ECOD relies on a combination of automatic and manual weekly updates to achieve its high accuracy and coverage with a short update cycle. ECOD classifies the approximately 120 000 depositions of the PDB into more than 500 000 domains in ∼3400 homologous groups. We show the performance of the weekly update pipeline since the release of ECOD, describe improvements to the ECOD website and available search options, and discuss novel structures and homologous groups that have been classified in the recent updates. Finally, we discuss the future directions of ECOD and further improvements planned for the hierarchy and update process.Evolutionary Classification Of protein Domains (ECOD) ( Protein three-dimensional structures continue to be determined at an exponential rate, both due to improvements in structure determination techniques and the increase of involved investigators –3. AdditWe developed the Evolutionary Classification Of protein Domains (or ECOD)  as a hieFollowing the initial release of ECOD (v22), we implemented a weekly update pipeline for ECOD coupled to the release of new structures from the PDB. By quickly and efficiently classifying known structures, and through dedicated manual curation, we are able to classify all depositions in the PDB without overburdening manual curators or relying on solely classifying a reduced set of representative structures. Briefly, the ECOD update pipeline separates a weekly PDB release into a set of individual protein queries based on peptide chains within the PDB depositions (putative fragments or peptides are removed early in the process and either placed into the peptide/fragment categories or reincorporated into ECOD as segments of multi-chain domains). Each member of this set of peptide chains is then individually queried against ECOD reference libraries using a combination of sequence  and structural (DALI) aligners –12. BLASWe initiated weekly updates in February 2014 with ECOD version 33. In the following 123 weekly PDB releases, 187 ± 51 structures were classified each week on average Figure . These cThose chains that cannot be partitioned into domains and assigned automatically are manually curated (8). The result of manual curation is either to partition a peptide chain into domains and to assign those domains to H-groups within the ECOD hierarchy, or to assign a chain, in part or wholly, to one of the ECOD special architectures. In either case, the manual assignment of these domains or special architecture regions helps to automate subsequent assignments. Over time, our manual curation workload has remained steady in the face of the gradually increasing rate of structure releases, and the ratio of domains to non-domain  regions has decreased. We attribute these shifts to the change in focus from small, structurally novel, globular domains, to larger complexes wherein associated domains may contain more extended regions in the deposited coordinates increasing numbers of extended structures in recent PDB releases likely result from their physical ordering by protein–protein interactions within large complexes, or from a shift from X-ray crystallography refinement techniques, where disordered regions are more likely to be excluded from modeled regions, to microscopy refinement techniques, where large disordered regions are more likely to be included in the deposited structural coordinates.http://prodata.swmed.edu/complete/distributions).ECOD is highly redundant with respect to sequence and structural similarity. Since the release of ECOD we have departed from our F-group model of Pfam HMM-based families mixed with HHsearch-based single-linkage clusters. Now we use families described by Hidden Markov Models (HMMs), some of our own creation. With the increased week-to-week stability and performance that this change provided, we were able to recalculate ECOD representative sets on a weekly basis and provide them as a standard distribution. Domain clusters are generated using BLASTCLUST at 99%, 70% and 40% sequence filtering over 90% alignment coverage of both the query and the reference sequences. Because H-groups can contain multiple T- and F-groups, filtering only occurs between members of the same F-group. This rule ensures that each F-group is represented at least once, although representatives may be sequence-similar above the level of filtering to members of other F-groups. Representatives are selected for each version from clusters preferring (i) existing F40 representatives (i.e. from previous versions), (ii) manually curated domains, (iii) provisional manual domains, (iv) domains from structures determined by X-ray crystallography, (v) domains from structures with higher X-ray crystal resolution, (vi) and domains from more recently released structures. These representative sets are made available, along with their PDB structure sets, at our webpage  and potentially homologous groups (X-groups). In addition, we partition groups of homologous domains into sequence families of closer relatives, frequently with similar functions. Because family classification in itself is to a large degree reliant on manual identification of functional motifs and interaction partners, we rely on the well-established classification provided by the Pfam sequence database to the extent possible . Putativhttp://hmmer.org) searches against the UniRef90 non-redundant protein database  must be created through manual curation and populated with at least one representative. Although not all curation results in the creation of new groups, on average 2.5 new X-groups (no clear homology to any known domain) and 1.9 new H-groups (some inconclusive evidence of homology to known domains) were created and populated in each weekly update. Proteins assigned to these new groups were generally partitioned as single-domain (60%), with a smaller fraction partitioned into more than one domain (40%). Domains in newly created X-groups are not homologous to any other ECOD domain; however, they may gain new homologs in subsequent versions. Of the 273 X-groups and 220 H-groups defined by manual curation during the weekly updates, 75% of both have received additional domains subsequent to their inception. Many of these additions are redundant, considering only the addition of new F40 domains, only 14% and 12% have received new additions, respectively. These novel PDB chains are generally not classified by other structural domain classifications. Only proteins observed in 19 and 27 of the new ECOD X- and H-groups are observed in SCOPe v2.06. Similarly, only proteins within 31 of the new ECOD X-groups and 34 of the new ECOD H-groups are observed in CATH v4.1.Plasmodium falciparium. The structure of RH5 from Plasmodium was determined and found to have a novel alpha-helical fold that may form an obligate multimer  protein family is necessary for invasion of host erthyrocytes in multimer ,21. The multimer . A subsel domain .New groups in ECOD can contain domains from viral proteins. The fast evolution of viral proteins can be responsible for significant structural variation and elaboration. The initial 2.3 A X-ray structure of the hantavirus glycoprotein Gn (5fxu) ectodomain  was manuThe mamallian mitoribosome, although descendant from alpha-proteobacteria, contains many unique structural features . The strThe mission of ECOD is to provide timely and consistent domain classification of proteins released by the PDB. We maintain a unique database uniquely focused on the full classification of the available structures, and by full coverage of the residues in those structures. Here, we have presented some of the improvements to website search and the result of more than 18 months of consistent weekly updates. In the future we intend to focus on making quality-control data available through the same portal, formalizing our description of sequence F-groups, and describing the inter-domain interfaces present in the protein complexes in which ECOD domains participate. We anticipate that the types of structures classified by ECOD will continue to shift toward large complexes determined by microscopy methods, and that the fraction of single-domain X-ray crystal structures will continue to decrease. This shift will require a continued focus on the automated treatment of these structures, and the curation tools used to classify them manually.


Efforts have multiplied in the past decade to underline the importance of pain management. For both acute and chronic pain management, various barriers generate considerable treatment accessibility issues, thereby providing an opportunity for alternative intervention formats to be implemented. Several systematic reviews on Web-based interventions with a large emphasis on chronic pain and cognitive behavioral therapy have been recently conducted to explore the influence of these interventions on pain management However, to our knowledge, the specific contribution of tailored Web-based interventions for pain management has not been described and their effect on pain has not been evaluated.The primary aim of this systematic review was to answer the following research question: What is the effect of tailored Web-based pain management interventions for adults on pain intensity compared with usual care, face-to-face interventions, and standardized Web-based interventions? A secondary aim was to examine the effects of these interventions on physical and psychological functions.We conducted a systematic review of articles published from January 2000 to December 2015. We used the DerSimonian-Laird random effects models with 95% confidence intervals to calculate effect estimates for all analyses. We calculated standardized mean differences from extracted means and standard deviations, as outcome variables were measured on different continuous scales. We evaluated 5 different outcomes: pain intensity (primary outcome), pain-related disability, anxiety, depression, and pain catastrophizing. We assessed effects according to 3 time intervals: short term (<1 month), medium term (1-6 months), and long term (6-12 months).P=.003) and pain-related disability . No other improvements were observed at follow-up in the medium and long terms. Compared with the active control group, no improvements were found for the primary outcome (pain intensity) or any of the outcomes except for a small effect size on pain catastrophizing  immediately after the intervention.After full-text review, we excluded 31 articles, resulting in 17 eligible studies. Only 1 study concerned acute pain and was removed from the meta-analysis, resulting in 16 studies available for quantitative assessment. Compared with standard care or a waiting list, tailored Web-based intervention showed benefits immediately after, with small effect sizes (<0.40) for pain intensity : CRD42015027669; http://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42015027669 (Archived by WebCite at http://www. webcitation.org/6uneWAuyR) Efforts have been made in the past decade to underscore the importance of pain management and its status as a human right -3. AcuteOver the past decade, more Web-based interventions for pain management have been developed and, as opposed to non-Web-based interventions, they have been shown to positively influence health behaviors ,12. Many...a primarily self-guided intervention program that is executed by means of a prescriptive online program operated through a website and used by consumers seeking health- and mental-health[-]related assistance. The intervention program itself attempts to create positive change and or improve/enhance knowledge, awareness, and understanding via the provision of sound health-related material and use of interactive Web-based components.This definition is composed of three types of health-related interventions: educational, self-guided therapeutic, and human-supported therapeutic interventions .Several systematic reviews on Web-based interventions for pain, with a large emphasis on chronic pain issues and cognitive behavioral therapy (CBT) interventions, have been conducted to explore their influence on pain management -19. OverExperts in health behavior change have shown that conveying health information without considering individual differences may inhibit behavior change -26. TailTherefore, this systematic review aimed to answer the following research question: What is the effect