Despite global vaccination efforts, influenza virus continues to cause yearly epidemics and periodic pandemics throughout most of the world. Many of us consider the generation of broader, potent and long-lasting immunity against influenza viruses as critical in curtailing the global health and economic impact that influenza currently plays. To date, classical vaccinology has relied on the generation of neutralizing antibodies as the benchmark to measure vaccine effectiveness. However, recent developments in numerous related fields of biomedical research including, HIV, HSV and DENV have emphasized the importance of Fc-mediate effector functions in pathogenesis and immunity. The concept of Fc effector functions in contributing to protection from illness is not a new concept and has been investigated in the field for over four decades. However, in recent years the application and study of Fc effector functions has become revitalized with new knowledge and technologies to characterize their potential importance in immunity. In this perspective, we describe the current state of the field of Influenza Fc effector functions and discuss its potential utility in universal vaccine design in the future. Despite our efforts in controlling the spread and health toll that influenza plays on a global level, influenza virus continues to cause yearly epidemics and intermittent pandemics. The degree of morbidity and mortality is astounding; with 10–20% of the world’s population infected and 290–650,000 deaths per year , (2) theThe “holy grail” of influenza vaccine development for many is a universal influenza vaccine that provides broad and effective protection against both influenza A and B viruses. Such strategies to date include but are not limited to eliciting broadly neutralizing antibodies to the surface hemagglutinin stem region (HA-stem), the M2-protein, the and NA protein. These vaccines all show great promise, and some have been tested in humans. These vaccine approaches have relied on a distinct immunological mechanism for the protection afforded. Furthermore, with testing of these vaccines in human clinical studies we are likely to gain a greater understanding of the immunological mechanisms of vaccine-mediated protection which will be further beneficial for rational immunogen and vaccine design. Vaccination or infection principally generate antibodies that target the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) proteins. These HA-specific antibodies neutralize virus by binding to regions proximal to the receptor binding site and inhibiting the ability of the virus to either enter or egress from the host cell. The sites targeted by neutralizing antibodies have mapped to five-distinct sites within the HA head region  ,16,17,18Fc-mediated effector functions provide an essential link between the role of innate immune system and the adaptive immune system. These effector functions rely not only on the binding of the antibody to the antigen but also engagement of the antibody constant-region. The Fc-receptor functions have long been characterized during influenza infection and vaccination, these functions include:Antibody-dependent complement mediated-lysis (ADCL), are antibodies  which bind to the surface of virus-infected cells. Subsequently, C1q binding triggers the complement cascade, and the formation of the membrane attack complex at the surface of the target cell leading to a number of antiviral effects including killing of the virus-infected cell and promoting a pro-inflammatory environment. Complement fixation has been shown to play an important role in controlling influenza infection with studies showing that mice deficient in C3 have impaired viral clearance and increased viral loads in the lungs ,29,30. AAntibody-dependent phagocytosis (ADP) involves antibodies  opsonizing the surface of virus-infected cells or immune complexes forming with virus. Innate phagocytic cells bearing either, CD32, CD64, or CD89 receptors then bind to the antibodies and proceed to uptake the cell or antigen complex. This can also lead to the activation and secretion of some pro-inflammatory mediators and the presentation of peptide antigen on the surface MHC receptor of the phagocyte. Phagocytes have been shown to intake the influenza virus in an antigen-dependent fashion, either using beads ,38,39 orAntibody-dependent cellular cytotoxicity (ADCC), involves the FcγRIIIa (or ortholog FcγRIV in mice) bearing cells such as NK cells, monocytes/macrophages and neutrophils engaging the Fc-region of antibodies , which specifically bind to antibodies presented on the surface of virus-infected cells. Cross-linking of the CD16 (FcγRIIIa) receptor leads to phosphorylation of the C-terminal immunoreceptor tyrosine-based activation motif (ITAM) to activate downstream calcium-dependent signaling pathways. This results in the release of granzyme B and perforin from preformed lysosomes, which together facilitate DNA fragmentation and apoptosis of the target cell. Activation of innate immune cells such as NK cells can have a number of other consequences including the secretion of antiviral cytokines and chemokines, such as IFN-γ and TNF, which have substantial antiviral and immunopathological properties.Antibodies should not merely be considered as those that “mediate ADCC” or those that “neutralize”, rather antibodies can have a number of these effector functions available to them, given their isotype and the ability to engage Fc-receptors. Some of the factors that seem to impact Fc-effector functions include the concentration, isotype and the binding specificity. Additionally, Fc-glycosylation has been shown to influence the ability of Fc-receptors to mediate ADCC. In this perspective, we concentrate our attention on highlight important findings in influenza ADCC, and discuss some of the limitations and potential areas for future investigation. Human IgG antibodies are composed of two heavy-chains and two light-chains linked via disulphide bonds. The structure of IgG antibodies are separated into distinct regions, and the antigen recognition is mediated by “fragment antigen binding” (Fab)_domains, comprising the complementarity-determining regions (CDRs) located at the N-terminal end of the heavy and light chains that binds an epitope . The C-tEach IgG molecule contains a highly conserved IgG-Fc N-glycan in CH2 domains. In particular, the highly conserved asparagine 297 (N297)-glycosylation site is conserved in all IgG subclasses and amongst many species . This gl−/− deficient mice [Fc-receptor function is vital to the utility of stem-specific antibodies in particular, ADCC function. The isolation of stem-specific antibodies has brought new hope to the development of a universal influenza vaccine. Early studies showed that mice administered an FcR-binding deficient (FI6-LALA) version of the broadly neutralizing antibody FI6, were less likely to survive a lethal dose of influenza virus compared to the unmutated form of FI6 mAb . This fient mice . More spent mice . These sThe importance of Fc-receptor function in protecting mice from lethal influenza challenge has been exemplified in a number of mAbs targeting different proteins of the influenza virus. There are some broadly conserved epitopes within the HA-head region that are non-neutralizing but facilitate potent ADCC activity. A study by DiLillo et al. exemplifies two broadly neutralizing anti-head mAbs (4G05 and 1F05) and three non-neutralizing head-specific mAbs  that can protect from lethal influenza challenge in mice . LikewisIn accordance with these findings, similar findings have been found for candidate universal vaccines developed to the influenza-stem. The most notable are stabilized stem-immunogens which provide broad protection from influenza infection in various influenza animal models  and are associated with antibodies that mediate Fc-receptor functions ,65. FurtLastly, some studies have suggested that ADCC may provide some level of protection from influenza infection in humans. A recent study by Jacobsen et al. showed that passive transfer of human serum from H5 vaccinated individuals protected mice from heterologous challenge with the H1N1pdm09 virus, this antibody-mediate protection observed correlated with binding antibodies, in particular, those that mediate in vitro ADCC . NotablyThe next generation influenza vaccines will likely require different assays to measure immunogenicity. Typically, influenza vaccine immunogenicity has been measured using standard HAI or microneutralization assays, whereby a fold-change in titer is used to determine whether a vaccine is immunogenic. Such assays would not be effective when the vaccine primarily relies on non-neutralizing effector functions for their activity , such as some HA stem vaccines or M2 based vaccines. These vaccines will require alternative assays to show immunogenicity, such as using in vitro ADCC assays. We have briefly provided a description and features of commonly used in vitro ADCC assays for testing human clinical samples, with the hope that they may be of value in testing immunogenicity