducing angiogenesis and to contribute to the tumour immune escape. We had previously identified up-regulation of Gal-1 in preclinical models of aggressive neuroblastoma (NB), the most common extracranial tumour of childhood. While Gal-1 did not confer a survival advantage in the absence of exogenous stressors, Gal-1 contributed to enhanced cell migratory and invasive properties. Here, we review these findings and extend them by analyzing Gal-1 mediated effects on immune cell regulation and radiation resistance. In line with previous results, cell autonomous effects as well as paracrine functions contribute to Gal-1 mediated pro-tumourigenic functions. Interfering with Gal-1 functions in vivo will add to a better understanding of the role of the Gal-1 axis in the complex tumour-host interaction during immune-, chemo- and radiotherapy of neuroblastoma. Glycan-binding proteins were first described in the 1960s by Ashwell and Morrell ,3. They −/− mice are healthy and fertile. Interestingly, targeted infection of Gal-1−/− mice results in severe autoimmune disease, pointing to a role of Gal-1 in regulating and terminating inflammatory responses. This process has been attributed to increased Th1 and Th17 responses in Gal-1−/− mice. Interestingly, Th2 cells…seemed to be protected from the anti-proliferative effects of Gal-1 as a consequence of different glycosylation patterns [As a member of the prototypical group, Galectin-1  is divalent and its ability to cross-link carbohydrate chains on cell surfaces has been recognised early on . Gal-1 ipatterns . Thus, GAnother important physiological function of Gal-1 has been described in mediating feto-maternal tolerance . It has In tumours, mainly the intracellular functions of Gal-1 have been linked to pro-tumourigenic process, as Gal-1 can interact with oncogenic H-RAS to promote angiogenesis , while GHowever, accumulating evidence suggest that both Gal-1 is linked to many hallmarks of cancer  and that2+-dependent protein kinases to stimulate cell growth. Secondly, TrkB induces the PI-3 kinase signalling pathway to activate AKT. Additionally, induction of the MAP/ERK pathway downstream of Ras/B-Raf promotes differentiation or cell survival depending on signal strength and duration [Neuroblastoma (NB) is the most common extracranial solid tumour of childhood and arises from the sympathetic nervous system . Interesduration ,32. The To further understand the biological consequences of the differently expressed neurotrophin receptors, TrkA and TrkB, we previously selected a Trk-negative human neuroblastoma cell line, SH-SY5Y, and designed subclones with either high TrkA or TrkB expression. Analysis of transcriptome and proteome changes revealed that Gal-1 is up-regulated only in SY5Y cells with ectopic TrkB expression (SY5Y-TrkB) ,34. As s−/− mouse strain was generated by Poirier and co-workers back in 1993. They used homologous recombination techniques in embryonic stem cells to disrupt and inactivate the L14 lectin gene . Animals with a global knock-out of Gal-1 were indistinguishable from control littermates. The mice were fertile and developed normally with an unimpaired lifespan indicating that other highly related lectins may be able to compensate for the function of Gal-1 in these animals [T. cruzi infected mice, in which Gal-1 is produced and secreted by B cells [Several mouse models have been developed to study neuroblastoma development in vivo as a consequence of MYCN or ALK overexpression in neural crest cell progenitors. While recent models used Cre-Lox technology to induce MYCN or ALK ,38, an e animals . As poin animals . Impaire animals . A toler animals  and in T B cells . While G−/− mice. We demonstrated that the Gal-1 gene dosage did not affect tumour incidence, growth rate, or survival probability, but was correlated with alterations in the immune phenotype exemplified by a reduction of CD4+ T cell infiltration in tumours of Gal-1−/− mice [To further investigate the impact of Gal-1 on neuroblastoma aggressiveness, we crossbred TH-MYCN mice to Gal-1−/− mice . Tumour −/− mice . Here, G−/− mice  requirin−/− mice  but also−/− mice . Additio−/− mice . Hence, −/− mice . Consequ−/− mice . Remarka−/− mice, these animals suffer from splenomegalies indicated by a significantly higher spleen weight observed in tumour bearing Gal-1−/− mice. These splenomegalies could be explained by a lower migratory capacity of Gal-1 deficient CD4+ T cells, while proliferation and apoptosis of CD4+ T cells was unaltered [−/− mice present with higher infiltration of CD11+ dendritic cells compared to Gal-1+/− or Gal-1 wt animals suggesting an enhanced tumour antigen presentation as a response to Gal-1 knock-out. However, tumours derived from mice lacking Gal-1 showed a reduction in CD31 endothelial cell staining confirming a role for Gal-1 in tumour angiogenesis depending on the Gal-1 gene dosage [In addition to the altered immune phenotype of TH-MYCN/Gal-1naltered . TH-MYCNe dosage . These fe dosage . It has e dosage . Furthere dosage .Standard therapies for aggressive neuroblastoma include surgery as well as chemo- and radiotherapy. Response to the latter therapeutic modalities is strongly modulated by the tumour microenvironment and development of resistance with subsequent disease relapse is frequently observed in aggressive type 2 neuroblastoma . As bothFor neuroblastoma, standard of care includes surgery, chemotherapy and also radiotherapy. Since Gal-1 is overexpressed in the chemo- and radioresistant subtype of neuroblastoma, it can be considered as a potential therapeutic target for neuroblastoma therapy. Still, tumour irradiation is mainly performed by indirectly ionizing electromagnetic photons generated by an X- or γ-ray source, while only specialised centres make use of charged particles including protons, which are directly ionizing. Radiotherapeutic approaches mainly aim to induce DNA damage to kill malignant cells, as unrepaired DNA double-strand breaks subsequently induce apoptosis, mitotic catastrophes or autophagy. Thus, DNA damage repair pathways play a crucial role in determining the clinical outcome of radiotherapy, rendering it essential to find predictive biomarkers and novel targets for radiotherapy ,54. Gal-2O2 hydroxyl radicals requires oxygen. Thus, hypoxia within tumours causes a three-fold increase in the radiation dose required to generate the same amount of DNA damage when compared to normoxic conditions [Additionally, Gal-1 is not only an important player in the tumour microenvironment by modifying the immune response and angiogenesis, but it is also affected by tumour hypoxia. Several reports indicate a positive feedback loop expression between Hif1α, the major transcription factor involved in adaptation to hypoxic condition, and Gal-1 via the H-RAS oncogenic pathway . Gal-1 enditions . In addinditions ,58. Our nditions . While iGal-1 is a multifaceted protein regulating different aspects of tumour biology. While necessary for balancing immune responses and angiogenic processes in physiological settings, tumours exploit these functions to escape from attacks of the host immune systems and to better cope with hypoxic conditions. Several strategies are currently evaluated to identify the best method to interfere with Gal-1 functions in therapeutic settings. One promising approach is to combine radiotherapy and Gal-1 blockade with the aim to reactivate immune responses towards the tumour and to limit angiogenic responses. Combining immune checkpoint inhibitors with blocking Gal-1 functions might also be an option to boost immunogenic anti-tumour responses. On the other hand, radiation-induced upregulation of Gal-1 could be used to selectively deliver drugs to Gal-1 positive cells in the tumour microenvironment. The results of these studies are anticipated to inform us about the optimal use of Gal-1 directed strategies in tumour therapy.