of tailored Web-based pain management interventions for adults on pain intensity compared with usual care, face-to-face interventions, and standardized Web-based interventions? We also examined secondary outcomes related to the effects of these interventions on physical and psychological functions.This systematic review protocol has been developed based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines for reporting systematic reviews evaluating health care interventions -33. The We include solely randomized controlled trials (RCTs) in this systematic review. Other inclusion criteria were studies with patients 18 years of age or older and experiencing any type of pain . We selWe selected outcomes according to pain clinical trials recommendations -38. PainWe searched MEDLINE, Embase, CINAHL, PsycINFO, Web of Science, and the Cochrane Library for articles published from January 2000 to December 2015. Reviewing the reference lists of relevant articles and previous systematic reviews helped identify 9 additional articles. Moreover, an experienced research librarian used subject headings to avoid missing nonindexed concepts. Search terms were “pain,” “pain management,” “program,” “intervention,” “Internet,” “Internet-based,” “online,” “Web-based,” and “mobile OR mobile applications” .Eligibility was assessed independently in an unblinded standardized manner by 2 team members  and results were then compared. Titles and abstracts were screened. If a trial was potentially eligible, the full text was reviewed. The 2 reviewers are researchers in the field of pain with a clinical background in nursing. Disagreements between reviewers at the full-text level were discussed until consensus was reached.Data were extracted independently by 2 teams of 2 reviewers  composed of 1 doctoral student and 1 researcher using the software DistillerSR v2 (Evidence Partners Inc). We developed the data extraction form we used based on the Cochrane Consumers and Communication Group’s data extraction template , pilot tRisk of bias for the 17 selected studies was assessed by the 2 teams of reviewers using the Cochrane Collaboration tool -43, and 2 statistic above 50% to indicate high heterogeneity, values between 25% and 50% to indicate moderate heterogeneity, and those below 25% to indicate low heterogeneity.We used Review Manager  software  for statAfter a full-text review of 48 articles, we excluded 31 for the following reasons: 3 studies were not RCTs, 18 studies did not involve 1 of the tailoring mechanisms, and 10 studies did not measure pain intensity using a VAS or NRS or a pain index calculated with scores obtained on an NRS over a period of time. We included 17 studies in the qualitative synthesis and 16 in the meta-analysis see . Of noteAs The total number of participants entering chronic pain trials was 4103 . All studies described the total number of participants providing data at the end of the interventions. The mean completion rate for studies that provided such data was 77.6%, with the proportion of completers ranging across studies from 44% to 95.2%. The mean age of participants entering the studies was 48.2 years . The average proportion of female participants was 71%.A total of 5 studies used usual care as the comparator arm ,55,57-59A total of 13 studies evaluated Web-based interventions using a CBT or behavioral approach -54,57-60A total of 6 potential biases were evaluated according to the Cochrane Collaboration tool: selection, performance, detection, attrition, reporting, and other . RegardiWe included 16 studies in the quantitative analysis, having removed the single study on acute pain . We condWe removed the control group that received the standardized Web-based intervention with optional contact from the meta-analysis, as it was impossible to evaluate the amount of contact with a therapist that was received. Overall, we included 16 studies in the 2 main meta-analyses: 11 in the tailored Web-based intervention versus standard care or waitlist control , and 4 in the tailored Web-based intervention versus active control -48,52,56P=.003; I2=29%). P=.45; I2=48%) and long term . We entered 10 studies (n=1310) into an analysis of the short-term effect of Web-based tailored interventions on pain intensity. The overall effect of Web-based tailored interventions on pain intensity was beneficial, with a small effect size and no significant heterogeneity . P=.49; I2=0%). The meta-analysis for long-term effect on pain-related disability could not be conducted due to the lack of assessments available at this time point.We entered 6 studies (n=953) into the meta-analysis of the short-term effect of Web-based tailored interventions on pain-related disability. The overall effect was significantly beneficial with a small effect size, although heterogeneity was high  or depression  with the tailored Web-based intervention compared with standard care or waitlist control. Meta-analytic statistics could not be run for medium-term and long-term effects due to the lack of assessments available at these time points. Meta-analysis could also not be run for pain catastrophizing, which was measured in only 1 study. We found no significant short-term reductions in anxiety  or medium term . We could not analyze the long-term effects on pain because of lack of assessments available at this time point. The tailored Web-based intervention did not relieve pain significantly better than active control in the short term  or medium term . The long-term effect on pain-related disability could not be analyzed due to a lack of assessments available at this time point.The tailored Web-based intervention did not relieve pain significantly better than the active control in the short term . In regard to medium-term effect on anxiety, we entered 2 studies (n=411) into the meta-analysis, and the overall effect was not significant . Long-term effect on anxiety could not be explored due to a lack of assessments available at this time point.We entered 3 studies (n=450) into the meta-analysis of the short-term effect on anxiety. The overall effect was not significant . We explored the medium-term effect on depression with 2 studies (n=411). The overall effect was not significant . Long-term effects on depression could not be analyzed due again to a lack of assessments at this time point.We entered 3 studies (n=450) into the meta-analysis to analyze the short-term effect on depression. The overall effect was not significant . Medium-term and long-term effects could not be explored due to lack of assessments available at this time point in this subgroup of studies. The only significant effect of the tailored Web-based intervention was observed for pain catastrophizing in the short term  was reported by the experimental group, as well as fewer negative pain beliefs and attitudes (P=.02).We could not include 1 study in the meta-analysis because it was the only study targeting acute pain after cardiac surgery . The WebWe examined funnel plot asymmetry for the meta-analysis of the effect on pain intensity when comparing tailored Web-based interventions versus standard care posttreatment (short term) solely because other analyses included too few studies (n<10)   used a hybrid format including telephone or face-to-face contact with a therapist. Most of the studies compared the tailored Web-based intervention versus standard or waiting-list control (n=12). Only 1 study concerned acute pain, which we removed from the meta-analysis, resulting in 16 studies available for quantitative assessment. We evaluated 5 different outcomes: pain intensity (primary outcome), pain-related disability, anxiety, depression, and pain catastrophizing. We assessed effects according to 3 time intervals .Compared with standard care or waiting list, pain intensity  and pain-related disability  were improved immediately after the tailored Web-based intervention with small effect sizes . No other improvements were observed at follow-up in the medium and long terms. Other systematic reviews and meta-analyses on Web-based CBT ,18,19, aWhen comparing the active control group, we found no improvements for the primary outcome (pain intensity) or any of the outcomes except for a small effect size on pain catastrophizing  immediately after the intervention. The 2 studies included in this analysis were quite different ,52. AlthLastly, based on our results and previous results, a general observation can be made pertaining to the effects of Web-based interventions on chronic pain. Regardless of the type of comparison used , effects were redundantly small, which poses a question concerning the adequacy of pain reduction as an outcome. Previous authors  have sugAlthough our results are consistent with other meta-analyses, a major difference in our results is related to the heterogeneity of intervention approaches that we found in tailored Web-based interventions for pain management as opposed to traditional CBT. Selected studies included interventions profoundly influenced by CBT, but approaches were oftentimes multimodal and varied from wearing a pedometer and physical activity coaching to relaxation and mindfulness. Many of the interventions also included an educational aspect related to management of a specific chronic disease and wellness. The dosage of interventions was also very diverse, ranging from 2 sessions per week for 3 weeks  or 5 sesAttrition and fidelity of intervention delivery are challenges in Web-based interventions. The attrition rate ranged from 5% to 56% with an average of 22%, even though all interventions included some kind of feedback, with half of them using human interaction. This rate is similar to the rate found in another meta-analysis, underlining that there was no difference in attrition even when participants received more guidance as opposed to no reminders or feedback . QuestioIn an effort to decrease heterogeneity in measures and their involved concepts when looking at pain-related disability, we focused on the 2 most commonly used measures: the Roland Morris Disability Questionnaire and the Brief Pain Inventory. This could have influenced our results. Although our study is 1 of the few making a distinction between comparators, we could include only 5 studies in the meta-analysis focusing on active control groups. Our results are in that sense to be interpreted with caution.Lastly, we found only 1 intervention for acute pain, which highlights the need to explore the avenue of tailored Web-based interventions for this type of pain and for the prevention of chronic pain. The context and settings of acute pain are very different from the chronic spectrum and could lead to different outcomes. Hence, it has been shown that Web-based tailored interventions that are preventive in nature and targeting a general population, not a specific condition, were more successful . AnotherAlthough several meta-analyses and systematic reviews of Web-based interventions for pain management have been conducted, this is, to our knowledge, the first examining tailored Web-based interventions and using active control groups as a comparator. Tailored Web-based interventions did not prove to be more efficacious than standardized Web-based interventions in terms of pain intensity, pain-related disability, anxiety, and depression. Similar findings to other meta-analyses on Web-based interventions for pain management were generated, meaning that these interventions may have a short-term effect on pain intensity and disability compared with usual care. An interesting finding was that some efficacy was shown in pain catastrophizing compared with active control interventions.Tailored Web-based interventions are a recent field of research among behavioral change interventions. Considering the diversity of approaches used in tailored Web-based interventions for chronic pain management, their efficacy is yet to be explored. Moreover, the scarcity of tailored Web-based interventions available for acute pain management reflects the multitude of possibilities for intervention development. Feedback was used in all studies but content matching, the most important tailoring ingredient, was used in only half of the studies. More studies would improve our understanding of the efficacy of these interventions, enabling subgroup analyses according to their therapeutic content and their level of tailoring .