of candidate universal vaccines.51Cr) release assay [51Cr-labelled infected target cells , which are incubated with serum and either purified NK cells or donor PBMCs. Following incubation, the level of 51Cr released from dead or dying cells is assessed in the media . This assay detects the release of enzyme lactate dehydrogenase from dead or dying cells and via measuring the absorbance of LDH reaction products ,80 FiguB. Such aOther approaches to measuring Fc-receptor activity has involved the utility of flow-cytometry to measure either the markers on target cells or effector cells. A flow-cytometry based method to measure target cell killing has been to label target cells  with a membrane dye (PKH-67 or similar) and measure their killing via the increase in fluorescence of the target cells upon uptake of 7-AAD dye by dead or dying cells ,83,84. TUpon stimulation of NK cells with either antibody-coated or MHC devoid target cells (direct killing), there is significant upregulation of a number of surface markers and expression of an number of antiviral cytokines ,86. The Since healthy adult blood donors are typically exposed to influenza throughout their lifetimes; we found that this form of assay made it challenging to measure influenza-specific ADCC-activity of donor plasma samples. To overcome this problem, our laboratory coated antigen onto a plate and subsequently added plasma and NK cells (in the form of purified NK cells or PBMCs). This allowed antibodies from serum or plasma to bind to the antigen-coated onto the plate and for purified PBMCs/NK cells to then be used. The NK cells bound to immobilized antibody-complexes and become activated, which was measured via intracellular cytokine (ICS) staining and collected via flow-cytometry  Figure D. This aIt is evident that antigen coated onto a plate may not reflect the density and conformation of antigen exposed on the surface of a virus-infected cell. To determine the “total” influenza-specific ADCC response, we and others have measured NK cell activation upon exposure to infected A549 cells ,93. SimiTo facilitate the running of a large number of clinical trials samples simultaneously, many groups have turned to using more high-throughput methods. Such an assay was developed by Promega (ADCC Bioassays), wherein Jurkat T cell lines have been transfected with the individual Fc-receptors from either humans or mice. These cell lines activate via a NFAT-response element which drives luciferase expression when there is Fc-receptor engagement F. The luVery recently, there has been the development of dimeric Fc-receptors ligands. These ligands contain two identical Fc receptors that are linked by a FcγRIIa membrane proximal stalk region ,104,105.Numerous studies have shown that ADCC-mediating antibodies can be generated by both influenza vaccination and influenza infection in adults and children. Early studies on influenza ADCC demonstrated that vaccination of healthy adults with an inactivated influenza vaccine or experimental infection with live influenza virus could generate antibodies capable of mediating cytotoxicity ,107. RecThe study of cross-reactive ADCC-Abs in serum has been of great interest to our laboratory, due to their potential utility in providing broader immunity. Further, these cross-reactive ADCC-Ab were present even when neutralizing antibodies to the given virus-strain are not present . This haCross-reactive ADCC-Abs are generated early in life and expand throughout a lifetime. It is clear that even in cord-blood and early during infancy, these cross-reactive ADCC-Abs are generated and increase as you become older ,94,110. A number of remaining questions hinders the utility of Fc-receptor function to aid in the design of universal influenza vaccines. Firstly, some studies have suggested that ADCC may have an immunopathological role during influenza infection rather than a role in protective immunity ,111,112.+ antibodies cannot [The ability of antibodies to mediate Fc-receptor function is thought to be dependent on the epitope in which it binds. Stem-specific mAbs can engage FcRs and mediate ADCC activity, whereas head-specific mAbs  are limited in their ADCC capacity . This has cannot ,114,115.The analysis of influenza-specific ADCC-Abs in human serum has provided some key insights into the generation of these antibodies following vaccination and infection. However, many questions remain that will require detailed analysis of both serum antibody and B cell responses including: (1) Are all cross-reactive antibodies encompassed by stem-antibodies or are there other distinct cross-reactive epitopes? Studies by others would suggest that there are additional H1–H3 cross-reactive epitopes that can be targeted by antibodies ,117. TheThe functionality of NK cells and their ability to mediate ADCC in humans is determined by a number of factors, including the concentration of antibodies, isotype, activating and inhibitory co-receptors on the surface of the NK cells. An interesting study performed by Goodier et al. illustrates a particular example of this whereby the CD16 (FcγRIII) receptor on NK cells is significantly downregulated following TIV, partially via ADAM17 matrix metalloprotease mediated cleavage . This daThe culmination of the information described in this perspective hopefully shows that there is still a great deal to be learnt about Fc-mediated effector functions and their potential utility in universal vaccine design. Indeed, as greater interest is drawn into this exciting area of research, this will hopefully aid in its development and serve in the generation of a universal vaccine in the future.


Pulmonary veins (PV) are often the trigger to atrial fibrillation (AF).Occasionally, left PVs converge on a common trunk (LCT) providing a simplerstructure for catheter ablation.To compare the clinical characteristics and outcomes of ablation inparoxysmal atrial fibrillation (PAF) of patients with or without LCT.Case-control study of patients undergoing first-ever catheter ablationprocedure for drug refractory PAF. The information was taken from patients'records by means of a digital collection instrument, and indexed to anonline database (Syscardio(r)). Clinical characteristics and procedures werecompared between patients with or without LCT (LCT x n-LCT), adopting alevel of statistical significance of 5%. The primary endpoint associatedwith efficacy was lack of atrial arrhythmia over the follow-up time.One hundred and seventy two patients with PAF were included in the study, 30(17%) LCT and 142 (83%) n-LCT. The clinical characteristics, comorbidities,symptoms scale and risk scores did not differ between the groups. There wasAF recurrence in 27% of PAF patients in the n-LCT group and only 10% ofpatients in the LCT group (OR: 3.4 p: 0.04) after a follow-up of 34 ±17 months and 26 ± 15 months respectively.Patients with a LCT have a significantly lower recurrence rate when comparedto patients without this structure. It is mandatory to report the results ofAF catheter ablation as a PV anatomical variation function. As LCT can be easilyidentified by computed tomography, knowing the clinical outcome of ablation in thispopulation may be relevant in clinical decision-making when an ablative procedure isindicated. Therefore, the objective of this study was to compare the clinicalcharacteristics and outcomes of patients undergoing PAF ablation with and withoutVPs common left trunk.In most patients, four pulmonary veins reach the left atrium. However, previousstudies suggest that PV anatomical variations are related to a higher incidence ofAF.This is a single-center, case-control study conducted between January 2011 andDecember 2015, with the inclusion of patients (≥ 18 years old) undergoingthe first catheter ablation procedure to treat PAF that does not respond toantiarrhythmic drugs with a minimum follow-up of 12 months. The information wascollected and indexed in a digital database aimed at AF ablation (SysCardio(r)software). Along with computed tomography (CT), the presence of the LCT wasconfirmed through a 3D atrial model constructed during the procedure with anelectroanatomic mapping system by (NavX(r)). Patients with persistent orlong-term persistent FA, patients with previous ablations and FA of reversibleetiology, hypertrophic cardiomyopathy, rheumatic heart disease, congenital heartdisease and prior catheter ablation were excluded from the sample .All procedures were performed under general anesthesia, orotracheal intubation,and invasive monitoring of blood pressure by radial or left femoral puncture,under the care of an anesthesiologist. Transseptal punctures were performed withechocardiography assistance, which was maintained throughout the procedure.bolus of 100mg/kg was performed,followed by continuous infusion to keep activated coagulation time between 350and 450s.All patients underwent circumferential isolation of the PVs through a 3.5-mm tipirrigated catheter ablation without contact force measurement, usingradiofrequency energy with applications of up to 35 watts and 43ºC for30-45 seconds, and demonstration