The Illumina 450k array has been widely used in epigenetic association studies. Current quality-control (QC) pipelines typically remove certain sets of probes, such as those containing a SNP or with multiple mapping locations. An additional set of potentially problematic probes are those with DNA methylation distributions characterized by two or more distinct clusters separated by gaps. Data-driven identification of such probes may offer additional insights for downstream analyses.We developed a procedure, termed “gap hunting,” to identify probes showing clustered distributions. Among 590 peripheral blood samples from the Study to Explore Early Development, we identified 11,007 “gap probes.” The vast majority (9199) are likely attributed to an underlying SNP(s) or other variant in the probe, although SNP-affected probes exist that do not produce a gap signals. Specific factors predict which SNPs lead to gap signals, including type of nucleotide change, probe type, DNA strand, and overall methylation state. These expected effects are demonstrated in paired genotype and 450k data on the same samples. Gap probes can also serve as a surrogate for the local genetic sequence on a haplotype scale and can be used to adjust for population stratification.The characteristics of gap probes reflect potentially informative biology. QC pipelines may benefit from an efficient data-driven approach that “flags” gap probes, rather than filtering such probes, followed by careful interpretation of downstream association analyses. Our results should translate directly to the recently released Illumina EPIC array given the similar chemistry and content design.The online version of this article (doi:10.1186/s13072-016-0107-z) contains supplementary material, which is available to authorized users. DNA methylation (DNAm) is a type of epigenetic mark and term commonly used to denote the covalent addition of a methyl or hydroxymethyl group to a cytosine nucleotide base in the DNA sequence, typically at cytosine–guanine dinucleotide sequences, or CpG sites. DNAm is a necessary component to cellular differentiation during development and is a leading mechanism for the plasticity of the genome in response to various environmental stimuli during the life course . There iThe Illumina HumanMethylation450 BeadChip (450k) has largely enabled the fast growth of epigenetic epidemiology because it effectively balances sample throughput and cost with epigenome coverage. Specifically, the 450k allows for the efficient interrogation of roughly 485,000 CpG sites in the human genome, covering 99% of RefSeq genes, CpG islands, lower-density CpG regions, termed shores and shelves, shown to be associated with differentiation and disease , and othAs use of the 450k has become increasingly widespread, there have been several contributions that have increased our general understanding of probe behavior on the 450k. One frequently cited example is that of ambiguously mapping probes, or probes that can hybridize to multiple places in the genome. A list of these probes has been made publicly available, and they are often removed prior to association analysis . SeveralRecently, Daca-Roszak et al. overcame these reference annotation limitations on a small scale through the analysis of combined study-specific genotype and 450k array data on 96 probes that distinguished European and Chinese populations. 69% of these probes contained study-specific SNPs that were ancestry informative. They specifically note the existence of tri- and bimodal beta value distributions at many of these 96 probes, and carefully delimit, through consideration of bisulfite conversion and probe chemistry, how each possible SNP at the C and G sites of interest  can affect methylated and unmethylated signal and the subsequent beta value calculated. Ultimately, the authors recommend a careful consideration of the potential influence of genetic polymorphism on DNAm signal when interpreting epigenome-wide association study (EWAS) results .The clustered distributions for some probes had been addressed previously with lesser detail , 14, butIn our exploration of 450k data, we first noticed such clustered distributions by the “gap” pattern apparent when methylation signals per mode clustered into non-overlapping groups. In this paper, we present a method, termed “gap hunting” to identify 450k probes that result in such a distributional “gap.” Identification of 450k probes with clustered methylation values using the empirical approach we propose here overcomes previous limitations with other probe removal approaches , 17 becaWe developed a simple, computationally fast algorithm, called “gap hunting,” to flag probes on the 450k exhibiting distributions of percent DNAm that cluster into discrete groups. We applied this to DNAm data from 590 whole-blood-derived samples from the Study to Explore Early Development I (SEED I) at various combinations of user-supplied arguments to this procedure termed “threshold” and “outCutoff”  and methylation data, we found that 5453 gap probes (49.5%) contain a SNP from our SEED GWAS dataset, and thus direct evidence for SNP influence. Of the remaining gap probes, 3746 (34.0%) have an (dbSNP13) annotated SNP, in-del, microsatellite, or multi-nucleotide polymorphism or map to a University of California, Santa Cruz (UCSC)-annotated repeat (and thus have indirect evidence for SNP/variant influence), and 1808 gap probes (16.4%) did not contain a SNP from our SEED GWAS dataset and also were not annotated as containing a variant from the dbSNP138 database or a UCSC repeat. Given the large proportion of SNP-associated gap probes, we first sought to examine the role of SNPs in producing gap signals. Our approach to understanding the role of SNPs in producing gap signals consisted of two main elements. First, we theoretically conceptualized how various types of SNPs located at different locations in the probe, including the measured C and corresponding G loci, the SBE site, as well as elsewhere in the probe would affect 450k signal based on our knowledge of the measurement chemistry. Second, we performed empirical analyses using our joint GWAS and 450k DNAm data from SEED. We also examined the remaining ~16% of gap probes that do not have an associated SNP or variant, according to the SEED GWAS data and reference annotations.Based on the underlying 450k probe chemistry, we predicted how SNPs influence 450k signal. Our predictions are summarized in Fig. Next we predicted the influence of nucleotide changes for SNPs that overlap the G nucleotide of the measured CpG site on signal detection Fig. . For TypFinally, we predict the influence of SNPs that overlap the SBE site on signal. For Type II probes, the SBE site overlaps with the interrogated C site; therefore, the influence of SNPs is the same as for C site SNPs, as described above and shown in Fig. SNPs can also occur elsewhere in the probe length; however, it is less straightforward to develop theoretical rules or principles guiding how these may affect probe signal. Similarly, it is unclear how probes with multiple SNPs may behave with respect to methylation signal. Therefore, we do not provide a theoretical framework for these types of probes, but instead provide the results from our empirical analyses below.n = 5129)   that have a T/G SNP at the SBE site, where G is the a priori defined base at the SBE according to the genome reference sequence . We limited our analysis to probes that contained a single SNP to determine the relationship between SNP distance to the interrogated C site and gap signal. The probes were binned by SNP distance to the interrogated C site, and samples were grouped by genotype: homozygous for the reference allele, heterozygous, and minor allele homozygous. When we plotted the signal intensities, for both the methylated and unmethylated channels, which represent the mean intensity for all probes with a SNP at that particular distance to C site, we observed differences in mean signal intensities, and inter-quartile ranges (25th–75th percentiles), between heterozygotes and homozygotes that were consistent with allelic dosage  non-gaps with measured or imputed SEED SNPs, (2) non-gaps with annotated variants or repeat elements, (3) non-gaps with no associated variants, (4) gaps with measured or imputed SEED SNPs, (5) gaps with annotated variants or repeat elements, and (6) gaps with no associated variants. As shown in Fig. p value, and 210 were found to discriminate blood cell types [We were interested in quantifying the degree to which other factors, aside from a measured/annotated SNP or annotated repeat element, could lead to gap-like behavior. For example, it is possible that some of these 2-cluster gap signals ambiguously mapped to sex chromosomes and clustered according to sex; however, we only observed sex-specific clusters in 6 (0.3%) of these probes. 