Based on mathematical analysis a subsequent study stated that death of normal or neoplastic cells could be a predestined, genetically determined phenomenon2. In 1972, Kerr, Wyllie, and Currie coined the term “apoptosis” to describe a basic biological phenomenon with wide-ranging implications in tissue kinetics3. They postulated that apoptosis can occur spontaneously in untreated malignant neoplasms, and is involved in therapeutically induced tumor regression. To date, more than ten different cell death modalities have been recognized by the Nomenclature Committee on Cell Death4. Disturbances in cell death pathways at the molecular level can be linked to the pathogenesis not only of cancer, but also other diseases of enormous social importance, such as HIV, atherosclerosis, ischemia, reperfusion injury, infection, inflammation, autoimmune, and neurological disorders5. An accumulation of data testifies the existence of cross talk between the various cell death mechanisms. There is also considerable evidence that suppression or activation of one type of cell death can influence the activity of another and that the balance between them can affect the response of cells to treatment. Nowadays there is evidence that tissue-specific and organ-specific activation of cell death mechanisms influences how various diseases respond to treatment. Thus, the therapies for kidney-associated diseases are linked to efficient necroptosis or ferroptosis, therapies for neutrophil-associated disorders are linked to necroptosis etc. Tiny perturbations in the regulation of cell death pathways could be potential targets for improvements of therapy. With this in mind, many research groups and pharmaceutical companies are focusing on development of new compounds that act against specific diseases by targeting cell death pathways. In the last decade FDA approved many drugs targeting cell death processes for treatment of various diseases. Amongst these are anti-cancer drugs. In 2016, the FDA approved a highly selective small-molecule Bcl-2 inhibitor, venetoclax (ABT-199) for the oral treatment of AML and CLL. A selective inhibitor of Bcl-W, navitoclax (ABT-263), has been proposed as a treatment for advanced and recurrent small-cell lung carcinomas. An antibody against programmed cell death protein 1 (anti-PDL1) was approved for treatment of bladder cancer and nivolumab, which also targets PD-1, for non-small-cell lung cancer, melanoma, and renal cell cancer. A number of p53-Mdm2 inhibitors, including Nutlin and MI-773, as well as reactivators of mutant p53, including APR-246 and SCH529074, are at different stages of clinical trials involving various cancers.More than 50 years ago, Anna Kane Laird suggested that the retardation of tumor growth observed by researchers and clinicians might be due to an active decrease in the tumor growth rate or to cell death. She postulated that “stimulation of natural growth-retarding factors might be of practical importance in reducing the incidence of clinical cancer, i.e., the systemic illness and death of the host, if tumor growth could be retarded enough to bring it to an upper limit that is within the physiological tolerance of the host”6. However, activation of necroptosis does not always imply successful therapy. Necroptosis is involved in the regulation of pathogenesis of inflammatory diseases, suggesting that triggering necroptosis in cases of Crohn’s disease or inflammatory skin disease might have off-target effects, leading to pathological inflammation of normal tissues. These concerns should be taken into consideration during the planning of treatment. With these concerns in mind, Infliximab (Remicade) was successfully tested as a potential treatment for Crohn’s disease, which is associated with inflammasome dysfunction.Interestingly, the combination of a novel caspase inhibitor IDN-7314 with 5-fluorouracil synergistically blocked tumor growth compared with 5-fluorouracil alone, via induction of necroptosisRecent achievements in development of cell death-based approaches to treatment of various diseases are summarized in a series of eleven review articles published in this issue of Cell Death & Disease.7. The authors highlight the key inducers of necroptosis in neutrophils. Thus, inhibition of caspase-8 activates apoptosis in response to the presence of the TNF ligand and RIPK3-dependent necroptosis can be triggered by phagocytosis of S. aureus. However, under some conditions, TNFR1 stimulation via TNF triggers other cellular pathways, such as activation of NF-kB signaling or apoptotic cell death. Since neutrophils are involved in various types of tissue inflammation and disease, it is important to understand the different mechanisms regulating neutrophil necroptosis in order to uncover novel drug targets for treatments of autoimmune and inflammatory diseases.The role of necroptosis in neutrophil cell death is discussed by Wang and colleagues8. By contributing to AKI, regulated necrosis causes antibody-mediated rejection. AKI promotes loss of the majority of tubular cells by ferroptosis, resulting in peroxidation of membrane lipids. Although elimination of cells by apoptosis is beneficial in many physiological conditions, the integrity of plasma membrane during apoptosis prevents it being immunogenic. It seems that pyroptosis is the most immunogenic pathway due to its role in maturation of IL-1 and IL-18 alongside the release of inflammatory caspases. However, production of IL-33 and CXCL1 during necroptosis results in inhibition of innate immunity.In addition to necroptosis there are another two forms of regulated necrosis, namely ferroptosis and pyroptosis, which have been intensively studied in the context of acute kidney injury (AKI) and transplantation. The immunological consequences of kidney cell death are discussed by Sarhan and colleagues9 describe the role of different cell death modalities in the pathogenesis and therapy of urinary tract-associated diseases. In cancer of urinary tract there are several key processes that lead to escape from apoptosis, including autophagy, downregulation of Fas and caspase-3 as well as upregulation of Bcl-2. A deficiency of VHL in renal cell carcinoma makes it resistant to NK cells and hypoxia-induced cell death, whilst inflammatory signaling induced by NF-kB promotes necroptosis. Tumors of the urinary tract develop resistance to cell death, but infection of urothelial cells with uropathogenic E. coli may, depending on the bacterial strain, prevent host cell death or promote cell death by apoptosis, necrosis or an iron-dependent mechanism.Martin-Sanchez and colleagues10. Hyperglycemia-mediated upregulation of ROS is associated with activation of several metabolic routes, including diacylglycerol—protein kinase C—and NADPH-oxidase pathway. The authors suggest that inhibitors of this pathway could be used to control ROS and prevent cell death in diabetes.It is known that diabetes is characterized by hyperglycemia and involves significant loss of insulin-producing pancreatic β cells via several cell death mechanisms, including apoptosis, necroptosis and pathological necrosis. Oliveira Volpe and colleagues describe the therapeutic targets for controlling this hyperglycemia-triggered metabolic pathway, which is one of the causes of diabetic complications, by down-regulating oxidative stress in β cells11. It has been shown that inflammatory mediators released by microglia and astrocytes contribute to the pathogenesis of neurodegenerative disease. Under healthy conditions microglial cells generate and secrete factors  essential for neuronal survival but, after activation, they secrete various pro-inflammatory cytokines, such as IL-1β and immunomodulatory factors e.g., nitric oxide, which have a negative impact on neuronal function, structure and survival.Christina Ising and Michael Henneka focus on the role of innate immune mechanisms in damage of neurons during neurodegeneration12. Activated CD8+ T cells can recognize tumor-associated antigens at the surface of tumor cells and promote tumor cell death via the perforin-granzyme and/or FasL/TRAIL systems. However, the tumor vasculature creates physical and functional barriers to infiltration of immune cells. The authors describe the mechanisms by which tumor-associated blood vasculature promotes an immunoresistant tumor microenvironment. At present, several methods of stimulating anti-tumor immunity with IL-2, TNF-α, and IFN-γ are subject to intensive investigation.The role of the tumor vasculature in anti-tumor immunity and immunotherapy is a hot topic in cancer biology and is summarized by Schaaf and colleagues13. It has been suggested that MYCN plays a role in regulation and expression of p53 and Tap73 and their relation to apoptosis in NBs. Hypoxia promotes activation of several factors, including HIF-1 that can block apoptosis by inhibiting p53 or promote tumor growth by stimulation of glycolysis and angiogenesis. Hence, it has been proposed that inhibitors of HIF-1 could promote apoptosis in NB cells. Since many NB cells do not express caspase-8, treatments that increase expression of necroptotic proteins are amongst the promising strategies for triggering necroptosis in NBs.Amplification of MYCN and mutations in anaplastic lymphoma kinase (ALK) are considered the main drivers of neuroblastoma (NB). Valter and colleagues discuss various pathways that allow NB cells to escape death14 discusses potential routes to reactivation of pathways triggering cell death in glioblastomas. Current treatment regimens for glioblastoma include radiation and chemotherapy with the alkylating agent temozolomide (TMZ). It has been suggested that SMAC mimetics operate synergistically with TMZ by promoting apoptotic cell death. This mechanism would engage NF-κB-dependent upregulation of interferon-β, which promotes apoptosis by upregulating Bax and Puma. Other treatments for glioblastoma include targeting TRAIL death receptors in combination with kinase or HDAC inhibitors as well as BH3 mimetics. Thus, reactivation of apoptosis is one of the main aims of glioblastoma treatments.Simone Fulda15. Increased expression of the DNA repair protein O6-alkylguanine DNA alkyltransferase (MGMT) leads to melanomas resistance to alkylating agents. The necroptosis pathway is often deregulated in melanoma because of low RIPK3 expression. Targeted therapies for melanomas includes BRAF and MEK inhibitors. Immunotherapies with the antibody blocking CTLA-4 prevents immunosuppression of lymphocytes, and reactivation of effector T lymphocytes. Another approach to sensitization of tumors is to restore the functions of tumor-suppressive cell death pathways by applying epigenetic compounds.Cell death-based therapy of melanoma is discussed by Mattia and colleagues16. Lung adenocarcinoma is characterized by mutations in epidermal growth factor receptor (EGFR) and KRAS, fusion in the ALK oncogene and alterations in different kinase signaling cascades. The FDA has approved several tyrosine kinase inhibitors (TKI) for therapy of lung adenocarcinoma. However, resistance to TKI inhibitors may be developed via enhancement of antiapoptotic signals such as NF-kB. Therefore, several combinations of chemotherapy plus antibody-targeted therapies are now in clinical trials.Denisenko and colleagues discuss signals driving growth of lung adenocarcinoma and cell death pathways induced by various therapeutic approaches to this tumor type17. These authors describe the ways in which malignant cells escape pharmacological inhibition of kinase, such as mutation or upregulation of tyrosine kinase receptors. Several kinase inhibitors have been suggested as potential treatments for pediatric tumors. These tumors often exhibit resistance to death-receptor targeted therapy due to downregulation of caspase-8. BH3-mimetics have been shown to be potent treatments in high-risk groups of patients with ALL and NBs, whereas Smac mimetics are to “sensitize” pediatric tumors to apoptosis. Thus, cell death-based treatments are one of the main forms of precision therapy for childhood cancers.Cell death-based therapies of childhood cancer are discussed by Westhoff and colleaguesCell Death & Disease contains reviews summarizing recent achievements in the field of cell death-based treatment of a number of diseases. We hope that this collection of articles will attract scientists’ attention to this growing area of research, which offers many opportunities to exploit cell death mechanisms to develop new treatments for human disease.In the almost 50 years since cell death was first proposed as a potential target for cancer therapy is has become obvious that targeted cell death-based treatment has an important role to play in successful therapy of various human diseases. Various approaches to regulation of cell survival and cell death are currently in use. This issue of