of electrical VPs entrance and exit block inrelation to the left atrium at the end of the isolation. After the demonstrationof entrance and exit block, patients received 18 mg of IV adenosine bolus. Incases of electrical reconnection, new mapping-guided radiofrequency applicationswere performed until adenosine-mediated reconnection no longer occurred. Theapplications in the left atrium posterior wall were performed with 20 watts forup to 15 seconds, and were interrupted in case of increased esophagealtemperature > 38ºC. Applications to the left atrium posterior wallwere monitored through an esophageal thermometer with multiple coated sensors(Circa(r)), and were stopped whenever there was a change in esophagealtemperature above 38ºC. During all procedures performed with anelectroanatomical mapping system based on thoracic impedance (EnSite Navx -Abbott(r)), IV heparin The vein anatomy was defined as normal when two right pulmonary veins and twodistinct left pulmonary veins were viewed, and the presence of the left commontrunk was defined when the two left pulmonary veins coalesced on a path > 10mm from before insertion into the left atrium in a common ostium .2DS2-VASc. There was a clinical follow-up of 1, 3,6 and 12 months after the procedure with ECG and at least two continuous 5-dayelectrocardiographic (Holter) monitoring throughout the wholeclinical follow-up. At the 10th week after ablation, patients were encouraged toundergo a 5-day Holter. Any atrial arrhythmia greater than 30 seconds durationdocumented after 1 month of blanking period indicatedarrhythmia recurrence.2Symptoms severity before ablation and during any recurrences was characterizedby the Canadian Cardiovascular Society Severity of AtrialFibrillation (CCS-SAF) score, and the score of atrial fibrillationrelated symptoms of the European Heart Rhythm Association(EHRA).7After the procedure, patients remained on antiarrhythmic drugs for 1 month, and anticoagulant for at least 3 months regardless ofCHAPatient characteristics and procedures, recurrence rates after a singleprocedure, and complication rates were compared according to the groups: LCT(case) or non-LCT (control). The sample size was determined by a 1: 4 ratio forcases and controls with study power of 80%.Continuous variables were described as mean and standard deviation and comparedusing unpaired Student's t-test (two-tailed), respecting the criteria ofnormality by Shapiro-Wilk test. Categorical variables were described by absolutenumber and percentages in relation to the total sample, and were compared usingthe χ² test or Fisher's exact test. The level of statistical significanceadopted was 5%. Kaplan-Meier curve was used to evidence the relapse-free ratesover the follow-up time, and Log-Rank test was used to evaluate the differencebetween the groups (LCT x non-LCT). Statistical analysis was performed using IBMSPSS Statistics Editor software, version 22.0.One hundred and seventy-two patients were enrolled between 2011 and 2015 in a singlecenter in Brazil. Thirty (17%) had LCT. There was no difference in follow-up timebetween cases and controls, with all patients completing a minimum follow-up of 12months.2D2-VASc) forstroke were similar among the samples. There was no significant difference in theseverity of symptoms associated with AF (CCS-SAF and EHRA scores) between cases andcontrols. Four percent of the patients had a previous history of stroke.There were no major complications related to procedures and / or hospitalization. Among the minor complications there were 4pseudoaneurysms and 1 inguinal hematoma, all in the non-LCT group, treatedclinically without surgical intervention . There w10 Our study suggests that pulmonary vein common lefttrunk patients have a more favorable clinical outcome after catheter ablation, witha clinical relapse around 10%. These results can be obtained without comprisingprocedure safety.The durability of PVs electrical isolation is directly related to the efficacy of thepercutaneous treatment of AF, so that PVs electrical reconnection seems to be themain mechanism for post-ablation AF relapse.11 In the past, whenthe need for ablation of the 4 pulmonary veins in the same procedure was stilldiscussed, and maneuvers were used to trigger AF, some authors suggested that whenAF originated in LCT, it was not necessary to perform right PV ablation.13 Over the years, this concept proved to be inadequatebecause some recurrences occurred from foci in the right PVs.8 For this reason, currently the information on the presence ofthe left common trunk helps more in the indication of the procedure rather than inthe definition of the ablation strategy, which, unless otherwise indicated, willinclude the ablation of the LCT and the right PVs.LCT, when present, has been indicated as the predominant origin of the triggers ofAF.In the present case-control/single-centered study of patients undergoing the firstablation procedure for PAF, it is suggested that - in comparison to the standardanatomy - the presence of the LCT favors the results in the percutaneous treatmentof AF, with lower recurrence rate and low complication rates in a long-termanalysis. These findings highlight the importance of knowing PVs anatomy for theefficacy and safety of AF ablation.The definition of LCT (approximately 20% of the sample) deserves discussion. Ingeneral, the diagnosis is quite simple, through detomography or cardiac resonance.In our study, we chose an unequivocal definition of common trunk, that is, whenthere was a minimum distance of 10 mm between the common ostium of the left PVs andthe bifurcation of their left lower and upper branches. Thus, the diagnosis of LCTwas intentionally simplified. This aspect has important clinical relevance. Whenidentifying LCT, the clinical decision for catheter ablation can be simplifiedbecause the patient with this type of anatomical change has an excellent clinicalresult after ablation. In fact, there was no comparison between ablation and the useof antiarrhythmic drugs in this subgroup, but it is worth remembering that allpatients ablated in our study were unresponsive to antiarrhythmic drugs.Holters, and even coronaryangiography with the intention of diagnosing a possible trigger for AF.Infrequently, however, these patients undergo heart CT/MRI that, if properlyperformed, could define the pulmonary venous anatomy and assist in the clinicaldecision facilitating the indication of catheter ablation.This observation has an important practical effect. In clinical practice, it is notuncommon that patients with AF and anatomically normal heart by transthoracicechocardiography undergo numerous diagnostic tests. As a rule, these patientsundergo multiple stress tests, 24-hour 15 In our results, the relapse rate in the LCT groupwas 3 times lower than in the non-LCT group, with patients' clinical characteristicsbeing similar and homogeneous in all analyzes.Our study is not aligned with evidence that the presence of PVs anatomical variations(number and disposition) would be associated with a higher and more advanced degreeof atrial remodeling from the electrical and structural point of view and,consequently, worse post-ablation outcome.16 Therefore, efficient tissue contact presumes agreater energy delivery through radiofrequency and formation of a more stable andhomogeneous scar,17 which wouldultimately result in a longer lasting isolation of PVs. That way, other studiesusing cryoablation also presented a more favorable result in patients with LCT. Incryoablation studies, however, the presence of LCT was not related to a moresatisfactory outcome when compared to patients without the structure.18From the technical point of view, the presence of LCT facilitates the manipulationand the contact of the ablation catheter in the left atrium. The simpler themanipulation, the better the contact with the region to be ablated. Recent studieshave shown that the tissue-to-contact relationship during ablation is crucial forlesion formation and is linked to better outcomes.The objective of this study was to describe the clinical characteristics andefficacy and safety outcomes in patients undergoing catheter ablation in thetreatment of AF. This is a case-control study and, certainly, there arelimitations. First, the sample size is small and may not be sufficient to detectdifferences between the two groups, particularly because of the low prevalenceof LCT. Second, the presence of observation bias is recognized, since PVsanatomy is revealed during the procedure. Third, no detailed tool was used toassess symptoms in the presence of AF, but rather CCS-SAF and the EHRA score,which are generic scales for symptomatic evaluation. Despite this, the studyprovides an interesting perspective on the invasive treatment of AF in patientswith LCT.In our sample, patients with LCT who underwent the first catheter ablation procedureto treat PAF presented a lower rate of relapse compared to patients without thisanatomical alteration. The research on LCT should be incorporated to theinvestigation of PAF patients because ablation is more effective in this group ofpatients.