161 of these 1808 probes in total were previously defined as ambiguously mapping , 14 werep values of 0.73. We were then interested in examining the performance of these methods at specific scenarios  as well as having 3 discrete clusters, consistent with our gap hunting approach.We assessed the potential for other methods to identify clustered DNAm distributions with respect to detection sensitivity and the specific types of sample clusters they identify. We tested these methods against a set of 5000 probes made up of gap signals (which functioned as positive controls) and 5000 probes which were not gap signals, had no measured, imputed, or annotated SNP, and had very low variability (and thus functioned as negative controls). First, using the beta values for these probes, we applied a Gaussian mixture model clustering algorithm, which selects the optimal number of clusters based on the Bayesian information criterion (BIC), and found that it had 100% sensitivity, but only 50% specificity, to distinguish between the gap and non-gap probes. Additionally, in cases where the mixture model predicted a gap signal to (correctly) have more than 1 cluster, it was only able to identify the correct number of clusters 43% of the time. We also examined the utility of the dip test, in which the null hypothesis is that the data are unimodal , and fouAfter gaining an understanding of gap signal properties, we were interested in highlighting the potential utility of gap hunting in EWA studies. A recent paper by Barfield et al.  demonstrOne approach used in EWA studies to address multiple testing burden is to subset the dataset to only those probes that are variably methylated. We sought to define the proportion of probes in the post-variably methylated filtering dataset that had gap signals identified using gap hunting. As expected, due to our gap signals having inherently high variability, we observed gap signal probe enrichment in the filtered dataset as we increased our standard deviation threshold for filtering  for infant arousal. Of these significant probes, 5 were gap signals  can likely be attributed to an underlying SNP(s) or other variant in the C site, G site, SBE site, or elsewhere in the probe length. We document the specific mechanisms by which SNPs at the C, G, and SBE sites lead to gap signals, which involve a consideration of the type of nucleotide change occurring, as well as the probe type, DNA strand of interrogation, and overall methylation state, and demonstrated that expected effects are met using paired genotype and 450k data on the same samples. We additionally demonstrate that distance between a probe SNP and the C site of the probe is a relevant factor influencing methylation distributions. Finally, we delimit the situations in which a SNP-affected probe does not produce a gap signal, highlight a subset of gap signals that cannot be attributed to an underlying SNP, and discuss various utilities of gap signal identification in an EWAS framework. We recommend using gap hunting to “flag” probes for special consideration when interpreting EWAS findings, rather than the common practice of removing probes annotated with SNPs (using reference annotations) prior to EWAS.identify these probes per se. In light of this conceptual framework, we selected a “threshold” argument of 0.05 and a “outCutoff” argument of 0.01 for our analyses because it balanced detecting too many probes versus too few probes for downstream characterization and provided good face validity to capture the distributions of interest when we visually observed the data.The focus of this manuscript was to characterize sources of clustered distributions and highlight implications of their removal or inclusion in an EWAS framework as opposed to developing an optimized statistical method to We recognize that changes to these parameters may influence the total number of gaps detected, indeed for our SEED dataset, the number of gap signals varied with different combinations of these parameters  are limited. For example probes of this nature, we applied Gaussian mixture models with BIC and dip statistics, but could not consistently identify them as multi-modal and could not estimate the proper number of clusters. These methods show similar (limited) performance on both the beta value and M-value scales; the advantage of using beta values in gap hunting, however, is that the “threshold” argument can be more easily informed by biological intuition. These alternative approaches could potentially be used to classify probes as having 1 versus more than 1 cluster, but this would result in many false positives, likely including many probes unaffected by SNPs, as described by Daca-Roszak et al. This is an unnecessary price to pay, considering that probes of these sorts of distributions do not seem to be prevalent to such a large degree, at least in SEED Fig. . To partOur work also emphasizes the utility of visual inspection of methylation distributions when considering the potential influence of SNPs on probe signals. Methylated and unmethylated signal intensities can be inspected with consideration of expected SNP effects  of children on whom the DNA methylation was measured (see “The Study to Explore Early Development (SEED) is a multi-site case–control study of 3–5-year-old children with autism spectrum disorder (ASD), conducted in localities within 6 US states. Approximately 2600 families participated in SEED phase I, and the children were classified into three groups according to neurodevelopmental outcomes, as previously described . SEED pared see “” sectionminfi R package for 450k Array data [minfi function “getSex” was discordant with self-reported sex. Cell composition estimates were obtained via the “estimateCellCounts” function, also in the minfi package. Additional samples with outlying cell composition estimates were removed. We did not perform any probe-level QC on these data, such as detection p value or ambiguously mapping probe filtering [minfi. These processing steps resulted in 607 samples for use in the downstream analysis. Beta values  + 100) were calculated and implemented in downstream analyses.Genomic DNA (gDNA) was isolated from whole blood samples using the QIAsymphony midi kit (Qiagen). For each of 610 gDNA SEED samples, 500 ng of DNA was bisulfite-treated using the 96-well EZ DNA methylation kit (Zymo Research). Samples were then processed on the 450k Array, randomized across and within plates to minimize potential confounding effects introduced by batch. Illumina.idat files generated from the array were processed using the ray data . Standariltering , becauseOf the 607 samples in our DNAm dataset, 590 had whole-genome genotyping data available, which was measured using the Illumina HumanOmni1-Quad BeadChip. Standard QC measures were applied: removing samples with less than 95% SNP call rate, sex discrepancies, relatedness (Pi-hat > 0.2), or excess hetero- or homozygosity. Markers with less than a 98.5% call rate, or are monomorphic were removed. Phasing was performed using SHAPEIT  followedgaphunter, that can be implemented using the minfi package [We identified gap signals using a function we developed, called  package .Order beta values sequentiallyCalculate consecutive pairwise differences for these values.Determine the number of difference values calculated in 2) that are greater than the “threshold” argument, defined herein as gaps. If one or more gaps exist, classify probe as gap signal and define the number of groups as the number of gaps plus one. If zero gaps exist, define probe as non-gap signal.For all gap signals, use location of gaps to classify individuals into distinct groups. For all probes defined as gap signals, sum the number of samples in all groups except that of the largest count. Define “outlier-driven gap signals” as those in which this sum does not exceed the user-defined “outCutoff” parameter, which is the proportion of the total sample size . Remove these outlier-driven gap signals from output.A matrix of beta values  is input to or calculated by the function. The method incorporates two user-defined arguments: “threshold,” and “outCutoff.” For each row in this matrix:We report the number of gap signals detected at all possible combinations of a series of threshold  and outCutoff  values. We chose to complete all subsequent analyses with results from setting the threshold argument to 0.05 and the outCutoff argument to 0.01. We also elected to implement this method on the Beta value scale because this allows for the threshold argument to be informed by biological intuition. In the case where a user only has M-values available, a simple transformation to the Beta scale can be performed .We developed an annotation of