Our approach identified informative associations between kinases and metabolic enzymes capable of predicting metabolic changes. We extended our analysis to two studies containing transcriptomics, phosphoproteomics and metabolomics measurements across a comprehensive panel of kinases/phosphatases knockouts and time-resolved perturbations to the nitrogen metabolism. Changes in activity of transcription factors, kinases and phosphatases were estimated in silico and these were capable of building predictive models to infer the metabolic adaptations of previously unseen conditions across different dynamic experiments. Time-resolved experiments were significantly more informative than genetic perturbations to infer metabolic adaptation. This difference may be due to the indirect nature of the associations and of general cellular states that can hinder the identification of causal relationships. This work provides a novel genome-scale integrative analysis to propose putative transcriptional and post-translational regulatory mechanisms of metabolic processes.Cells react to extracellular perturbations with complex and intertwined responses. Systematic identification of the regulatory mechanisms that control these responses is still a challenge and requires tailored analyses integrating different types of molecular data. Here we acquired time-resolved metabolomics measurements in yeast under salt and pheromone stimulation and developed a machine learning approach to explore regulatory associations between metabolism and signal transduction. Existing phosphoproteomics measurements under the same conditions and kinase-substrate regulatory interactions were used to  in silico estimated activity of transcription-factors and kinases/phosphatases. This provided functional information about the proteins important for the experimental conditions at hand that had not been uncovered before. We showed that activity profiles are predictive features to estimate metabolite changes in dynamic experiments, while the same was not visible in steady-state conditions. We also showed that dynamic experiments could be used to recapitulate and provide novel TFs-metabolite and K/Ps-metabolite regulatory associations. We believe these findings illustrates the usefulness of this approach for future integrative studies interested in studying metabolic regulation.Phosphorylation is a broad regulatory mechanism with implications in nearly all processes of the cell. However, a global understanding of possible regulatory mechanisms remains elusive. In this study, we examined the potential regulatory role of kinases, phosphatases and transcription-factors in yeast metabolism across a variety of steady-state and dynamic conditions. The main novelty of our analysis was to infer putative regulatory interactions from  The c changes –6. The i changes ,8. Currein silico estimated metabolic fluxes [Regulatory responses mediate the adaptation of many biological aspects of a cell, for example, metabolism may be regulated transcriptionally and post-transcriptionally. At present, most of the integrative analysis of metabolomics data-sets have focused on the role of transcriptional regulation –11. Prevc fluxes ,15. Nonec fluxes ,15.in vivo activity of TFs and K/Ps. For that purpose, we considered prior-knowledge on regulatory interactions and mathematical approaches that have been developed to predict the activity status of transcription factors [Transcriptional and translational regulatory interactions of metabolism can, in principle, be comprehensively explored using available high-throughput data-sets and methods ,5,11. Ho factors ,17 and k factors ,19 . On the untargeted data-set, 33 ions were defined as significantly changing in at least one of the time-points analyzed   approachNitrogen downshift and rapamycin are similar conditions that inhibit TOR1 activity; in contrast, nitrogen upshift displays increased TOR1 activity. Thus, it is reassuring that the predicted protein activities tend to have similar changes in time for the nitrogen downshift and rapamycin condition, and opposite changes for the nitrogen upshift . Severalp-value < 7.0e-3) with the relative growth rates of the knockout strains. Growth rate displayed stronger correlations  with PC 1 of K/P and TF activities   .In summary, the variance in molecular measurements for the steady-state genetic perturbation experiments is more strongly influenced by the growth rate than the measurements performed in the dynamical perturbations. For the steady-state conditions, the gene-expression changes are the molecular changes, confounded mostly by the growth rate.For the subsequent association analyses we tested the impact of removing the growth rate from each data-set to rule out any confounding effects it may have on the identification of direct functional interactions. To this end, we regressed-out growth rates from the original metabolite measurements and estimated TFs and K/Ps activities using linear regression models and growth as a covariate.Next we explored the correlations between TFs and K/Ps enzymatic activities and metabolic changes for each of the three experiments: genetic, nitrogen metabolism and salt/pheromone perturbations. To identify the relationships we used linear regression models that consider the estimated activities as features and metabolite fold-changes as observations . ConsideFirstly, we considered the genetic perturbations and assessed the capacity of TFs and K/Ps activities to predict the changes of a given metabolite across the panel of knockouts . This diA similar analysis as with genetic perturbations was applied to the dynamic experiments to estimate the metabolic variation. The trained models displayed in general higher predictive power than the genetic perturbations . OverallThe predictive difference between growth normalised and non-normalised K/Ps activities in the genetic perturbations . Furthermore, RIM15 is inhibited by TOR1 [RIM15 and TPK1 displayed the strongest associations with the metabolites and these play a key role in the regulation of the cellular growth and their adaptation to nutrient availability –55. TPK1 by TOR1  and cons by TOR1 . Moreove by TOR1 . The assp-value < 2.6e-2). Also, YAK1 is required for full activation of MSN2/4 TFs [p-value < 1.1e-1), for example, both have negative associations with dUDP and positive associations with L-Proline. However, this type of analysis is limited to the number of metabolites that can be well predicted with the TFs and K/Ps activities.TFs-metabolites and K/Ps-metabolites associations can also be taken together to elucidate associations between K/Ps and TFs. For example, TPK1 kinase inhibits the activity of TF ADR1  and thesN2/4 TFs  and thisIn summary, the estimated activity of TFs and K/Ps were capable of building predictive models to infer the metabolic adaptations of previously unseen conditions across different dynamic experiments. Specifically, TFs activities provided better predictive power than K/Ps, although K/Ps-metabolite associations are more likely to represent previously reported interactions.Signal transduction is an important cellular mechanism that allows cells to sense and respond to environmental cues. These mediate intracellular adaptations by regulating a variety of biological processes, including metabolism and gene expression. Thereby interactions among different biological processes occur and are very important to coordinate the whole phenotype of the cell. Nevertheless, the systematic identification and functional annotation of these regulatory interactions is still a challenge. Experimental data-sets covering different omics in similar conditions are becoming more available and it is likely that analyses like the one we propose here will be useful to systematically explore these regulatory events.The key novelty of the approach proposed here is that regulatory interactions are inferred from estimated activity of TFs and K/Ps, which are difficult to measure directly. This provides the possibility of considering the activity profile of regulatory proteins important for the experimental conditions at hand which has not been considered in previous studies of metabolism using phosphoproteomics and transcriptomics.Our results show that it is possible to use K/Ps and TFs activities to predict changes of several metabolites in time-resolved experiments. However, the predictive power does not extend to all conditions. For example, the models trained with K/Ps activities showed limited capacity in the pheromone perturbation experiment. This can arise from the higher technical variability in the data obtained and also be due to lower number of regulated metabolites. Additionally, the regulatory interactions are often condition specific. As such, if the proteins that are important to regulate the nitrogen or osmotic related conditions are not used for the pheromone response, then the associations learned cannot be predictive of the pheromone induced metabolic changes. Interestingly, protein-metabolite interactions inferred from the genetic perturbations experiment displayed poor predictive power to estimate the metabolic changes of a new condition, in contrast to the dynamic experiments . This suNevertheless, while the protein-metabolite interactions that we infer provide reasonable power to predict metabolic changes  of unseeThe increasing number of recorded interactions for regulators will provide important information to expand the coverage of TFs and K/Ps for which it is possible to estimate activities and increase the robustness of the estimated activity. The generic characteristic of the approach used to infer regulatory interactions allows it to be easily expanded to integrate other types of information and thereby augment its predictive power to infer causal and direct protein-metabolite interactions. For example, to account for other confounding effects, such as cell cycle as a covariate and thereby remove it as a possible source of interactions. Furthermore, other types of biological measurements can also be integrated, for example, protein abundance. This can provide information into other regulatory mechanisms but also provide information to possible associations between the different regulatory processes, for instance, phosphoproteomics measurements are intrinsically