METCAM/MUC18 is an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family. It can carry out common functions of CAMs which is to perform intercellular interactions and interaction of cell with extracellular matrix in tumor microenvironment, to interact with various signaling pathways and to regulate general behaviors of cells. We and other two groups previously suggested that METCAM/MUC18 probably be utilized as a biomarker for predicting the malignant tendency of clinical ovarian carcinomas, since METAM/MUC18 expression appears to associate with the carcinoma at advanced stages. It has been further postulated to promote the malignant tendency of the carcinoma. However, our recent research results appear to support the conclusion that the above positive correlation is fortuitous; actually METCAM/MUC18 acts as a tumor and metastasis suppressor for the ovarian carcinoma cells. We also suggest possible mechanisms in the METCAM/MUC18-mediated early tumor development and metastasis of ovarian carcinoma. Moreover, we propose to employ recombinant METCAM/MUC18 proteins and other derived products as therapeutic agents to treat the ovarian cancer patients by decreasing the malignant potential of ovarian carcinoma. Ovarian carcinoma is the fifth leading cause of female cancer death in USA . The reaCell adhesion molecules (CAMs) very likely play a substantial role in the malignant progression of carcinomas, since they govern the social behaviors, influence outlasting, proliferation and growth of tumor cells and modulate generation of new blood vessels in the tumor microenvironment . We haveIn this review, we show negative correlation of the level of METCAM/MUC18 expression in various human ovarian carcinoma cell lines with their malignant status. We indicate negative effects of METCAM/MUC18 over-expression on the epithelial-to-mesenchymal transition and on the tumorigenesis and metastasis of two human ovarian carcinoma cell lines. Then we propose preliminary detailed knowledge of how METCAM/MUC18 may induce suppression of the malignant tendency of human ovarian carcinoma cell lines. Finally, we describe perspectives of the studies and suggest possible clinical applications.CAMs participate in many significant normal biological functions, such as organ generation, tissue organization, de novo formation of endothelial cells from mesoderm cell precursors (vascularization) and formation of new blood vessels from pre-existing ones (angiogenesis), immune response, inflammation, wound healing and cellular general behaviors . An alteAltered expression of several CAMs, such as mucins ,10, inteMETCAM/MUC18 was first discovered to be profoundly expressed on the cellular surface of the majority of malignant human melanomas (thus named as MUC18) and suggested to play a central role in the progression of human melanoma  . Later, Human METCAM/MUC18 is expressed in about ten normal tissues: hair follicular cells, smooth muscle cells, endothelial cells, cerebellum, basal cells of the lung, activated T cells, intermediate trophoblasts , breast In contrast, over-expression of METCAM/MUC18 decreases tumorigenesis of a mouse melanoma cell line, K1735-9 ,35, nasoThe impact of METCAM/MUC18 over-expression on malignant tendency of ovarian carcinoma has not been systematically investigated. The recent findings in this aspect are outlined in the following section.METCAM/MUC18 overexpression was first observed to be significantly correlated with the ovarian carcinomas at advanced stage and with the serous and undifferentiated subtypes of the tumors. Since METCAM/MUC18 is expressed at a higher level in the carcinoma than residual disease, it was suggested to be useful for prognosticating tumor relapse and as a self-reliant predicting marker for poor prognosis of ovarian carcinoma  also repTo investigate if the above hypothesis is correct, we compared the expression levels of METCAM/MUC18 in one immortalized normal human ovarian epithelial cell line (IOSE) and five human ovarian cancer cell lines, which were established from primary adenocarcinoma or metastatic ascites . If we aR clones [To investigate if METCAM/MUC18 expression alters the in vitro and in vivo behaviors of the ovarian cancer cells, it should be useful to enforcedly increase the expression of the gene in the two cell lines, SK-OV-3 and BG-1, which weakly expressed METCAM/MUC18. For this purpose, we transfected the human METCAM/MUC18 cDNA, which was inserted in a mammalian cells-expressible vector, pcDNA3.1+, into these cell lines and selected for the high expressing G418R clones ,24. ThenR clones were subjected to the determination of the effects of enforced expression of METCAM/MUC18 on in vitro motility and invasiveness.Activation of the epithelial-to-mesenchymal transition (EMT) is critical for the acquisition of the malignant nature of carcinoma cells  clone, indicating that METCAM/MUC18 over-expression reduced the tumorigenesis of SK-OV-3 cells in the nude mouse model [After se model . We deduse model , whereasse model .IP) cavity). We found that the mice injected with cells from the control vector clone formed swollen abdomen, whereas the mice injected with the METCAM/MUC18-expressing clone did not develop swollen abdomen. After surgically opening the abdominal cavities, we found that the final weights of abdominal tumors and volumes of ascites in the group injected with the control vector clone were significantly heavier and larger than those injected with the METCAM/MUC18-expressing clone. We deduced that over-expression of METCAM/MUC18 decreased the tumorigenicity and ascites formation of SK-OV-3 cells at orthotopic (IP cavities) site in nude mice [Then, we further investigated effects of METCAM/MUC18 over-expression on tumorigenesis of SK-OV-3 cells after injecting the above clones at the orthotopic sites  . The in SC injection of the two METCAM/MUC18-expressing clones/cells and one empty vector control clone, the final tumor weights of the METCAM/MUC18-expressing clones and the empty vector control clone were found to be not statistically different, indicating that increased METCAM/MUC18 expression minimally impacted the final tumor weights via this injection route. We concluded that METCAM/MUC18 over-expression did not significantly induce in vivo tumorigenesis when non-orthotopic site was injected with the cells [We further carried our studies on impacts of METCAM/MUC18 expression on tumorigenesis of BG-1 cells in an athymic nude mouse model also via two injection routes, the (non-orthotopic) subcutaneous (SC) injection route and the (orthotopic) intraperitoneal cavity route . After She cells .In contrast, when the above clones were intraperitoneally injected in nude mice, the final tumor weight of the METCAM/MUC18-expressing BG-1 clone was significantly decreased in comparison to that of the empty vector control clone, indicating that increased METCAM/MUC18 expression significantly decreased in vivo tumorigenesis of BG-1 cells . Taken tTaken together, a higher expression level of METCAM/MUC18 significantly reduced the in vitro tumor formation and the in vivo tumor proliferation at the orthotopic site. Thus, in addition to SK-OV-3 cells , over-exAs shown above, precaution must be taken that the biological effects demonstrated in one cancer cell line may not be completely reproduced in other cancer cell line, because the origin from which each cell line was established is different, as each tumor or metastatic lesion is a mixed bag of tumor/cancer cells with slightly different mutated genotypes.To understand further the detailed knowledge of METCAM/MUC18-mediated suppression of the malignant propensity of ovarian carcinoma cells, perhaps some clues may be deduced from what we know from the METCAM/MCU18-mediated tumorigenesis of various tumor cell lines, such as cancers of breast and prostate, melanoma and nasopharyngeal carcinoma. METCAM/MUC18 could impact many downstream signaling pathways that regulate proliferation, survival pathway, apoptosis, metabolism and angiogenesis of various tumor cells ,9. For tWe offered solid evidence to strongly suggest that METCAM/MUC18 is a new suppressor for the tumorigenesis and malignant propensity of the two human ovarian carcinoma cell lines, SK-OV-3 and BG-1 ,24. (a) The first novel aspect of the above conclusion is that it appears to disagree with an apparently positive correlation of clinical prognosis with the increased expression of METCAM/MUC18 in malignant ovarian carcinoma specimens . We suggThe second novel aspect of the above conclusion is that the tumor suppressor role of METCAM/MUC18 in the human ovarian carcinoma cell lines ,24 is alThe third novel aspect of the findings is that METCAM/MUC18 seems to act a dual role, either as a promoter or suppressor, in the malignant propensity of several tumor cell lines . It eithThe fourth novel aspect is that a tumor and metastasis suppressor role played by human METCAM/MUC18 in the malignant propensity of human ovarian carcinoma cells and other cancer cell lines appears to suggest the probability that METCAM/MUC18 may trigger on tumor dormancy , which iThe current studies have laid an important biological basis for inspiring future intense investigation to further understand the detailed knowledge of METCAM/MUC18-mediated suppression of ovarian tumorigenesis and metastasis. However, the current research has not completely proven that the suppressor role of METCAM/MUC18 is generally applicable to all human ovarian cell lines, such as HEY, CAOV-3 and NIHOVCAR3 and other cell lines. To prove that the suppressor role of METCAM/MUC18 in all human ovarian carcinoma cell lines, human ovarian carcinoma cell lines other than the two used in the above research should also be tested by using siRNA to knock down endogenously expressed METCAM/MUC18. To further understand the role of this CAM in regulating cancer metastasis, other future experiments may include: (a) searching for the heterophilic ligands or partners of METCAM/MUC18 , (b) sysMetastasis of primary tumor to distant organs is responsible for 90% cancer-associated mortality. It would be a major success in cancer treatment if it is possible to arrest the metastatic tendency of cancer cells and keep cancer cells confined to primary site. Alternatively, it is also a major success if it is possible to keep the cancer cells at the condition of dormancy, as micro-metastatic lesions. Since metastasis suppressors are able to trigger tumor dormancy, the metastasis suppressor role of METCAM/MUC18 in the malignant propensity of human ovarian carcinoma cell lines may be useful for designing novel therapeutic methods to treat clinical ovarian carcinomas, similar to the clinical application of other metastasis suppressors, such as KISS1, KAI1, nm23, MAP2K4 and some micro-RNAs to arrest other cancers . Three gTo further design a novel therapeutic strategy to more aggressively stamp out ovarian carcinoma, we may be able to take advantage of both our METCAM/MUC18-derived recombinant proteins, oligopeptides, or small molecule mimetics and the highly effective anticancer platinum drugs to formulate a combinatory treatment strategy . For thi