all polymorphisms that mapped to probes on the 450k Array in order to have available flexible information on the site(s) to which a polymorphism mapped  and on the polymorphisms themselves . The Database for Single Nucleotide Polymorphisms version 138 (dbSNP138) was downloaded from the UCSC Genome Browser . All claWe were interested in analyzing the impact of specific SNPs  at specific locations in the probe  through joint analysis of our SEED genotype and 450k data. We again used the “findOverlaps” function in the “GenomicRanges” R package to find which of our measured SNPs overlapped to the C, G, SBE and probe length sites of all 450k probes. We performed these overlaps separately for all 4 probe locations and then pooled the overlap results together. We then removed probes that had more than 1 type of SNP mapping to them. For example, if in our overlap results we found that a probe had a mapping C site SNP and a probe-length mapping SNP, that probe was not considered in these analyses.Once we defined a “clean” set of probes with respect to the location at which they overlapped a SNP, we grouped together probes of similar relevant characteristics. For the C and G site SNP analyses, we grouped probes based on if they had the same: nucleotide change , probe design (Type I or Type II), SNP mapping location (C site or G site), and strand on which the CpG of interest is designed to be interrogated. All probe level information was found via the Illumina 450k manifest. For the SBE site SNP analyses, we grouped probes based on all of these criteria as well as the reference nucleotide of the SNP. This step was necessary in order to more easily understand in which genotypes to expect a loss of signal. Our groupings were done for all of the scenarios delimited in Figs. minfi function “getMeth” and “getUnmeth,” which we performed on an un-normalized (output of minfi function “preprocessRaw”) R object. The copy number metric was defined as:i” refers to each individual and “Meth” and “Unmeth” to the methylated and unmethylated signals, respectively. At each probe, the intensities are scaled by the mean values of the reference genotype of the SNP affecting that probe. This copy number metric therefore serves as way to jointly consider methylated and unmethylated signal and more explicitly evaluate the difference between genotypes in terms of overall signal.For each of these scenarios, we collected 4 metrics across all probes that fell into that scenario, grouping samples by their genotypes at each probe. The 4 metrics were: percent methylation, methylated signal, unmethylated signal, and a copy number metric. The methylated and unmethylated signals were derived from the We were interested in evaluating the effect of distance from the C site to the SNP for situations in which a SNP was located in the probe but outside of the interrogated CpG and SBE positions. We first performed a similar overlap evaluation and filtering process as described above. Once we were limited to probes that had only overlapped measured probe-length mapping SNPs, we further filtered to probes that only had a single SNP in the probe length. This step was done in order to control for the potential effect of total amount of the probe length affected by SNPs. Next we grouped probes into bins of equal distance from the C site to the SNP, which was from 1 to 50 base pairs for the Type II design and 1 to 49 base pairs for the Type I design. At each probe, we identified the reference homozygote, heterozygote, and non-reference homozygote genotypes and group samples accordingly. We performed this grouping across probes within a specified distance value. Next we plotted the means and inter-quartile ranges  of the methylated and unmethylated signals as a function of distance, separately for the three genotype groups. The greater the discordance between the means and IQRs of the three groups indicated a greater effect of the mapping SNP.We were interested in comparing the overall standard deviation distributions of non-gap and gap signals. Moreover, we were further interested in within-group differences relating to probe having an underlying SNP (measured or annotated) or not. For both non-gap and gap signals, we first identified probes that had at least one measured SNP anywhere in the probe body . From the remaining probes in each group, we identified which probes that overlapped at least one polymorphism in the dbSNP138 annotation described above, or a repeat element as defined by UCSC . Again, p value, or were previously determined to be cell-type distinguishing probes in whole blood. To define ambiguously mapping probes we used a previously defined list [p value of greater than 0.01, as determined via the detectionP function in the minfi package [p value <1E − 8 reported from a previous study investigating differential methylation according to blood cell types [We were also interested in quantifying the role of additional factors in producing gap-like behavior in the gap signals that did not have a measured/imputed SNP or a mapping annotated variant via dbSNP138 or a mapping repeat element. We determined the proportion of these probes that were known to be ambiguously mapping, were determined to fail via detection ned list . We defi package . We defill types .mclust R package [diptest R package [p value for each of the 10,000 probes and calculated the area under a receiver operating characteristic curve using the “auc” function in the MESS R package [p value thresholds against the “true” status of the 10,000 probes. We performed the mixture model and dip test experiments on both beta values and M-values  to examine if the performances of these methods were affected by the scale of the methylation values.We sought to investigate whether other methods that specifically identify multimodal distributions could overcome gap hunting’s insensitivity to distributions that appeared multimodal but did not cluster into discrete groups, but still retain the ability to identify methylation distributions that did have discrete groups. One key complication to this question is the fact that the “true” status of methylation distributions at every measured probe is not known, which hampers the ability to assess the classification properties of alternative methods. To overcome this problem, we constructed a subset of 10,000 probes in which half were classified as gap signals by gap hunting (and were thus positive controls) and the other half were non-gap signals, did not have a measured/imputed or annotated SNP, and whose standard deviation was in the lowest decile of standard deviations across all autosomal probes (and were thus negative controls). In this way, we could maximize our understanding of the true status of the probes we were testing as having a clustered distribution or not. The first method we tested was a Gaussian mixture model implemented via the “Mclust” function in the  package . We allo package ; we impl package . We reco package  and geneUpon confirming that gap signals were largely due to underlying SNPs, we were interested in exploring their potential to correct for population stratification. We calculated principal components (PCs) from gap signals and compared them to eigenvectors derived from GWAS, via the EIGENSTRAT method , and PCsWe were interested in exploring gap signals in the context of a typical step in the EWAS pipeline to filter out probes that are of low variability. We calculated the standard deviation of all 473,864 autosomal probes and calculated the percentages of gap and non-gap signals in the remaining probe set after imposing various standard deviation filters. Our cutoffs were ranged from the 5th to the 99th percentile of standard deviation across all probes.We sought to demonstrate the potential for gap signals to serve as a surrogate for the local genetic sequence, on a haplotype scale. We phased our genotype data using the SHAPEIT software . After dN = 4), and samples with a detection p value greater than 0.01 in more than 1% of probes (N = 7). We removed probes at which more than 10% of samples had a detection p value greater than 0.01 (n = 1959), and if they were previously identified as being ambiguously mapping  [gaphunter, and used the limma R package [p value < 1E − 4) hits, the number of these flagged as gap signals, and the number that would have been removed via SNP annotation, using a dbSNP137 annotation included in the minfi package.We sought to illustrate the utility of incorporating gap hunting into a typical EWAS pipeline. We downloaded 450k data from a previous study examining placenta methylation and newborn neurobehavioral outcomes in 335 samples  from the 29,233) . The res 29,233) , and per 29,233) , in the  package  to perfo