dependent on protein abundance.In this study we demonstrated the utility of phosphoproteomics and transcriptomics data-sets to estimate the enzymatic activity of K/Ps and TFs, respectively. The estimated activities recapitulated several previously expected regulatory events, such as HOG1 and PBS2 responses to osmotic stress, and RIM15 and MSN2/4 activation under TOR1 inhibition. This results emphasise the usefulness of this approach to explore functional implications in regulatory proteins. Our results also showed that activity profiles are informative features to estimate metabolite changes in dynamic experiments. Interestingly, the same was not visible across a large panel of K/Ps knockouts, supporting the idea that time-resolved experiments are a better experimental design for the identification of causal regulatory interactions. We expanded on previous work by developing a novel and rigorous framework to identify regulatory associations between the estimated activities and metabolite changes. Rigorous analysis of the putative TFs-metabolite and K/Ps-metabolite associations occurring in the dynamic experiments revealed that despite their regulatory implication these are most likely indirect. Further confirmation of these results with the integration of other experimental data-sets will provide deeper insights into the regulatory events mediating the metabolic phenotype.Saccharomyces cerevisiae strain used for the salt and pheromone dynamic experiments was BY4741 as in Vaga et al. [E. coli extract as internal standard and dried completely in a vacuum centrifuge . The dried extracts were resuspended in 100 μl MilliQ water before analysis.The a et al. ,23, thisTargeted metabolomics was performed by LC-MS/MS as described before . The masUntargeted metabolomics was performed by direct flow double injection of extracts on an Agilent 6550 series quadrupole TOF MS operated in negative mode. In total 11,190 ions were detected across all samples. The detected ions were then mapped against an existing ion mass library of metabolites generated from the yeast genome-scale model iMM904 . 452 outStatistical significance of the ion fold-changes for the QTOF-MS measurements was estimated with a two-sided t-test followed by multiple hypothesis correction with false-discovery rate. The list was then filtered and only ions with and an absolute fold-change higher than 1 were considered, resulting in 33 ions.For K/Ps knockouts in yeast, a total of 3,011 transcripts were measured across 1,484 deletion mutants, comprising approximately 26.4% of all protein-coding genes in yeast . PhosphoDynamic perturbations to nitrogen metabolism and TOR signalling were captured in a time frame from 0 to 79 minutes, where 0 minutes represents the unperturbed state. Transcriptomics measurements covered 5,620 transcripts across all the time-points of the conditions. Phosphoproteomics captured the profile of 1,660 single phosphorylated phosphosites  over the same time-points. Given the lack of complete coverage, only 50.8% of the whole matrix is measured. Intracellular metabolomics were acquired with QTOF-MS and quantified a total of 146 ions, after quality filtering, across all conditions and time-points.p-value and signed according to the direction of the enrichment, for example, regulators enriched towards negative fold-changes have a negative score. K/Ps target phosphosites were extracted from PhosphoGrid [p-value for each gene as a measure of over or under-expression, relative to the distribution of values for all genes. Two-tailed p-values were computed from Z-scores when a p-value was not provided [Kinases/phosphatases and transcription factor activities were estimated using GSEA approach  and statsphoGrid . SpecifisphoGrid . Weight sphoGrid , was usesphoGrid ,60,61, oprovided –65, via provided . 144/355Python module Sklearn  version To infer the predictive power within each data-set across all the measured ions , for eacp-value lower than 0.05 and an coefficient of determination higher than zero.Protein-metabolite associations were inferred only using the time-resolved metabolomics data-sets. For the TFs activities only the nitrogen metabolism perturbations were used considering that no transcriptomics data was available for the NaCl/Pheromone perturbations. For this analysis no filtering was applied in the metabolomics data-sets and K/Ps activities were filtered as before to consider only those consistently measured and estimated across 75% of the conditions. The capacity of predicting each ion fold-change in each condition was tested using k-fold cross-validation, where each fold corresponds to the time-points measured in each condition, thus 3 and 5 folds were used for the TFs and KPs activities, respectively. Elastic net models were used and the alpha was estimated using a bootstrap approach of ten iterations leaving out 20% of the samples. A range of 100 alphas was considered as default by the Sklearn python module . Train aFor the top predicted ions the feature importance was estimated using all the conditions together with two bootstraps. The first bootstrap was 20 iterations and leaves out 20% of the samples out, for each iteration an inner bootstrap with 10 iterations leaving out another 20% of the data is performed to estimate the alpha of the Elastic net. This estimates 20 coefficients for each feature-metabolite association. As before, train features and observations are standardised and centered. The most important features per ion are estimated by taking the median of the coefficients and the Pearson correlation between the protein activity and the ion fold-change.https://github.com/saezlab/yeast_phospho. All plotting was performed using Python modules Matplotlib version 1.4.3 [All data analysis was performed in Python (v 2.7.10) and all the code, preprocessed data-sets and generated plots are openly available in github under the GNU general public license version 3 in the following URL on 1.4.3  and Seabon 1.4.3 .S1 Figa) identifies the overlap between the TF activities, b) between the K/P activities and c) between the metabolite ions measured. For the overlap it was considered only TFs and K/Ps for which it was possible to estimate their activity in at least 75% of the samples in each data-set.(TIF)Click here for additional data file.S2 FigThe principal component with higher absolute correlation coefficient was picked and plotted.(TIF)Click here for additional data file.S3 FigList of metabolites that displayed a positive coefficient of determination and significant Pearson correlation between the measured and predicted fold-changes across the different conditions.(TIF)Click here for additional data file.S4 FigTrue-positive tables were built considering the specified resources.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


We present the three-dimensional (3D) spectral-domain optical coherence tomography (SD-OCT) findings of schisis-like maculopathy associated with structural changes of the optic nerve (ON) head as well as the treatment outcomes of a case of advanced glaucoma. In addition to ophthalmological examination, B-scan and 3D-SD-OCT images of the ON head, peripapillary retina, and the macula were obtained. The B-scan images only detected typical retinoschisis findings. However, the 3D-SD-OCT images of the ON head revealed defects of various sizes, shapes, and depths at the outer wall of the prelaminar and laminar regions of the ON canal. The 3D images were able to establish that these defects were both adjacent to and interconnected with the retinal layers. The patient successfully received 3D-SD-OCT-guided thermal laser treatment that is used in congenital optic disc pits complicated with macular schisis. In brief, 3D-SD-OCT is very useful for demonstrating the ON head defects that can lead to schisis-like maculopathy in cases of advanced glaucoma. Acquired optic disc pit typically appears as a pale area at the inner periphery of the optical disc margin. Unlike congenital optic disc pits, acquired pits may form as a result of neuroretinal rim loss secondary to glaucoma.Acquired optic disc pits, a finding specific to glaucomatous optic nerve (ON) head damage, develop in association with localized depressions in the lamina cribrosa (LC). This special condition was first described by Radius et al.5 In recent years, optical coherence tomography (OCT) imaging techniques have allowed the detection of these pathologies, which can now be visualized in the peripapillary area, ON head, and even the LC with the use of advanced techniques.8Macular retinoschisis (or schisis-like maculopathy), a complication of congenital optic disc pits, and subsequent serous retinal detachment may also develop in acquired optic disc pits and result in serious vision loss. In fact, even in the absence of a detectable pit lesion, peripapillary and macular retinoschisis and associated serous macular detachment have been reported in glaucomatous eyes with severe optic disc cupping.This study presents the three-dimensional (3D) spectral domain (SD) OCT features and treatment outcomes of prelaminar and laminar defects in the walls of the ON canal caused by advanced glaucomatous ON head damage and leading to macular retinoschisis.A 30-year-old man whose vision level exhibited diurnal fluctuations was referred to our clinic for bilateral macular retinoschisis. The patient underwent a detailed ophthalmologic examination including best corrected visual acuity (BCVA), slit-lamp examination, intraocular pressure measurement, fundus photography, and fluorescein angiography.In addition, B-scan and 3D SD-OCT  images of the ON head, peripapillary retina, and macula were obtained and analyzed.The patient’s history revealed he had been diagnosed with closed-angle glaucoma and undergone bilateral trabeculectomy 6 years earlier. His BCVA fluctuated periodically, and was measured as 2/10 in the right eye and 10/10 in the left eye. Substantial BCVA fluctuations between 1/10 and 4/10 were noted in the patient’s right eye prior to treatment. The patient also reported BCVA fluctuations in his left eye; the lowest measurement we obtained was 9/10. On slit-lamp examination, the anterior chambers of both eyes appeared quiet and the surgical iridectomies were open. The corneas and crystalline lenses of both eyes were clear. Right and left intraocular pressures were 12 and 13 mmHg with latanoprost and were measured as 11 and 15 mmHg during follow-up. Fundoscopic examination revealed cup-to-disc ratios of 1.0 and 0.9 in the right and left eyes, respectively, and no abnormalities were detected other than advanced glaucomatous cupping of the optic discs. Fundus fluorescein angiography was normal in both eyes. The OCT images were examined in detail in light of these findings. B-scan SD-OCT images showed retinoschisis-like cystic structures in the papillomacular and macular areas (an area about 4x4 mm between the ON and fovea) and separation of the outer retinal layers . FurtherPresuming that the intraretinal fluid was probably vitreous and may have leaked between the retinal layers through these defects, the patient was treated with thermal laser photocoagulation to the peripapillary areas adjacent to the defects. Under 3D-SD-OCT guidance, thermal laser settings similar to treat congenital optic disc pits  were used to produce the desired level of whiteness to the desired area using consecutive burns .Within 6 months after the laser therapy induced fibrosis and cicatrization of the peripapillary areas, the focal defects in the ON periphery were observed to markedly decrease in size, though some