The unprecedented bright X-ray beams have been applied to determine electron density distributions and electronic structures.In the past 10 years, the world has witnessed the revolutionary development of X-ray free electron lasers (XFELs) and their applications in many scientific disciplinaries . The comThanks to the femtosecond X-ray laser pulses, experiments can be carried out in the so-called ‘diffraction-before-destruction’ mode : the X-rThere are four essential components in XFEL protein crystallography: (1) batched microcrystal sample preparation; (2) sample delivery and manipulation; (3) XFEL and instrument operation for diffraction data collection; and (4) data analysis and model interpretation. Four articles nicely cover components (1), (2) and (4). The sample delivery methods are summarized by Nam, with a focus on the sample delivery medium that carries the crystals to the XFEL interaction volume . The samEach crystal usually results from a single diffraction pattern within tens of femtoseconds of exposure time, so the measured intensity for each reflection in a single pattern is incomplete (inaccurate) due to the excited volume defined by the overlapped region between the thin Ewald shell and the full reflection volume. Therefore, a high measurement redundancy is desirable for accurate diffraction intensity. This can be achieved by the high repetition rates of XFEL pulses. The processing of large data volumes requires special software for screening ,19,20, aWith the ‘diffraction-before-destruction’ approach, the cryogenic protection of crystals is not required, so the experiments can be done with the samples at room temperature (if the temperature changes during the injection can be neglected). Therefore, the structures might be different from those determined at synchrotrons in some regions, as discussed by Park et al. in the HIV-CCD structure analysis . Such coWith the femtosecond XFEL pulses, the temporal resolution can also reach the femtosecond time scale in theory, making it possible to study ultrafast dynamics. Light-triggered reactions are very suitable for time resolved SFX, revealing conformational changes down to a sub-picosecond time scale ,27. EnzyThe ultimate goal of structure studies is to probe structure information from single molecules using XFELs, similar to the cryogenic electron microscopy method. Even with the most powerful XFELs, the resolution for single particle imaging can only reach a few nanometers for viruses ,31. NoneCompared to conventional crystallography, the SFX at XFELs has a few differences:The protein crystallography at XFELs has shown encouraging results—127 structures were found using XFEL as the searching keyword in the protein data bank when this manuscript was prepared. We would anticipate more structures reported using the XFEL crystallography method as several X-ray lasers are commissioned. Two super-XFELs, the LCLS-II in USA and the SHINE in China, each designed to produce up to 1 million XFEL pulses per second, are under construction. More excitingly, the time resolved crystallography at XFELs will capture the conformations of proteins in-action, producing three-dimensional (3D) molecular movies to reveal the functioning state down to atomic levels.


What is the cost-effectiveness of short-course radiotherapy vs long-course chemoradiotherapy for patients with locally advanced rectal cancer?In this economic evaluation, short-course radiotherapy was the cost-effective strategy, with an incremental cost-effectiveness ratio of $133 495 per quality-adjusted life-year. However, for the subset of patients with distal tumors, long-course chemoradiotherapy was the cost-effective approach, with an incremental cost-effectiveness ratio of $61 123 per quality-adjusted life-year.Patients with locally advanced rectal cancer should be treated with preoperative short-course radiation therapy unless they require tumor downstaging prior to resection, in which case long-course chemoradiotherapy is cost-effective. This economic evaluation compares the cost-effectiveness of short-course radiotherapy vs long-course chemoradiotherapy for locally advanced rectal cancer. Although both short-course radiotherapy and long-course chemoradiotherapy have been practiced in parallel for more than 15 years, no cost-effectiveness analysis comparing these 2 approaches in patients with locally advanced rectal cancer has been published.To analyze the cost-effectiveness of short-course radiotherapy vs long-course chemoradiotherapy for the treatment of patients with locally advanced rectal cancer.This economic evaluation used a cost-effectiveness model simulating 10-year outcomes for 1 million hypothetical patients aged 65 years with locally advanced rectal cancer treated with either short-course radiotherapy or long-course chemoradiotherapy, followed by surgery and chemotherapy. Utilities and probabilities from the literature and costs from the Healthcare Bluebook and Medicare fee schedules were used to determine incremental cost-effectiveness ratios. It was assumed that long-course chemoradiotherapy would result in higher rates of low anterior resection (LAR). To model preference-sensitive care, a 2-way sensitivity analysis was conducted in which the utilities of the no-evidence-of-disease (NED) states with LAR and abdominoperineal resection (APR) were simultaneously varied. The analysis was repeated for patients with distal rectal tumors. Analysis was conducted from January to October 2018.Short-course radiotherapy and long-course chemoradiotherapy.Incremental cost-effectiveness ratios.Short-course radiotherapy was the cost-effective strategy compared with long-course chemoradiotherapy . Two-way sensitivity analysis revealed that the cost-effective approach for a given patient depended on the utilities for the NED-LAR and NED-APR states. Assuming that a greater proportion of patients with locally advanced distal tumors undergoing long-course chemoradiotherapy (39%) would proceed to LAR compared with those treated with short-course radiotherapy (19%), long-course chemoradiotherapy was the cost-effective approach .Short-course radiotherapy was the cost-effective strategy compared with long-course chemoradiotherapy for patients with locally advanced rectal cancer. The cost-effectiveness of short-course radiotherapy vs long-course chemoradiotherapy was sensitive to the utilities of the NED-LAR and NED-APR health states, highlighting the importance of care that is sensitive to patient preference. Long-course chemoradiotherapy was the cost-effective approach for patients with distal tumors. Recent prospective randomized trials have compared these 2 treatment approaches and suggested no difference in short-term disease outcomes.5Radiation therapy prior to total mesorectal excision has been shown to yield low rates of pelvic recurrence in the treatment of rectal cancer.6 Supporters of SCRT point to patient convenience, lower cost, and less acute radiation toxicity. Those favoring LCRT have emphasized increased likelihood of tumor downstaging, allowing for increased rates of sphincter preservation for low-lying tumors and R0 resection in the setting of threatened mesorectal fascia. Given the generally favorable outcomes for locally advanced rectal cancer, proponents of LCRT have noted the potential increased risk of late radiation-induced toxic effects in the setting of hypofractionated radiation. While the Trans-Tasman Radiation Oncology Group (TROG)3 and Bujko et al5 reported no significant differences in late toxic effects between these 2 regimens to date, the median follow-up of this study is limited.Although LCRT remains the standard of care in the United States, many countries treat locally advanced rectal adenocarcinoma with SCRT.7 The significant debate regarding the optimal neoadjuvant regimen for patients with locally advanced rectal cancer lends itself well to cost-effectiveness analysis. Cost-effectiveness analysis is a set of mathematical tools designed to compare the relative costs and benefits of treatment alternatives.8 Accordingly, the goal of this study was to perform a cost-effectiveness analysis to compare SCRT vs LCRT in patients with locally advanced rectal cancer.In the existing health care system, high costs without proportional improvements in quality or outcome have prompted demands for change, and value-based models have been suggested. In health care, value, as defined by health outcomes achieved per dollar spent, provides a framework on which health systems may base their resource allocation decisions.CHEERS) reporting guideline.9We developed a Markov model to analyze the cost-effectiveness of SCRT vs LCRT for patients with locally advanced rectal cancer. In a Markov model, hypothetical cohorts of patients transition between different health states in fixed time increments and at defined probabilities . Markov Two treatment strategies were