Tumour budding and podia formation are well-appreciated in surgical pathology as an aggressive invasion phenotype of colorectal carcinoma cells that is attained in the microenvironment of the invasive margin. In this study, we addressed how tumour budding and podia formation feature in xenografts. Primary colorectal carcinomas (N = 44) of various molecular types  were transplanted subcutaneously into T and B cell-deficient NSG mice, making possible immunohistochemistry with routine surgical pathology antibodies. Tumor budding and podia formation were both appreciably present in the xenografts. Quantitative evaluations of cytokeratin immunostains of primaries and their corresponding xenografts showed a reduction of tumour buds in the xenografts. Furthermore, in xenografts tumour cells were completely negative by pSTAT3 immunohistochemistry, indicating absence of cytokine/chemokine signalling, but nuclear β-catenin and SMAD4 immunostainings as read-out of wnt and BMP pathway activation, respectively, were maintained. Carcinoma cells in most xenografts retained immunostaining of at least some nuclei by immunohistochemistry with antibodies against pERK1/2. K-ras/B-raf mutational status did not correlate with tumour budding or podia formation in the xenografts. Our results indicate that tumour budding and podia formation can be modelled by xenografting, and in NSG mice it can be studied with the same immunohistochemical methods as used for primaries in surgical pathology. Dysregulation of wnt and BMP signalling appears to be transferred into the xenograft microenvironment, but not cytokine/chemokine signalling. For colorectal carcinoma, different phenotypes of invasion have been described, namely the expansive ormation . They arormation . This feormation , will noFor colorectal carcinoma, dysregulation/activation of wnt-signalling, and of the bone morphogenetic pathway (BMP) are known to be cardinal signalling transduction pathways compromised by genomic aberrations. These signalling pathways are amenable to morphologic study of tumour specimens by β-catenin or SMAD4 immunohistochemistry, respectively: as downstream-effectors in the signalling cascades, when activated, these proteins can be found in the nuclei of the tumour cells . SimilarTraditionally, morphologic study of colorectal carcinoma invasion is based on tissues from surgical resections. The above mentioned phenotypes of invasion have been delineated in the last decade by this type of study. However, in order to gain insight into cellular mechanisms of colorectal carcinoma invasion, studies of model systems that allow observation of tumours in a different microenvironment could be valuable. To this end, xenografts have several advantages: contrary to cell culture, xenografting is a procedure with fairly high success rates , thus alHere we report on a series of colorectal carcinomas and their xenografts in NSG mice that comprise all the major molecular subtypes. We addressed, how tumour budding and podia formation in xenografts compared to its primaries, how immunohistochemical studies would work in this setting, and tested if the expression of downstream-effectors of the cardinal potentially dysregulated signal transduction pathways would be altered in the xenograft microenvironment.3 or if 30 weeks after xenografting had elapsed. According to animal welfare regulations euthanization would have been performed if there had occurred tumour ulceration, motoric impairment, rectal bleeding or diarrhoea, or loss of >15% of animals' initial body weight, but this was not observed in this series. Times from implantations to harvesting the xenografts ranged from 4 to 30 weeks. Euthanasia was done using CO2 at an air-exchange rate of 20–25% per minute until breathing stopped, followed by cervical dislocation. One half of each xenograft was immersion fixed in buffered formalin (10%) immediately upon removal and processed for histology by routine methods.Resection specimens were received fresh from the operating theatre in the Institute of Pathology. Prior written informed consent was obtained from all patients, and all procedures were approved by the Ethics Committee of Rostock University (ref. II HV 43/2004). Small cubes (ca. 3 x 3 x 3 mm) were taken from the invasive margins for subcutaneous xenografting into NSG mice, either immediately, or after cryopreservation . All in The remaining surgical resection specimen was fixed overnight in buffered formalin (10%) and processed for a surgical pathology report. For each case, one of the archived paraffin-blocks of the primary tumour that included representative regions of the invasive margin was selected for this study.A total of 44 tumours were included in this study. Tumours were classified according to molecular features as sporadic standard type , sporadic microsatellite-unstable , CpG island methylator phenotype , or of the Hereditary Non-Polyposis Carcinoma Coli (HNPCC) Syndrome type (N = 5). Detailed information on the molecular studies can be found in .Primary tumours and xenografts were studied by immunohistochemistry with antibodies against cytokeratin (CK) 18, β-catenin, SMAD4, pSTAT3, and the dually phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) using an autostainer  with DAB as chromogen (details in http://rsb.info.nih.gov/nih-image) on digital microphotos of CK18 immunostains. Areas with maximum tumour budding or podia formation ("hot spots") at the invasive margin of the primaries and the xenografts were selected at scanning magnification and microphotographs covering an area of 0.292 mm2 (20x objective) were taken. Tumour buds were defined as CK18 positive cancer cells that, singly or in aggregates of up to four cells, were found discontinuous from the bulk of the tumours. Podia were recorded as a-nucleate CK18 positive globules or “wisps”with diameters of 0.5–5 μm.Counting of tumour buds and podia was done with the imageJ cell counter tool . Because the distributions of tumour budding and podia data were skewed , comparisons of between groups were done by the Mann-Whitney test.All immunohistochemical reactions on the paraffin-embedded xenograft tissues were straightforward to perform with the same primary antibodies and secondary reagents used in routine immunohistochemistry of human tumour tissues, producing crisp immunostains without background. Examples of xenograft immunohistochemistry are shown along with the results detailed below.Counting tumour buds and podia on CK18 immunostains see . The difOn a case-by-case basis, reduction of tumour buds or podia was not seen universally: in 10 and 16 of the 44 cases, tumour budding or podia, respectively, were more frequent in the xenografts than in the primaries .In order to explore if tumour cells were subject to cytokine and/or chemokine signalling, we performed pSTAT3 immunohistochemistry. In the xenografts, we observed complete absence of immunostaining in all tumour cells in all cases. Because the pSTAT3 antibody cross-reacts with murine pSTAT3, immunopositive stromal cells were found in all cases, thus providing a convenient internal control for the immunoreactions  was not observed. However, pERK1/2 immunohistochemistry worked for the xenografts. Nuclear immunostaining of stromal cells was seen in all xenografts, and tumour cell nuclei were positive in 36 of the cases . However, instead of an “intrinsic” dysregulation there also may be “extrinsic” pathway activation, presumably by receptor activation within the tumour microenvironment. Weather intrinsic or extrinsic, in all cases pathway activation entails nuclear translocation of β-catenin, SMAD4, or pERK1/2, which in primaries has been assayed successfully by immunohistochemistry as a “read-out” for activation in previous studies , 7. SincWe observed that the patterns of β-catenin and SMAD4 immunohistochemistry in xenografts mirrored the situation in the corresponding primaries (Tab 2). By conclusion, not only dysregulation of these pathways by intrinsic activation is transferred into the xenograft, which would be expected, but the xenograft microenvironment also emulates activation of these pathways by extrinsic factors.vide infra for discussion of this technical issue) did not permit confident evaluations to make primary-xenografts comparisons. pERK1/2 immunohistochemistry of xenografts, however, showed that nuclear immunostaining of stromal cells as internal controls was positive in all case, and tumour cells were positive in the majority of cases (81.3%). Published studies of pERK1/2 immunohistochemistry that used colorectal carcinoma biopsies  have shown that, regardless of K-Ras or B-Raf mutation, tumour cell nuclei are clearly immunopositive in 72.2% [Unfortunately, technical limitations of the pERK1/2 immunostains of the primaries  as well as stromal cells (black arrows). Image is from HROC183.(TIF)Click here for additional data file.