large defects did not resolve completely. The intraretinal fluid regressed substantially and BCVA stabilized . FurtherAlthough all examinations, imaging, and treatments were applied to both eyes, the findings were more pronounced in the right eye and these images are presented in this report. Due to inconsistencies in the patient’s visual field test, evaluations related to visual field are not presented here.9 This condition, also called optic disc maculopathy, may also be seen with acquired optic disc pits. Cases of macular retinoschisis without a visible pit have been reported recently in patients with advanced closed- or narrow-angle glaucoma.Macular retinoschisis, a term used to describe large separations of the retinal layers in the posterior pole, often accompanies congenital optic disc pits. Macular retinoschisis may also form due to various reasons such as tractional causes, venous occlusive diseases, cystoid macular edema, and juvenile retinoschisis.8 Using EDI-OCT, You et al.7 described LC holes and defects appearing as LC degeneration adjacent to the side walls of the ON canal in patients with advanced glaucomatous neuroretinal rim thinning. Takayama et al.8 used SS-OCT to demonstrate similar defects in the LC region in glaucomatous eyes. Kiumehr et al.10 examined the LC region of normal and glaucomatous eyes using EDI-OCT and found focal LC defects in 76% of glaucomatous eyes. They noted that a large proportion of these defects are not detected during routine examinations, and found that the LC of healthy eyes was intact. These advances in OCT imaging techniques have enabled the visualization of the optic disc pit and deeper optic disc structures such as the LC. These studies confirmed that focal defects may develop in the LC.10 In our patient, the focal glaucomatous defects in the peripheral ON and their relationship to the areas of macular retinoschisis were shown with 3D-SD-OCT. Due to the inadequate tissue penetration of SD-OCT, the LC area adjacent to these defects could not be clearly evaluated.In the present study, we closely evaluated optic disc, peripapillary, and macular OCT images in order to determine the primary cause of retinoschisis in our patient. While peripapillary and macular cystoid cavities and detachment of the outer retinal layers were detected on standard B-scan SD-OCT imaging, 3D-SD-OCT imaging revealed optically dark (empty) defects in the optic disc margins at the prelaminar and laminar levels. In our search of the literature to identify these defects, we found reports of pathologies with similar appearance in recent studies using enhanced-depth imaging (EDI) OCT and swept source (SS) OCT.There is a debate as to whether the intraretinal fluid seen in congenital optic disc pits is cerebrospinal fluid or vitreous. However, we believe that this fluid is vitreous that leaks in through focal defects in the outer layers of the optic nerve. Nevertheless, we found no OCT findings directly supporting the vitreous as the source of this fluid.In our patient, macular retinoschisis caused fluctuations in vision, especially in the right eye. In order to stabilize visual acuity, obtain a dry macula, and prevent possible serous macular detachment, we treated both of our patient’s eyes with thermal laser therapy as used in congenital optic disc pits, under 3D-SD-OCT guidance. Due to the lack of serous retinal detachment accompanying the macular retinoschisis and the advanced glaucomatous ON damage, pars plana vitrectomy was considered risky in this patient, whose clinical signs were relatively mild.Following treatment, the defects in the peripheral optic cup had substantially decreased in size, but larger and deeper defects did not close completely. Green argon thermal laser therapy resulted in fibrosis and scar tissue in the peripapillary regions adjacent to the defects which could prevent leakage of the vitreous between the retinal layers. Our patient showed substantial regression of the intraretinal fluid and stabilized visual acuity after treatment.In summary, 3D-SD-OCT imaging is useful and effective in detecting peripheral ON defects at the prelaminar and laminar levels which result in macular retinoschisis in advanced glaucoma patients, and in determining the efficacy of treatment.


Bamboo mosaic virus (BaMV), a single-stranded RNA virus. The genome of BaMV is approximately 6.4 kb in length, encoding five functional polypeptides. To reveal the host factors involved in the infection cycle of BaMV, a few different approaches were taken to screen the candidates. One of the approaches is isolating the viral replicase-associated proteins by co-immunoprecipitation with the transiently expressed tagged viral replicase in plants. Another approach is using the cDNA-amplified fragment length polymorphism technique to screen the differentially expressed genes derived from N. benthamiana plants after infection. The candidates are examined by knocking down the expression in plants using the Tobacco rattle virus-based virus-induced gene silencing technique following BaMV inoculation. The positive or negative regulators could be described as reducing or enhancing the accumulation of BaMV in plants when the expression levels of these proteins are knocked down. The possible roles of these host factors acting on the accumulation of BaMV will be discussed.To complete the infection cycle efficiently, the virus must hijack the host systems in order to benefit for all the steps and has to face all the defense mechanisms from the host. This review involves a discussion of how these positive and negative factors regulate the viral RNA accumulation identified for the  When a positive-sense RNA virus infects a host cell, it needs to produce its progeny and move it to the neighboring cells efficiently. In general, the entire infection cycle starts at the viral RNA entry, using the host translation system to produce the viral-specific replicase, transition the viral template from translation status to replication status, targeting the specific organelle for replication, rearrangement of the cellular membrane, recruitment of ancillary proteins to the replication site, viral RNA replication to synthesize the minus- and plus-strand RNAs, subgenomic RNA synthesis in some species, and finally, the viral-encoded movement proteins (MPs) and coat proteins accumulated for cell-to-cell movement and encapsidation, respectively. Some of the viral-encoded proteins that evolved not only fulfilled a specific role apart from amplification, but also performed counter-defense functions such as silencing suppressors against the virus-induced gene silencing system  or preveBamboo mosaic virus (BaMV) is a positive-sense, single-stranded RNA virus, belonging to the genus Potexvirus of the family Alphaflexiviridae. The genome of BaMV is approximately 6.4 kb in length with a 5′ m7GpppG structure and a 3′-end poly(A) tail and contains five open reading frames (ORFs)  . The 3′ movement . ORF 1 emovement , a helicmovement , and an movement . ORFs 2-movement . ORF 5 emovement . Furthermovement . The termovement .As mentioned previously, the positive-sense RNA virus has to establish an efficient replication after entry into a host cell; the host factors are usually required to join and form a multi-functional replication complex . The repA few strategies were used to identify the host factors involved in virus infection cycles. By screening the host cDNA library constructed in yeast with the two-hybrid technique, one can discover the specific host factor that interacted with the viral-encoded target protein, such as the replicase, MPs, or CP . The virTobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) technique . At this stage, the 3′ UTR is playing a critical role in trapping a few different factors that would lead to the next step of the replication process, targeting the replication site. At least four host proteins, glutathione transferase U4 (NbGSTU4), the eEF1a, chloroplasts PGK (chlPGK), and heat shock protein 90 (Hsp90), were discovered to interact with the 3′ UTR of BaMV. In particular, the eEF1a was shown to play a negative role in BaMV RNA replication (Brome mosaic virus (BMV) (Turnip yellow mosaic virus (TYMV)   and Turns (TYMV) . This ins (TYMV) ; howeverynthesis .Figure 1) . HSPs were shown to be assisting the viral RNA recruitment and viral replication complexes (VRCs) assembly (The nucleus-encoded chlPGK interacting with the 3′ UTR  was demoigure 1) . The genigure 1) . The advigure 1) . Heat shigure 1)  could heassembly . Accordiin vitro and in vivo  and Tobacco mosaic virus (TMV) (Arabidopsis (A 5′ to 3′ exonuclease (XRN4), a protein component enriched in BaMV RdRp preparation, was demonstrated to enhance the accumulation of BaMV . The memus (TMV) , the prebidopsis , downregFigure 1).Some host factors identified by cDNA-AFLP could influence the replication of BaMV in an indirect manner so that these proteins could not be revealed by using the strategy of detecting the direct interaction with viral products. NbRabG3f was demonstrated to be a positive regulator in BaMV replication by loss- and gain-of-function assays . Rabs arFigure 1).Based on the host factors identified so far for BaMV replication, BaMV RNA entry into the host cell would require chlPGK to usher the viral RNA into the chloroplasts. The transport of the viral RNP complex needs the chaperon Hsp90 to cross the membrane and assemble the functional replication complex. During this process of trafficking from the cytoplasm to the chloroplasts, NbXRN4 might be involved in reducing the activities of siRNA-mediated silencing. Once the viral RNP is transported into the chloroplasts, the VRC needs the anti-oxidant enzyme NbGSTU4 to neutralize the oxidative stress inside the chloroplasts for an efficient minus-strand RNA synthesis , similar to those revealed in Chinese wheat mosaic virus (Potato mop top virus (A few host factors were identified to participate in the process of BaMV movement. A RabGTPase-activating protein (GAP) designated as NbRabGAP1 was demonstrated to participate in BaMV cell-to-cell and systemic movements . Rabs, aic virus , or to sop virus .N. benthamiana (NbSTKL), an upregulated gene that is post BaMV inoculation, was demonstrated to be critical in the movement step of the BaMV infection cycle . Furthermore, another kinase, casein kinase 2α (CK2α), which interacts with BaMV CP in PD, might assist the release of viral RNA from the RNP movement complex during the virial RNP complex passage through the PD  (A serine/threonine kinase-like protein from on cycle . The resigure 2) .Figure 2). Obviously, some other factors are also required for this process such as the target of NbSTKL.Taken together, the identification of NbRabGAP1 involved in the movement of BaMV supports the idea that the movement could be made through the vesicle trafficking path . To reacFigure 1)  once interacted with BaMV RdRp, screened by a yeast two-hybrid technique, and displayed an inhibitory effect on the RdRp activity with a dosage-dependent fashion (igure 1) . PMTS1 cFigure 1). The purified recombinant, GAPDH, could specifically inhibit the synthesis of minus-strand RNA of BaMV/satBaMV in an in vitro replication assay. GAPDH is a multifunctional enzyme involved in quite diverse activities in cells, including glycolysis, cellular dysfunction, cell death, apoptosis, association with cytoskeleton and vesicles transport, exportation of nuclear RNA, and DNA repair  found in the purified RdRp complexes could bind the stem-loop C/poly(A) in the 3′ UTRs of satBaMV and the pseudoknot/poly(A) in the 3′ UTR of BaMV . FurtherA repair . In Arabxidation , but couxidation . AlthougRegarding the movement of potexviruses, both MP and CP are vital for efficient cell-to-cell movement and vascular transport . Post-trFigure 2). Also, the two conserved cysteins forming the disulfide bond were demonstrated to play a key role in BaMV movement (One of the thioredoxin proteins, NbTRXh2, an upregulated gene post BaMV inoculation, was demonstrated to restrict the movement of BaMV . NbTRXh2movement . TherefoTaking all the available results into account, it can be concluded that some of the host factors are unique to BaMV, while some of them could be applied to other viruses. Some host factors could assist virus replication and movement, but some of them are involved in resisting virus infection. This review summarized a few host factors identified with different strategies and their possible roles in BaMV infection. However, to complete an accurate understanding of BaMV infection, more host factors need to be identified. Based on our current understandings, a few processes in BaMV infection cycle are still unclear. One of the most challenges is to uncover the process of how the newly synthesized RNAs are transported out of the chloroplasts where BaMV replicates. Hopefully, a much clearer picture of the infection cycle of BaMV can be obtained in the near future by knowing how of these factors involved.Y-PH wrote the viral replication part of the manuscript. I-HC wrote the viral movement part of the manuscript. C-HT wrote the rest part and edits the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eventually, a three-dimensional (3D) FE model in small-hole drilling of Al6063/SiCp composites, using multi-objective identified constitutive formalism, is developed. Comparison with the experimental observations in thrust force, torque, and chip morphology provides valid evidence on the applicability of the developed multi-objective identification strategy in identifying constitutive parameters.Discrepancies in capturing material behavior of some materials, such as Particulate Reinforced Metal Matrix Composites, by using conventional ad hoc strategy make the applicability of Johnson-Cook constitutive model challenged. Despites applicable efforts, its extended formalism with more fitting parameters would increase the difficulty in identifying constitutive parameters. A weighted multi-objective strategy for identifying any constitutive formalism is developed to predict mechanical behavior in static and dynamic loading conditions equally well. These varying weighting is based on the Gaussian-distributed noise evaluation of experimentally obtained stress-strain data in quasi-static or dynamic mode. This universal method can be used to determine fast and directly whether the constitutive formalism is suitable to describe the material constitutive behavior by measuring goodness-of-fit. A quantitative comparison of different fitting strategies on identifying Al6063/SiC Particulate Reinforced Metal Matrix Composites (PRMMCs) present low-temp properties, high strength-to-weight ratio, good wear resistance, and low thermal expansion coefficient, but poor machinability ,2,3. DueMost engineering materials exhibit varying deformation behavior at low (quasi-static) and high (dynamic) deformation rates or temperatures. This brings great challenges to the establishment and identification of the material constitutive model that can describe the mechanical response under different loading conditions. The Johnson-Cook (J-C) constitutive model is one of the most commonly used semi-empirical phenomenological ones for describing the plastic deformation behaviors at high strain, high strain rate, and high temperate, especially suitable for the simulation of machining processes . Its disIn the J-C constitutive equation, the flow stress and Cook .(1)σ= are investigated to focus on the mechanical responses in quasi-static compression and SHPB tests. p/65p composites, with uniformly distributed SiC particles in the absence of obvious particulate clustering and preferred orientation. At low strain rates, the temperature rise caused by plastic strain at room temperature is considered to be negligible, while at high strain rates, there exists coupling between strain rate and deformation temperature since a great amount of heat is not timely transferred. To deduce the uncoupled relationship between temperature and strain rate, the isothermal mechanical behavior at elevated temperature is to be considered in the dynamic tests at high strain rates [Al6063 matrix composites reinforced with 65% volume fraction of SiC particulate . The specimens were machined into the cylinders of Φ6 mm given in . In SHPBp/65p composites in quasi-static and dynamic SHPB compression tests, respectively. It is to note that in quasi-static mode, Al6063/SiCp/65p composites show rate dependence to stress response, which differs from pure Al6063 alloy with no rate-dependent mechanical behavior in quasi-static compression [−4 to 10−2 is not significant, so the reference strain rate in fitting constitutive model for Al6063/SiCp/65p composites is identified as 10−2. As illustrated in p/65p composites is shown to be of significant rate- and temperature-dependence. The higher the deformation rate is, the greater the data fluctuation of the measured stress-strain curves is; the higher the deformation temperature is, the less the fluctuation is. Therefore, the measurement noise at high strain rates and low temperatures become more considerable in dynamic mode, which influences the reliability and accuracy of fitted data in constitutive identification.pression . This ispression . The strσ(εp = 0.002) is within a range of 10%, The J-C model in Equation (1) involves a set of five parameters  and (11), an overall objective function is represented as additive relationship with different weighting factors by Equation (12).The fluctuation in some flow stress curves corresponding to high strain-rate loading conditions observed in Since the measure error covariance Similarly,So, based on the definition of weighting matrix, this seemingly bi-objective optimization actually in practice is a multi-objective minimization problem relying on the number of loading condition combinations. Thus, the following aim is to minimize the bi-objective reduced Chi-squared error summation for the flow stress data experimentally determined from different loading conditions. An updated Levenberg-Marquardt formula based on the gradient scaling is employed in solving Equation (12) .(19)[JTInitialization and update of damping factor p/65p material model identification are summarized in R2). This indicates that it is not always necessary to modify such a classic constitutive model under scientific hypothesis that the parameter coefficient A is a variable to be optimized rather than offset yield point. Besides, the overall fit standard error of 21.88 MPa indicates the model fitting errors are of the same order as the measured ones. That is to say, the multi-objective optimization model can be relatively capable of fitting experimental noise.Based on the relationship of −1 and 2200 s−1 is mainly attributed to the experimental measurement resulting from poor signal-to-noise ratio, which easily occurs in the SHPB tests for particulate reinforced metal matrix composites. Hence, the important significance of introducing the varying weighting factors under different loading conditions into formulating multi-objective function is manifested in this aspect. When compared to the ad hoc strategy, the multi-objective optimization one enables the identified J-C model to fit the observations under high temperatures and high strain rates more accurately, in An accurate and reliable set of J-C material parameters under various combination of strain rates and temperatures could give rise to similar chip morphologies and cutting forces to experimental observations in cutting ,29,30. Tp/65p composites is given in The J-C material model for Al6063/SiCIn terms of macro- and microscopic strain-stress relationships under homogeneous strain boundary conditions shown in fined by .(22)ε‾=p/65p composites are roughly considered macroscopically isotropic. The plastic strain part on both sides for Equation (22) should be equal, and SiC particulate is elastically deformed, and therefore the failure criterion for Al6063 matrix composites can be approximately deduced using Equations (21) and (22) asIn macro scale, due to the polycrystalline aggregates characteristic and randomly oriented distribution of SiC particle in as-cast Al6063 composites, the mechanical responses of Al6063/SiCEquation (23) is normalized to obtain the failure parameters.p/65p composites are deduced by Equation (25), as shown in The Johnson-Cook damage parameters given in . The JohThe shear criterion is a phenomenological one for describing shear band localization, with the following form (26)εpS, haveWhen the damage factor Provided that material damage is initiated, the stress-strain law originally based on J-C material model no longer describes the material deformation behavior accurately . To avoiThe overall damage variable A linear softening law of flow stress with equivalent plastic displacement After damage initiation, the effective flow stress Therefore, when the value of D reaches 1, the material failure occurs, accompanied by element deletion.p/65p composites involving α-SiC hard phase and Al6063 matrix, the specific heat capacity can be estimated according to the rule of mixtures by Equation (35).p/65p composites for material modeling are listed in For Al6063/SiCp/65p composites. For workpiece modelling, the workpiece part neighbored to the tool tip are accounted for in drilling model, and a cone-like concave machined surface is prefabricated on the workpiece surface, such that stable drilling can be arrived at as soon as possible, as shown in A three-dimensional (3D) drilling model based on FE-based appoach has been built for simulating small-hole manufacturing process of Al6063/SiCIt is worth noting that at least five-layer locally meshed elements along the feed direction are allocated for feed per revolution, only by such meshing strategy can the chip be formed, as shown in The stick/slip contact model across tool-chip interface is adopted, as suggested by Zorev , for toop/65p composites based on the constitutive formalism determined using multi-objective optimization strategy. The thrust force is the applied resultant force on cutting tool along the z direction.The cutting forces and chip morphology were obtained numerically by running FE model of drilling Al6063/SiCp/65p composites, the drilling experiments of Al6063/SiCp/65p composites were performed on a CNC machining center DMU 80 monoBLOCK , as shown in To validate the built constitutive equation and drilling model for Al6063/SiCDue to semi-enclosed characteristics and the deformed complexity of drilling operation, the contact between the newly-formed chips by element deletion and machined surface in 3D drilling is difficult to define, so the chips flow away from the workpiece side wall, and do not impede the tool operation. This is in contradiction to the fact that in realistic drilling the newly-formed chips is inconvenient to remove, thus increasing cutting forces. To better approximate the simulation conditions, a peck drilling operation (with 0.05 mm step size) that facilitates the chip removal is employed to reduce the interference from the chips.Based on special consideration of drilling modelling, only the force signals at stable cutting are