compared. In strategy 1, patients with locally advanced rectal cancer underwent LCRT followed by delayed total mesorectal excision (at 6-12 weeks) and adjuvant chemotherapy. In strategy 2, patients underwent SCRT followed by surgery 1 week later and subsequent adjuvant chemotherapy. For the base case analysis, we assumed 3-dimensional (3-D) conformal radiation treatment.2 Patients then either remained in one of the NED health states or proceeded to the local recurrence or distant recurrence health states.2 We assumed that these rates were equivalent for SCRT and LCRT. At first transition to local recurrence, patients had the opportunity to undergo salvage treatment with possibility of entering a salvaged local recurrence state. Patients in the local recurrence and distant recurrence health states were able to transition to the death state as cancer-related deaths.10 Patients were also able to transition to the death state based on causes unrelated to cancer; the probability of dying of causes unrelated to cancer was based on Social Security actuarial life tables.11Hypothetical patients began in 1 of several no-evidence-of-disease (NED) health states, having undergone either LCRT or SCRT with low anterior resection (LAR) or abdominoperineal resection (APR). Low anterior resection represents sphincter-sparing surgery with intrapelvic anastomosis, whereas APR is performed when the tumor is low-lying and sphincter sparing is not possible. Low anterior resection is typically accompanied by temporary ileostomy to allow adequate postoperative healing of the irradiated anastomosis and to minimize risk of anastomotic leaks. Ileostomy reversal is typically performed after completion of postoperative chemotherapy. Unlike LAR, APR requires a permanent colostomy. Hypothetical patients were distributed among these NED health states based on previously published rates.3 This assumption was based on the TROG study,3 Bujko et al,5 and a recent meta-analysis,12 which reported no significant differences in late toxic effects between SCRT and LCRT. Given that these rates were assumed to be equivalent between the 2 strategies and would therefore not affect the incremental cost-effectiveness ratio (ICER) (the main outcome), the probabilities, cost, and utilities associated with these health states were excluded from the model. We further assumed that LCRT resulted in higher rates of LAR through tumor downstaging.1 Utilities were derived from a study explicitly evaluating patient utility values of stage II/III rectal cancer treated with resection, chemotherapy, and radiation therapy with or without permanent ostomy.13 Utilities and costs were discounted at 3% annually. Costs are presented in 2018 US dollars.We assumed that rates of late toxic effects were equivalent for SCRT and LCRT.14 and costs for hospital outpatient services on the basis of Current Procedural Terminology codes specific to radiation, including costs of consultation, simulation, weekly treatment management, treatment planning, and delivery. Medicare reimbursement was also used to calculate colostomy supply costs. Given the variability in postoperative care, hospital admission length, and anesthesia costs based on time in the operating room, which cannot be accurately calculated using National Medicare fee schedules, surgical costs were determined based on the fair price from the Healthcare Bluebook for rectal resection and closure of stoma.15 The fair price is the price that consumers should reasonably expect a medical service to cost if shopping for care. The fair price is calculated from actual amounts health plans have paid on claims and includes hospital services, physician services for the procedure and routine postoperative care, and anesthesia. Costs of acute toxic effects from treatment were not included as they were assumed to bias the model toward SCRT, with results to be taken as robust if SCRT was found to be cost-effective under this assumption (SCRT with early surgery is associated with lower severe acute toxicity than LCRT5). A payer perspective was used to derive costs in this study. Radiation costs were calculated using 2018 National Medicare fee schedules2 and were converted to 1-year probabilities to inform our model. The model was created and analyzed with TreeAge Pro 2017 . To ensure convergence in model outcomes, we simulated 1 million patients, and the history of events for each individual was tracked over the time span of the study. Our model was validated by comparing our model outputs with the expected local recurrence, distant recurrence, and overall survival rates of the German Rectal Cancer Study.2We developed a discrete-time Markov model to simulate 10-year outcomes for hypothetical patients aged 65 years . Analysi8 We defined cost-effectiveness at an ICER of $100 000 or less per quality-adjusted life-year (QALY).We report the ICER when one strategy is more effective but more costly compared with the other. A treatment strategy with an ICER value less than the societal willingness to pay (WTP) is considered to be cost-effective.13 To model preference-sensitive care, we conducted 2-way sensitivity analyses in which we simultaneously varied the utilities of the NED-APR and NED-LAR states. To explore whether our findings were sensitive to variations in surgical and radiation costs, we conducted 2 additional 2-way sensitivity analyses varying these costs. Because Medicare costs for radiotherapy may underestimate costs as compared with private payers, we performed an additional 2-way sensitivity analysis in which we varied the surgical costs assuming that radiation costs were 3 times higher than in the base case analysis, with SCRT costing $21 669 and LCRT costing $57 933. In addition, we performed a probabilistic sensitivity analysis with 100 000 samples to consider the combined uncertainty related to multiple parameters in the model. Beta distributions were used for probability and utility variables, and normal distributions were used for cost variables.Sensitivity analyses were used to evaluate the effect of adjusting the assumptions of the model. We performed 1-way sensitivity analyses varying key parameters. Clinical parameter ranges were based on data obtained via literature review where possible; otherwise, parameters were varied over a range of 0.5 to 1.5 times the base case value.Our base case analysis assumed that all SCRT and LCRT were planned and delivered using 3-D conformal radiation treatment. To model the current practice of many radiation oncologists, we repeated the analysis assuming 3-D conformal treatment for LCRT but intensity-modulated radiation therapy for SCRT.1 irrespective of tumor location within the rectum. To determine the cost-effectiveness of SCRT as compared with LCRT for patients with distal tumors only, we reran the analysis assuming that all patients had distal tumors. For this analysis, we assumed that 39% of patients who underwent LCRT where APR was initially deemed necessary were able to undergo LAR, compared with 19% of those undergoing SCRT.1Our base case analysis included all patients eligible for the German Rectal Cancer Study,Finally, we also calculated the cost per permanent colostomy prevented and the number of patients needed to undergo LCRT to prevent a permanent colostomy. These values were calculated by the following formulas: cost per permanent colostomy prevented = (cost of strategy 1 − cost of strategy 2)/(number of colostomies in strategy 1 – number of colostomies in strategy 2); and number needed to treat with LCRT to prevent a permanent colostomy = 1/(proportion of patients with colostomy in strategy 1 – proportion of patients with colostomy in strategy 2).We found that SCRT and LCRT were associated with QALYs of 4.72 and 4.79, respectively. In the base case analysis, SCRT was the cost-effective strategy compared with LCRT  .In the base case analysis, the utility of NED-LAR was 0.59 and the utility of NED-APR was 0.50. To model how different individuals value living in a health state with no evidence of disease with and without permanent ostomy, we performed 1-way sensitivity analyses varying the utilities of the NED-APR and NED-LAR states. On 1-way sensitivity analysis, LCRT became the cost-effective approach when the utility of NED-APR was below 0.47 . On 1-waTo model the current practice of many radiation oncologists, we performed an analysis where 3-D conformal treatment was used for LCRT but intensity-modulated radiation therapy was used for SCRT. Under this assumption, SCRT remained the cost-effective strategy (ICER of $125 632/QALY) .To further explore whether our findings were sensitive to variations in cost between SCRT and LCRT, we conducted 1-way sensitivity analyses and a 2-way sensitivity analysis varying these costs. To explore whether our findings were sensitive to variations in cost between APR and LAR, we conducted 1-way sensitivity analyses and a 2-way sensitivity analysis varying these costs. 