Herein, a label‐free multiplex photoluminescent silicon nanoprobe (PLSN‐probe) is introduced as a potential substitute for quantum dots (QDs) in bioimaging. An inherently non‐photoluminescent silicon substrate is altered to create the PLSN‐probe, to overcome the major drawbacks of presently available QDs. Additionally, crystallinity alterations of the multiplane crystalline PLSN‐probes lead to broad absorption and multiplex fluorescence emissions, which are attributed to the simultaneous existence of multiple crystal planes. The PLSN‐probe not only demonstrates unique optical properties that can be exploited for bioimaging but also exhibits cell‐selective uptake that allows the differentiation and diagnosis of HeLa and fibroblast cells. Moreover, multiplex emissions of the PLSN‐probe illuminate different organelles such as the nucleus, nucleolemma, and cytoskeleton, depending on size‐based preferential uptake by the cell organs. This in vitro study reveals that cancerous HeLa cells have a higher propensity for taking up the PLSN‐probe compared to fibroblast cells, allowing the diagnosis of cancerous HeLa cells. Additionally, the fluorescence intensity per unit area of the cell is found to be a reliable means for distinguishing between dead and healthy cells. It is anticipated that the multifunctionality of the PLSN‐probes will lead to better insight into the use of such probes for bioimaging and diagnosis applications. However, the applicability of QDs for live cell imaging has been severely restricted because of cytotoxicity problems, blinking, particle size dependency, and reliance on bioconjugation for delivery. Hence, methods for labeling as well as biocompatible coatings are essential for QDs.A wide variety of quantum‐scale materials have been studied for diverse bioimaging usage; among them, semiconductor quantum dots (silicon‐based QDs), noble nanoparticles (gold), super‐paramagnetic oxides (iron oxide), and carbon materials Single‐walled carbon nanotubes (SWCNT) have been extensively exploited for optical bioimaging purposes.The surface chemistry of Si‐based QDs (Si‐QDs) is of great interest because of their considerable difference from heavy metal QDs. In addition, the utility of Si‐QDs in bioimaging lies in their optical properties, which are size‐ and crystallinity‐dependent. Si‐QDs have shown to be a substantial breakthrough as a bioimaging agent due to their nontoxicity and biodegradability; one byproduct of degradable Si‐QDs, silicic acid, can even be readily excreted via the urine.Moreover, QDs are highly sensitive to surface defects, which can reduce the quantum yield of QDs by affecting the recombination of electrons and holes, resulting in blinking of the QDs.Here, we report the first development of multiplex emissions from photoluminescent silicon nanoprobes (PLSN‐probes). The transition from a single (polyhedron) to multiplanar  silicon crystal structure yields a higher absorption and hence a superior FL throughput. Unlike QDs, the PLSN‐probes not only exhibit superior optical properties including higher brightness and narrower emission and broader absorption spectra, but also cell‐selective uptake, which eliminates the need for coating and bioconjugation. This unique nanoprobe emits a higher FL intensity when there is a greater number of crystal planes , and it is hypothesized that the multicrystal orientation is the cause of the multiplex emission of the PLSN‐probes, which has never been observed with QDs.The use of ultrashort pulsed laser (USPL)‐assisted ionization to synthesize PLSN‐probes under inert atmospheric conditions resulted in impurity‐free and well‐characterized 3D structures. The PLSN‐probes were created based on kinetic self‐assembly activated by multiphoton ionization. By using USPL, we were able to systematically transform a nonluminescent bulk single‐crystal silicon wafer into multiplex photoluminescent nanoprobes with tunability via synthesized crystal orientations.22.1The PLSN‐probes were synthesized via USPL‐assisted ionization. The absorption of one or more photons by electrons in atoms of the substrate  causes spatial electron separation of the excited atom or ion species. To prevent any compositional changes, this process was carried out in the presence of an inert gas. The USPL has shown a capability of overcoming the ionization threshold of the silicon substrate by inducing sufficient photon energy. The accumulated energy of multiphoton absorption exceeds the ionization potential of the silicon and hence leads to atomic ionization. Therefore, the kinetics of the self‐assembly process were initiated by multi‐photoionization. The experiment showed that multi‐photoionization can be governed by precisely tuning the laser fluence. When a sufficiently higher laser fluence interacts with the substrate, it results in the formation of a 3D self‐assembled structure consisting of multiplanar crystal Si . Likewise, a lower laser fluence resulted in fewer crystal planes (polyhedron). The synthesis of the PLSN‐probes can be precisely tuned by regulating the laser parameters. While the laser pulse duration and laser power were kept constant at 214 fs and 16 W, respectively, high, intermediate, and low laser fluences were applied at a 4, 12, and 26 MHz repetition rate to synthesize polyhedron, intermediate, and polyhedral PLSN‐probes. Based on the polycrystallinity of the synthesized probe, they categorized in polyhedron, intermediate, and polyhedral PLSN‐probes.FigureTransmission electron microscopy (TEM) analysis Figure1A was −1, which is the characteristic of crystalline silicon, has been identified in all the synthesized PLSN‐probes. All of the evidences demonstrate the successful formation of pure 3D structured PLSN‐probes with varied crystal planes at the same size distribution range.Additionally, X‐ray diffraction (XRD) analysis Figure D further2.1.1FigureFigureThe PLSN‐probes were first investigated in terms of ultraviolet–visible–near‐infrared (UV–vis–NIR) reflection and absorption at room temperature, as shown in TableThe results imply that by varying the excitation wavelength, different FL emissions ranging from 500 to 800 nm can be achieved. Furthermore, for excitation at a longer wavelength, a redshift emission at 650 nm can be interpreted from the excitation and emission profile. Since the FL domain of the PLSN‐probes is wavelength‐dependent, the presence of a diverse size distribution was neutralized by narrowing the excitation wavelength. This phenomenon also revealed that size variations in the PLSN‐probes may allow one to precisely target different organs of the cell, which can then be distinguished at distinct excitation wavelengths. Figure The acquired result from excitation and emission spectra fits well with observations from confocal FL microscopy. The pronounced variational excitation features evince that the PLSN‐probes are indeed multiplex emissive and wavelength‐dependent. These attributes may be useful in exploring the implementation of polyhedral PLSN‐probes for bioimaging.FigureElemental mapping of HeLa and fibroblast in presence of polyhedral PLSN‐probes, as shown in 2.2FigureWe investigated the possible use of PLSN‐probes for optical imaging of cancerous HeLa and mammalian fibroblast cells. Confocal FL microscopy of the HeLa and fibroblast cells at four excitation wavelengths, 405, 488, 555, and 633 nm Figure5A,B, rThe self‐internalization of the PLSN‐probes is impelled by adsorption of proteins upon dispersion in the cell culture medium.The purple emission at the 405 nm excitation wavelength of the PLSN‐probes was primarily observed from the nuclear region, while the blue and green emissions, excited at 488 and 555 nm, correspond to the nucleolemma and cytoskeleton of the cell. The red emission displayed PLSN‐probes that had not been taken up and, therefore, showed only the periphery of cells. In this case, however, this phenomenon was found to be restricted to the HeLa cells. Figure FigureFigureTo determine the application prospects of these novel PLSN‐probes as efficient bioimaging tools, they were compared to two commonly used dyes, 4′,6‐diamidino‐2‐phenylindole (DAPI) and fluorescein isothiocyanate (FITC) (control), in the absence of the PLSN‐probes, as shown in FigureThe varied size distribution of the PLSN‐probes not only makes them wavelength‐dependent but also results in a bifunctionality by which different organs of the cell can be differentiated Figure8A,B. AAside from using the PLSN‐probes for bioimaging purposes, the unique label‐free cellular uptake attributed of these probes has demonstrated their potential use for diagnosing cell health. The diagnostic application of PLSN‐probes relies on the concentration of internalized PLSN‐probes in the cytoplasm, by which the intensity