extracted for comparison. To eliminate the interference when the forcing frequencies approach natural frequencies of piezoelectric dynamometer a, the TyBased on our drilling modelling strategy, the simulation results during stable cutting is take into account for comparison with the experimental data. When compared to the experimental observations in The mechanical behaviors of the workpiece play a significant role in mechanics of chip formation and evolution where the workpiece material is subjected to plastic deformation and shear along the primary shear zone by dint of cutting tool . The detp/65p composites in quasi-static compression and SHPB tests are investigated to obtain the stress-strain responses for identifying J-C constitutive constants. Aimed at hard identification of material parameters using conventional ad hoc method, a weighted multi-objective optimization strategy for identifying J-C constitutive parameters is proposed, coupled with updated Levenberg-Marquardt implementation algorithm. When compared to ad hoc optimization strategy, multi-objective strategy applied for J-C parameters optimization leads to a higher predictability of experimental observations and better capability of fitting experimental noise. It is found that it is not always necessary to modify such a classic J-C constitutive model under scientific hypothesis that parameter the coefficient A is a variable to be optimized rather than offset yield point.In this paper, the mechanical behaviors of Al6063/SiCp/65p composites was implemented, based on the constitutive model determined using weighted multi-objective strategy and failure criteria in a maximum sense. The maximum prediction error of no more than 11.00% for thrust forces indicates the reliability and accuracy of the proposed drilling model. It can be found from numerical and experimental results that there are the better machining conditions at rotational speed of 2000 rpm and feed of 75 mm/min. The developed FE model allows for presenting the thrust force dependence on the drilling conditions in the much more visual and usable form. Accurate prediction of thrust force is of great importance for investigating drilling induced edge defects at hole exit and entrance in PRMMCs or delamination defects in laminated composites. Hence, this proposed FE model can be used to optimize cutting processes to improve drilling performance and hole quality, based on the cutting forces effect on drilling induced defect. Accurate prediction of chip formation contributes to the disclosure of material removal mechanism. Comparative results of thrust force, torque, and chip morphology also verify the feasibility of the developed multi-objective constitutive identification strategy with varying weighted consideration to each combination of deformation rates and temperatures.A 3D FE simulation in small hole drilling of Al6063/SiC


The renaissance of cancer immunotherapy is, nowadays, a reality. In the near future, it will be very likely among the first-line treatments for cancer patients. There are several different approaches to modulate the immune system to fight against tumor maladies but, so far, monoclonal antibodies may currently be the most successful immuno-tools used to that end. The number of ongoing clinical trials with monoclonal antibodies has been increasing exponentially over the last few years upon the Food and Drug Administration (FDA) approval of the first immune-checkpoint blockade antibodies. In spite of the proved antitumor effect of these reagents, the unleashing of the immune system to fight cancer cells has a cost, namely auto-inflammatory toxicity. Additionally, only a small fraction of all patients treated with immune-checkpoint antibodies have a clinical benefit. Taking into account all this, it is urgent new therapeutic reagents are developed with a contained toxicity that could facilitate the combination of different immune-modulating pathways to broaden the antitumor effect in most cancer patients. Based on preclinical data, oligonucleotide aptamers could fulfill this need. Aptamers have not only been successfully used as antagonists of immune-checkpoint receptors, but also as agonists of immunostimulatory receptors in cancer immunotherapy. The simplicity of aptamers to be engineered for the specific delivery of different types of cargos to tumor cells and immune cells so as to harvest an efficient antitumor immune response gives aptamers a significant advantage over antibodies. In this review all of the recent applications of aptamers in cancer immunotherapy will be described. The recent success in clinical trials of different approaches to elicit protective immunity in cancer patients, especially immune-checkpoint blockade antibodies, have brought to prominence the importance of the immune system in the control of tumors ,2,3. CanO-alkyl nucleotides, among others [There is evidence in nature that nucleic acids, especially RNA, can fold into complex globular structures with protein-like functions. In fact, the ribosome, one of the most complex enzymes found in nature and responsible for protein translation, is made of RNA. Thus, it is reasonable to consider the feasibility to select high-affinity and specific oligonucleotide ligands that exhibit complex globular tertiary structures. The term “aptamer” was coined by Elligton and is derived from the Latin word “aptus,” which means “fit,” and the Greek word “meros,” which means “particle.” The first oligonucleotide aptamers were described several years ago by Elligton and Gold ,9. They g others . Some reg others ,17. ThisOligonucleotide-derived aptamers display some important advantages when compared with monoclonal antibodies. Monoclonal antibodies, as cell-based products, have a much higher production cost and a complex regulatory approval to reach good manufacturing production (GMP), whereas aptamers are chemically-derived products, which implies a reduced production cost. As mentioned before, cancer immunotherapy is leading towards multipronged approaches aimed at combining several immune-modulatory reagents. Therefore, it would be desirable to have simpler regulatory approval and more cost-effective translational reagents to combine them in future clinical trials. Protein molecules, as monoclonal antibodies, are more likely to induce T cell-dependent neutralizing antibodies after repeated administrations that could be critical in long-lasting treatments of chronic diseases. In cancer patients that have been treated with immune-checkpoint monoclonal antibodies there is still no data on this mechanism of resistance, but it should be taken into consideration for study as a possible mechanism of resistance, especially in responder patients that relapse and need to reinitiate treatment. The presence of neutralizing antibodies that reduces or abrogates the therapeutic efficacy has been documented even in whole humanized monoclonal antibodies . So far,The induction and maintenance of the immune response is tightly regulated by constant immune checkpoints. Currently, the most relevant immune checkpoints in the field of cancer immunotherapy are Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4) and Programmed cell Death protein 1 (PD1), which are expressed on the surface of T lymphocytes. The FDA-approved Ipilimumab (anti-CTLA4), Nivolumab, and Pembrolizumab (anti-PD1) monoclonal antibodies are able to unleash the antitumor immune response in cancer patients, showing unprecedented responses in aggressive tumors . We are CTLA4 is highly homologous to the co-stimulatory receptor CD28; both receptors compete for the same ligands CD80 and CD86, but CTLA4 has a much higher affinity. CTLA4 blockade would probably reduce the threshold of T-cell activation, perhaps favoring the priming of tumor-reactive T cells . CTLA4 aOn the other hand, PD1 is one of the main receptors expressed in exhausted T cells. An anti-PD1 DNA aptamer has been recently described. In this case, the aptamer exhibited a more modest Kd (167 nM), but was still enough to block PD1-PDL1 interaction and showed a potent antitumor effect in the PEGylated form in a colon carcinoma murine model . PolyethRecently my team has developed the first antagonist TIM3 aptamer. TIM3 is another receptor expressed in exhausted T cells; the blockade of PD1/ Programmed death-ligand 1 (PDL1) and TIM3 has been shown to display a synergistic antitumor effect . The aptO-methyl groups, conserving its activity. The aptamer was tested in vivo eliciting a therapeutic effect in tumor-bearing mice [There are also immunosuppressive cytokines secreted in the tumor microenvironment that are recognized by the tumor-infiltrated lymphocyte acting as immune checkpoint. An example of that is IL10. A 2′F-RNA aptamer that blocks the IL10 receptor on the T lymphocyte was described; the aptamer was truncated and selectively modified with 2′-ing mice .Immune checkpoints are not only limited to extracellular receptors; there are also very important ones intracellularly. Aptamers can be used to deliver specific cargos  to the intracellular compartment by targeting cell-specific receptors ,45,46,47Another very important intracellular immune checkpoint that regulates the function of Treg is Foxp3 ,27. We mAnother cancer immunotherapy approach to boost the antitumor immune response is to provide artificial stimulatory or co-stimulatory ligands to tumor antigen-specific lymphocytes . AgonistHowever, with these types of reagents we are at high risk of triggering important side effects, as they would activate indiscriminately all of the lymphocytes in the body that are expressing the co-stimulatory receptor. That could have very serious deleterious effects, as it was observed with the superagonist CD28 antibody clinical trial in which several patients suffered massive cytokine storms . 4-1BB aThe major advantage of this approach is that we were able to reduce the effective therapeutic dose over the non-targeting agents very significantly (aptamer and antibody), which would be translated into lesser toxicity. In fact, we did not observe any of the toxicity associated with the treatment 4-1BB agonistic antibodies. Either the targeting or non-targeting 4-1BB agonistic aptamer at their therapeutic dose did not display the toxicity observed with the 4-1BB antibody; that may be related with the bio-distribution and the higher half-life of the antibody in the blood (likely attributed to the Fc region or isotype). As an extension of this work Schrand et al. developed a bi-specific aptamer to target 4-1BB co-stimulation to the tumor stroma, by using an aptamer against Vascular Endothelial Growth Factor (VEGF), which is a soluble cytokine overexpressed in several types of tumors . This isYet, a main limitation factor in cancer immunotherapy is the lack of tumor antigenicity; several tumors do not express enough potent tumor neo-antigens to be recognized by the immune system as a foreign rejectable tissue. The use of immune-checkpoint blockade agents or co-stimulatory agonists on low antigenic tumors would elicit poor antitumor immune responses ,70,71,72Randomized, Partially-Blinded, Multi-Center, Active-Controlled, Dose-ranging Study Assessing the Safety, Efficacy and Pharmacodynamics of the REG1 Anticoagulation System in Patients with Acute Coronary Syndromes), it turned out to be that the toxicity was due to a PEG allergic reaction and not to aptamers, as the small fraction of patients that show signs of toxicity have anti-PEG antibodies [Since the selection of the first aptamer by Tuerk et al. in 1990 [tibodies ,74. In stibodies ,48,64 (F