5 To explore whether our findings were sensitive to variation in the benefit of SCRT vs LCRT radiation, we conducted a sensitivity analysis varying the probability of local recurrence conferred by SCRT. We found that LCRT became the cost-effective approach when the 10-year probability of local recurrence risk after SCRT was 10.4% or higher  . On sens16 To date, there have been 2 prospective randomized trials that have compared these 2 radiation treatment approaches.5The treatment of locally advanced rectal adenocarcinoma is unique in that 2 neoadjuvant approaches, SCRT and LCRT, have been studied in parallel and found effective in improving local control.Although the convenience and cost benefit of receiving 5 fractions of radiation therapy instead of 28 are self-evident, LCRT is thought to result in higher rates of sphincter preservation. Under the stated disease and treatment assumptions, we found that SCRT was the cost-effective strategy compared with LCRT for patients with locally advanced rectal adenocarcinoma. Sensitivity analyses that varied costs of surgery and costs of radiation therapy over a reasonable range showed that SCRT was the higher-value treatment over the vast majority of the ranges tested.There were several scenarios in which LCRT became the cost-effective approach compared with SCRT. For instance, we found that LCRT became the cost-effective approach when it was associated with a 3.3% absolute improvement in locoregional disease control. However, it is critical to note that currently available published data do not support any degree of improved locoregional control of LCRT over SCRT.13 However, utilities may vary significantly among patients, and such variations can create different optimal treatments for individual patients.8 These results confirm the importance of eliciting patient preferences in decision making, as preference determination would allow for more tailored and cost-effective treatment.We also found that lowering the utility of the NED-APR health state by more than 0.03 below the utility assumed in the base case drove the ICER to favor LCRT. In other words, the benefit of LCRT depends on how patients value living with vs without a permanent ostomy. We used published population-based utility values to inform the analyses of our model.2 Our results therefore suggest that LCRT is the cost-effective approach for patients with distal tumors.Although SCRT was the higher value treatment when including all patients with locally advanced rectal cancer, we reran the analysis with the assumption that all patients had distal tumors that would require APR prior to undergoing radiation. For this analysis, we assumed that 39% of patients who underwent LCRT where APR was initially deemed necessary were able to undergo sphincter-preserving surgery as compared with 19% of those undergoing SCRT.17 patients who underwent APR reported worse body image and worse sexual enjoyment at 1 year as compared with patients who underwent LAR. In a different study that included 336 patients with rectal cancer with a stoma and 117 patients without a stoma, patients with a stoma had significantly worse mental health, body image, and sexual function compared with those without a stoma.18 In addition, those patients with a stoma experienced more fatigue and loss of appetite than those who underwent LAR. Thus, whether potentially avoiding a stoma by undergoing LCRT is a benefit sufficient to justify its increased cost should be openly discussed by all stakeholders.Although LCRT was not cost-effective in the base case scenario, it reduced the number of patients needing a permanent ostomy. Quality of life is often impaired in patients with a stoma. In a study that evaluated LAR vs APR in patients with rectal cancer using patient-reported outcomes in the National Surgical Adjuvant Breast and Bowel Project randomized trial R-04,3 Although late toxic effect rates in the TROG study,3 Bujko et al,5 and a recent meta-analysis12 were no different, basic radiobiologic principles dictate that larger fraction size has a potential for higher risk of late toxic effects that can manifest in subsequent decades. However, despite limited follow-up at this time, although nonsignificant, the absolute rate of late toxic effects in the TROG study appears lower for SCRT than for LCRT, giving a sense that it is unlikely that the longer-term follow-up for the TROG data will show significantly higher late toxic effects in the SCRT group. Given that the time frame for this model was 10 years, we felt that our assumption was reasonable. Additionally, it is important to acknowledge that there are many factors that should play a role in determining the optimal treatment strategy for any individual patient with rectal cancer. Some of these factors, including subjective impact of radiation therapy on ease of achieving a complete total mesorectal excision resection and impact of radiation on surgically induced nerve injury, are beyond the scope of our analysis but should be discussed in the context of optimal care. Consequently, the treatment paradigm of rectal cancer is best decided in a multidisciplinary tumor clinic setting, with robust discussion between the surgeon, medical oncologist, and radiation oncologist. There are newer emergent treatment strategies for locally advanced rectal cancer, including definitive chemoradiation (with nonoperative management) and total neoadjuvant therapy, both in the setting of SCRT and LCRT, that are out of the scope of this analysis.21Several limitations of our study must be acknowledged. First, we made some simplifying assumptions about the natural history and treatment of disease to specify a finite number of clinical events. Second, we may not account for differences in late toxic effects of SCRT vs LCRT with modern techniques, as follow-up data are limited.We found that among patients with locally advanced rectal cancer, SCRT was the cost-effective approach compared with LCRT given current parameters. We found that LCRT became the cost-effective approach when the utility of a patient with permanent colostomy was lower by more than 0.12 than that of a patient without a permanent colostomy. Therefore, physicians should elicit individual patient preferences to maximize quality-of-life outcomes. For the subset of patients with distal tumors, LCRT was the cost-effective approach.


This paper aims to raise awareness of the different disease courses, comorbidities, and therapy situations in patients with giant cell arteritis (GCA), which require a differentiated approach and often a deviation from current treatment guidelines. With the approval of tocilizumab (TOC), which specifically binds to both soluble and membrane-bound IL-6 receptor and inhibits IL-6 receptor-mediated signaling, the spectrum of available effective treatment options has been significantly broadened. TOC yields an extensive range of possible applications that go beyond a glucocorticoid-saving effect. In this context, the treatment of GCA is dependent on the disease course as well as the associated comorbidities. The different stages of GCA in association to co-morbidities require a detailed treatment strategy. Giant cell arteritis (GCA) is an inflammatory disorder of medium- and large-size arteries affecting people older than 50 years. Classically involved vascular sites include the external carotid branches, the ophthalmic, vertebral, distal subclavian, and axillary arteries as well as the aorta. Segmentary inflammation leads to the occlusion of the vessel and to ischemic complications . On the *04 is to be evaluated as a genetic risk factor for the manifestation of GCA  remain the mainstay treatment of this disease. Unfortunately, relapses are common when the GC dose is tapered, leading to prolonged treatment duration and increased incidence of adverse events . MethotrInterleukin (IL) 6 plays a central role in the pathogenesis of GCA, and IL-6 serum levels correlate with disease activity and the likelihood of recurrence .Tocilizumab (TOC) is a humanized monoclonal antibody that blocks IL-6 signaling by binding to the alpha chain of the human IL-6 receptor . The firThe initial treatment objective of GCA is rapid disease control by reducing the concentrations of serum acute-phase reactants and freedom from symptoms as well as the prevention of ischemic organ damage. Treatment guidelines have been published by the European League Against Rheumatism (EULAR) , the BriWithin the scope of GCA treatment, different disease courses, and therapy situations can be observed, which require a differentiated approach. In addition, existing co-morbidities or GC-induced side effects may demand a deviation from therapy recommendations.The present article will discuss these aspects and present possible treatment options, especially regarding the approval extension of TOC.TOC does not only hold potential as a GC-sparing therapy but has already been used in numerous GCA therapy situations. While many case reports primarily focus on the use of the IL-6-receptor blocker in patients with refractory disease, GC dependence or intolerance , the ranIn the registration study GiACTA, 119 patients with newly diagnosed GCA and 132 patients with relapsed GCA were enrolled . HoweverTreatment at new-onsetGC-taperingRecurrence after therapy break.Various therapy settings can be divided into three main categories:In addition, further therapy situations such as GC-resistant or refractory disease (lack of response to GC-induction therapy), flare , GC-dependency (flare during GC reduction), or patients with co-morbidities and GC-induced side effects can be differentiated Figure .Table GCA is not a uniform disease. The latest GEFA guidelines differentiate two patient groups at the time of initial diagnosis: .Uncomplicated GCA without ophthalmic involvement and without arteritis of the aorta or its branchesGCA with ophthalmic involvement and GCA with aortoarteritis (non-complicated and asymptomatic involvement of the aorta or its branches)Accordingly, the GC dosage is adjusted to the risk profile; GCA patients with aortitis require a longer GC therapy due to increased flare rates .Although comparative studies on the most effective GC dosage are lacking, the guidelines recommend a GC dosage of 0.7 mg/kg body weight to 1 mg/kg body weight (maximum 60 mg/day) for this patient group , 39. WitFifty percent of patients show a good response to GC therapy . Both thIn the GiACTA study, 14% of patients with a GC taper over 26 weeks and 18% of patients with a GC taper over 52 weeks achieved a sustained remission of week 12 to week 52 . An incrHigh, fast-acting intravenous GC pulse therapy  is indicated especially in cases of imminent vision loss , 23. GC However, in some cases permanent blindness cannot be avoided, because it takes up to five days after the start of a high-dose GC therapy to control the inflammation of the Aa. ciliares posteriores .Furthermore, a recent published study presented on a small study cohort the usefulness of TOC in the treatment of visual symptoms in GCA. The abstract clearly highlighted the non-effectiveness