of the emitted light drastically increases. The overall image Figure E shows tFigureAs numerous studies have proposed, the current QDs used as contrast agents suffer from weak dispersion in aqueous base solutions such as cell culture medium, and hence, effortless dispersion of a contrast agent in cell culture medium is ideal for bioimaging applications.In addition, the fluorescence stability of the PLSN‐probes was examined at variant pH and temperatures. However, the bioimaging including in vivo and in vitro was carried out at 37 °C. The influence of pH alteration on FL stability of PLSN‐probes were inspected at pH values ranging from , after 6 h, the FL were measured (under a 100 mW 488 and 555 nm continuous wave (CW) excitation wavelengths).3Here, we presented a novel approach using polyhedral PLSN‐probes with a broad range of absorption and long Stokes shifts that exhibited multiplex emissions. This label‐free method has shown a precise capability of cell uptake that strongly improves upon the lengthy labeling process of other methods. However, this attribute was found to be cell‐selective, i.e., the HeLa cells demonstrated significantly extended internalization, whereas the fibroblast cells showed a weaker tendency to uptake the PLSN‐probes. The multicrystallinity of the polyhedral structure allows higher absorption for the PLSN‐probe, and hence, a greater FL emission was observed. The PLSN‐probes demonstrated excellent dispersion in aqueous cell culture medium and systematic division into smaller parts that internalize into different organs of the cell, including the nucleus, nucleolemma, and cytoskeleton by which 3D visualization of intercellular organs of the cell is made possible. Moreover, we found that the FL intensity of the PLSN‐probes per cell area increases upon shrinkage of the cytoskeleton of the cell. Hence, this characteristic allows one to conclusively differentiate healthy cells from dead cells. In addition, the proposed probes can be used to diagnose HeLa cells as well as to screen dead cells. Further investigations are required to study the interaction of different cell lines with these PLSN‐probes.4−1, which are characteristics of crystalline PLSN‐probes. UV–vis–NIR absorption and reflection spectra were obtained with a Hitachi UV‐3100 UV–vis–NIR spectrophotometer. PL spectra were recorded on a Shimadzu RF‐5301PC spectrofluorophotometer at room temperature.To investigate the morphology and dimensions of the synthesized PLSN‐probes, a JEOL JSM‐4800 field emission scanning electron microscope and a JEOL JEM 2100 HR‐TEM were used. HR‐TEM was performed to determine the size distribution and morphology of the PLSN‐probes; for this method, a droplet of suspended PLSN‐probes was placed on a copper grid for direct observation after drying. Image analysis software was used to analyze the HR‐TEM images, perform size measurements, and compile a distribution histogram. An HR‐TEM diffraction pattern (FFT) was also obtained to confirm the formation of crystalline PLSN‐probes. Energy‐dispersive X‐ray spectroscopy (EDX) analyses using Oxford Instruments further corroborated the formation of pure PLSN‐probes and the elemental mapping evidenced the self‐internalization of the synthesized PLSN‐probes into the cells. The Raman spectra were recorded with a Bruker SENTERRA dispersive Raman microscope using a wavelength of 325 nm. The Raman spectra showed a strong band and sharp peaks at 521 cmSynthesis of PLSN‐Probes: A single‐crystal N‐type silicon wafer with a (100) crystallographic was sliced into 2 cm2 square‐shaped samples, which were ultrasonically cleaned in a 50 °C acetone bath for 15 min, followed by rinsing and drying. To synthesize the PLSN‐probes, silicon substrates were exposed to a diode‐pumped Yb‐doped femtosecond laser beam . This laser system is capable of producing central wavelengths of 1040 nm with a pulse width that can be varied, ranging from 214 to 1428 fs, as well as a tunable laser pulse repetition rate that can vary between 4 MHz (low rep. rate) and 26 MHz (high rep. rate). A maximum average operating power of 16 W was attainable. Utilizing the ultrashort (femtosecond range) pulsed laser is advantageous since the absorption of multiphoton excitation energy delivers the required energy of ionization and also, given the localized laser beam spot within the focal point, results in precise control over the synthesized PLSN‐probes. The samples were, therefore, irradiated at the beam focal point, with a computerized galvano scanner precisely implementing the synthesis processes. The self‐assembled PLSN‐probes were synthesized by single‐step femtosecond laser processing of the silicon substrate. For the purpose of this study, the polyhedron, intermediate, and polyhedral PLSN‐probes were fabricated with a pulse width of 214 fs and repetition rates of 4, 12, and 26 MHz, respectively. The laser fluences at 4, 12, and 26 MHz were measured as 1.57, 3.39, and 10.18 J cm−2, respectively  To ensure reproducibility of the synthesized PLSN‐probes, the synthesis method was performed three times at the given conditions. Figures S1–S3 (Supporting Information) further illustrate average particle size distributions and HR‐TEM images of the polyhedral and polyhedron PLSN‐probes.Cell Culture and Seeding: The synthesized PLSN‐probes were sterilized under UV light for 20 min prior to their addition to cell culture medium. Fibroblasts (NIH3T3) and human cervical cancer cells  were employed in cell experiments to ascertain the comparative functionality of mammalian and cancer cell lines in response to the samples. Fibroblast cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat‐activated fetal bovine serum with 1% penicillin–streptomycin antibiotics (Pen‐strep). HeLa cells were grown in DMEM‐F12 supplemented with 10% fetal bovine serum and 1% Pen‐strep. Subsequently, the cells were separately cultured on substrates placed in Petri dishes with a seeding density of 750 000 cells cm−2. The Petri dishes were placed in an incubator for 24 and 48 h at 37 °C. Following the PLSN‐probe cell response study, the cells were studied via scanning electron microscope (SEM) and confocal FL microscopy.Cell Imaging: The morphology of the cells was observed using a SEM . For this aim, after the prescribed time period, the spent medium was aspirated. The samples were then fixed in 2% glutaraldehyde in (0.1 m) pH 7.3 sodium cacodylate buffer for 1 h. Next, the samples were dehydrated at increasing concentrations of alcohol for 20 min, followed by immersion in (0.1 m) sodium cacodylate buffer with (0.2 m) pH 7.3 sucrose for 20 min. The samples were then critical point dried. After the experiment, the cells were prepared for direct observations via SEM.Fluorescence Microscopy: For FL microscopy, the samples were first fixed in methanol‐free paraformaldehyde followed by incubation in skim milk to prevent nonspecific binding. To stain the actin and cytoskeleton, the samples were incubated with Alexa Fluor 488 (Life Technologies) followed by DAPI (Life Technologies) to stain the nucleus. The samples were studied using a FL microscope . Seeded cells were also fluorescence‐imaged on a Zeiss LSM 710 META upright confocal laser‐scanning microscope using 60× magnification water‐dipping lenses for the monolayer cultures. Wavelengths of 405, 488, 555, and 633 nm were used. Image data acquisition and processing were performed using a Zeiss LSM Image Browser, Zeiss LSM Image Expert and ImageJ.Phosphorescence Lifetime and Intensity Measurement: Photoluminescence lifetime imaging was performed on a fluorescence microscope (Zeiss LSM 710 META) equipped with confocal laser scanning system. The synthesized PLSN‐probes were excited at 405, 488, 555, and 633 nm and detected through center wavelength of 447, 525, 617, and 635 nm and bandwidth of 60 nm, 45 nm, 36 nm, and longpass, respectively.Statistics: All experiments were performed in triplicate, and the data points were averaged unless otherwise indicated. The error bars show standard deviations.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Optic disc pit (ODP) is a rare congenital anomaly of the optic disc, which can be complicated by a maculopathy associated with progressive visual loss. Optic disc pits are usually unilateral and sporadic in occurrence, and the development of maculopathy is unpredictable with no known triggers. Optic disc pit maculopathy (ODP-M) is characterized by intraretinal and subretinal fluid at the macula, causing visual deterioration. The source of this fluid is still unclear, and several competing theories have suggested it may be vitreous fluid, cerebrospinal fluid, leakage from blood vessels at the base of the pit or leakage from the choroid. The mechanism of pathogenesis of ODP-M has not been fully elucidated, but vitreous liquefaction and traction and pressure gradients within the eye have been implicated to be involved. There are no clear guidelines on the management of patients with ODP-M, and numerous techniques have been described, including laser photocoagulation, intravitreal gas injection, macular buckling and pars plana vitrectomy with many different modifications. The majority of reports describe small series, and as there are no comparative studies there is no consensus regarding the optimal treatment for ODP-M. This review discusses the literature on the possible sources of fluid and mechanisms of pathogenesis in ODP-M, as well as the wide array of treatment modalities and their results. Based on these, a set of recommended key concepts for the timing and choice of treatment for these challenging are presented. Optic disc pits are considered as part of a spectrum of congenital cavitary anomalies of the optic disc, which also includes optic disc coloboma, morning glory and extrapapillary cavitation . HistoloODPs are rare, and occur equally in men and women with an estimated incidence of 1 in 11,000 people , 4. TheyODPs are usually seen as single, oval-shaped depressions at the optic disc. They are most commonly found at the inferotemporal aspect of the optic disc, but may also be found elsewhere, including centrally , 8. OccaAn ODP by itself is usually asymptomatic, and may be found incidentally. Nevertheless, the presence of this defect has been demonstrated to cause visual field defects, most commonly an enlarged blind spot and a paracentral arcuate scotoma , 9, 10. When ODP-M is present, visual acuity (VA) is usually reduced to 20/70 or worse . AlthougAlthough ODP-M is a well-known pathology for several decades, the nature and origin of the fluid that is found intra- and sub-retinally in these patients is still controversial , 19. SevOver the years, four different possible sources have been proposed for the fluid seen in the intraretinal and subretinal spaces in ODP-M.The first possible source of fluid is the vitreous. It was first suggested that vitreous fluid can enter the subretinal space through the formation of a macular hole , 20. ThiThe second possible source of fluid is the cerebrospinal fluid (CSF), which has been proposed to enter the intra- and sub-retinal spaces from the subarachnoid space through the ODP defect. Several optical coherence tomography (OCT) studies have shown direct communication exists between the subarachnoid space and the subretinal space –28. FurtThe third possible source of fluid is leakage from blood vessels at the ODP . This coThe fourth possible source of fluid is from the choroid, through the Bruch’s membrane and peripapillary atrophy . This soBeyond the controversy regarding the source of fluid in ODP-M, the mechanism of pathogenesis for this pathology is also unclear. ODPs are congenital, but the development of ODP-M has no known triggers, and can occur at any age, from early childhood to the ninth decade of age . SeveralAs previously mentioned, ODP-M typically occurs in the third and fourth decades of life , 12, whiAnother theory is that pressure gradients within the eye cause migration of fluid from the vitreous into the subretinal space. A normal eye is a closed system, without significant differences in pressure between various compartments. In an eye with an ODP, a pressure gradient can exist because the intracranial pressure is transmitted to the ODP via the CSF. Thus, when the intracranial pressure is low vitreous fluid is drawn into the ODP, and when it rises the fluid is pushed back into the eye, and can dissect under or within the retina , 47. ThiRegardless of the source of fluid and exact pathophysiologic mechanism of ODP-M, a sequence of retinal fluid accumulation and progression of its formation has been described by Lincoff et al. , which iTreatment for ODP-M is warranted as the majority of cases suffer gradual deterioration with significant visual loss –18. A muEarly reports described treating ODP-M with oral corticosteroids. This treatment was not effective, and resorbed fluid tended to recur following discontinuation of the corticosteroids , 50. TheLaser photocoagulation at the temporal disc margin has been proposed as a treatment for ODP-M, with the reasoning that the laser scars will create a chorioretinal adhesion which will act as a barrier between the ODP and the subretinal space . InitialIntravitreal gas injection has been proposed as a treatment option for ODP-M, with the reasoning that pneumatic displacement will cause reattachment of the macula and improve VA . This teAn alternative approach proposed for the treatment of ODP-M is macular buckling surgery. This procedure includes an implant that is fixed to the posterior aspect of the globe along the 6-to-12 o’clock meridian, creating a buckling effect under the macula . This teThe predominant approach for the treatment of ODP-M is pars plana vitrectomy (PPV). The majority of the published literature on ODP-M is focused on PPV techniques, which have evolved along with the advances made in surgical technology. Several anecdotal reports from the late 1980’s and early 1990’s described successful anatomical and visual restoration in patients with ODP-M who underwent PPV with or without endolaser to the temporal disc margin and gas tamponade –63. In aIt has been proposed that induction of complete PVD during surgery is essential for relief of traction required to achieve macular reattachment . This waOne of the early reports on PPV for ODP-M described internal submacular fluid drainage . AlthougAnother controversial aspect is the necessity to peel the internal limiting membrane (ILM) in patients with ODP-M. It has been suggested to be in important component of the surgical treatment of ODP-M , and a fSome authors have proposed that in addition to inducing PVD, the surgeon should look for any glial tissue overlying the ODP and carefully peel it off during surgery. This maneuver has been described infrequently, and the ODP-M had resolved . In one A recent series by Ooto et al.  reportedA summary of the major published series of PPV treatments for ODP-M is provided in Table Patients with ODPs that are diagnosed incidentally should be counseled on the risk for development of ODP-M. Although rates vary from 25 to 75% , 9, 11 aAlthough spontaneous resolution is possible in as many as 25% of cases and visual improvement is possible , 15, 93,The most commonly used procedure for the treatment of ODP-M is PPV, especially in recent years. Treatment with laser alone was not very successful , 50, 51,When PPV is performed, it is worthwhile to induce complete PVD to relieve any traction on the ODP –67. TriaAdditional elements of surgery, such as peripapillary laser, ILM peeling, subretinal drainage, peeling of glial tissue and sealing of the ODP are controversial. Both success and failure of surgical management have been reported with and without them, and available data is not enough to conclude that any of them are associated with better outcomes. Therefore, these should be performed at the discretion of the surgeon, following a discussion of their potential advantages and risks with the patient.Limited vitrectomy with intraretinal fenestration is a recently described technique that has promising results . Future The most important issue to discuss with ODP-M patients in whom surgery has been recommended is that the visual recovery is a slow and long process. All surgical techniques have been reported to achieve complete resolution of fluid and significant visual improvement, but these were documented after at least 3 months and generally only after 6–12 months from surgery –84, 90. The majority of reports describe successful outcomes that have been maintained long term, with relatively low rates of ODP-M recurrence and very few postoperative complications.Although ODPs are rare, it is likely to assume that every vitreoretinal surgeon will encounter several patients with ODP-M during his or her career. These cases are challenging to manage. Compared to other indications for vitreoretinal surgery, there is a paucity of literature on this unique pathology, the majority of which includes case reports and small series with no comparative studies. This makes patient counseling, expectation setting and decision making regarding the timing and choice of surgical intervention very difficult. There are no established guidelines for the treatment of ODP-M, and no consensus on the mechanism of pathogenesis or the optimal surgical technique. Additionally, many of the proposed surgical techniques are challenging to perform. Based on the literature review, we suggest the following key concepts for the timing and choice of treatment of patients with ODP-M.