of TOC in blinded patients . In this18F-fluorodeoxyglucose under TOC therapy .In patients showing GC contraindications at the time of diagnosis, an initial combination of GC and an immunosuppressive agent should be considered. For these patients etanercept and TOC was examined Table . In the First-line therapy with GC does not always lead to disease control (which is not defined in GCA). Numerous articles have been published concerning GC-refractory disease , and somIf intolerance or GC-typical side effects occur during GC therapy, the question of GC-sparing therapy arises. In particular, substances and studies that had the goal of completely renouncing GC after a certain treatment period should be taken into account. Publications that included patients in this therapy situation were found for TOC  and etanAfter an initial high-dose GC therapy, GC is slowly reduced (GC tapering), depending on the response. Once again, a uniformly accepted and in clinical studies preferred tapering schedule is not existing. Ideally, a planned GC tapering succeeds as intended and there is no flare-up of the disease. These patients also have the best chance of achieving a very low daily GC dose (< 5 mg) or finishing GC completely after 1 year. In the GiACTA study, 49% of patients with GC monotherapy had a flare within 52 weeks . These rA flare (relapse) under existing therapy should be distinguished from a recurrence  . In someA flare during GC-tapering in the GiACTA study is an important criterion as in this case the GC dose could be increased at the physician's discretion. The GC monotherapy arms showed higher flare rates  than the TOC study arms including a GC taper over 26 weeks . Consequently, patients under GC monotherapy had almost twice the cumulative GC doses than patients under combination therapy with TOC . Of specIn 40.8% of patients, a certain GC dose cannot be undercut without a new increase in disease activity . EspeciaThe current guidelines of EULAR , BSR 2222, and GQoL and PRO should be a central component of RCT in view of the frequent GC-induced side effects and its long-term application in GCA. In fact, only two studies  assessed SF-36 Table . MTX didSince fatigue is a common side effect of MTX, its general use as GC-sparing therapy might be problematic in GCA patients .A recurrence after a therapy break occurs in about one third of patients .Patients with recurrent disease, no contraindications to GC as well as no high cumulative CG-dosages  can again be treated with a GC monotherapy according to the published guidelines –23.Patients suffering a recurrence after 1 or 2 years of GC therapy usually have a correspondingly high cumulative GC dosage and therefore require GC-sparing therapy. In this context, the EULAR guidelines recommend the same “initial” therapy with GC as for new-onset patients . AccordiIn everyday clinical practice, however, patients with recurrent disease are a particular challenge as they can develop typical co-morbidities within the first 2–4 years . This isThis subgroup evaluation of the GiACTA study suggests that patients with recurrent disease benefit from therapy intensification, as sustained remissions were more frequently achieved in the two TOC study arms compared to the GC monotherapy arms  .RCTs with this patient population are only available for abatacept, leflunomid, and TOC , 24, 33.To monitor GCA under the treatment with TOC, traditional acute phase reactants cannot be used to control the disease activity, whereas GCA patients under the treatment with GC present also normal acute phase reactants at the time of disease flare . In thisThe IL-6-receptor inhibitor has been approved for the treatment of rheumatoid arthritis (RA) in Europe in 2009 and its safety profile has been published several times . The GiAThe TOC-therapy in GCA is significantly increased compared to GC-therapy, where at the TOC-therapy is comparable to other biologic drugs in the treatment of rheumatoid arthritis. With ending of the TOC patent protection in 2020 and the implementation of TOC biosimilars the price of TOC will be marked down.In the course of GCA treatment, numerous situations arise in the clinical routine in which a deviation from therapy guidelines seems to be necessary. However, treatment options as well as co-morbidities are not sufficiently addressed in the recommendations. The approval extension of TOC results in an extensive range of possible applications that go beyond a GC-saving effect. Future studies are necessary to obtain better validated results in efficacy and safety for these treatment situations. New guidelines should also take into account the specific patient profile with regard to the therapy situation and co-morbidities.AP, PO, and PH performed the literature research. AP and PH wrote the first draft of the manuscript, which was then reviewed and edited by PO.PH is an employee of Chugai Pharma Europe LTD, Frankfurt am Main, Germany. The remaining 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.


Negative image-based (NIB) screening is a rigid molecular docking methodology that can also be employed in docking rescoring. During the NIB screening, a negative image is generated based on the target protein’s ligand-binding cavity by inverting its shape and electrostatics. The resulting NIB model is a drug-like entity or pseudo-ligand that is compared directly against ligand 3D conformers, as is done with a template compound in the ligand-based screening. This cavity-based rigid docking has been demonstrated to work with genuine drug targets in both benchmark testing and drug candidate/lead discovery. Firstly, the study explores in-depth the applicability of different ligand 3D conformer generation software for acquiring the best NIB screening results using cyclooxygenase-2 (COX-2) as the example system. Secondly, the entire NIB workflow from the protein structure preparation, model build-up, and ligand conformer generation to the similarity comparison is performed for COX-2. Accordingly, hands-on instructions are provided on how to employ the NIB methodology from start to finish, both with the rigid docking and docking rescoring using noncommercial software. The practical aspects of the NIB methodology, especially the effect of ligand conformers, are discussed thoroughly, thus, making the methodology accessible for new users. Negative image-based (NIB) screening  is a rigIn the NIB screening, a negative image is built based on the target protein’s ligand-binding cavity shape and electrostatics . The NIBWhereas standard flexible docking relies on estimating the favorability of ligand-receptor complexes by summing up the weak interactions, such as hydrogen bonding and the hydrophobic effect, the NIB focuses squarely on the shape/electrostatics similarity of the molecular recognition process. Despite the apparent simplicity of this shape-centric approach, the benchmarking has shown thatthe NIB produces high enrichment as indicated by the area under curve (AUC) values and early enrichment factors with various targets ,3,8. TheApplying 3D similarity- or shape-based methods in the virtual screening schemes increases the diversity of the discovered compounds . With thIn general, flexible docking is better positioned to sample the possible ligand poses than the rigid docking approach. Therefore, the NIB methodology was recently repurposed for rescoring existing molecular docking solutions . The NIBThe study provides simple step-by-step instructions on how to perform rigid docking  or dockiThe negative image-based NIB;  screeninThe practical aspects of the NIB and R-NiB methodologies are discussed below using a virtual screening or benchmarking example, i.e., the screening is performed using the directory of useful decoys (DUD) test set ,19 and aThe terminal commands and further practical information are given in the In the NIB screening , the rigThe DUD  ligands Ultra-fast speed and computational efficiency are hallmarks of both the NIB screening  and the The protonated ligand 3D coordinates were used as an input to generate multiple conformers using the --confab option in OBABEL  command.The success of the NIB screening is dependent on the input protein 3D structure, especially its ligand-binding cavity conformation, used as a template for the negative image generation . The inphttps://www.rcsb.org/).Two PDB-entries were selected for the NIB screening with COX-2. The PDB-entry 3LN1  Figure C is usedThe extra chains and other non-peptidic residues do not necessarily have to be removed for building NIB models using PANTHER , althougThe A chain of the PDB-entry 3LN1  was seleThe NIB model generation using PANTHER  requiresThe COX-2 inhibitor ligand celecoxib, or CEL, , altogether 12 alternative NIB models would be outputted for COX-2. This is because the cavity houses several residues capable of H-bonding, and each proton of the H-bond donor groups, such as the hydroxyl group, is given an alternative position that affects the charge distribution of the model The NIB model must be restricted to the area of the cavity that facilitates the ligand binding.(2)The NIB model shape should resemble, however loosely, an envisioned or actual ligand molecule occupying the cavity.(3)The NIB model cavity points must contain crucial charge/electrostatics information required for mimicking the ligand-receptor H-bonding, as the shape similarity alone might not be enough for ensuring rigid docking success B.There are at least three major concerns regarding the model viability in the NIB screening:The preliminary model or Model I (command #9) outputted by PANTHER  matches The NIB screening is performed using the similarity comparison algorithm ShaEP  Figure , which wThe rigid docking is performed using the default settings of ShaEP  commandB,E is esFollowing the rigid docking, one should inspect best-ranked docking poses and fine-tune the NIB models with the benefit of hindsight . For exaThe third NIB model, or Model III H, was geThese enrichment metrics  indirectIn addition to the cavity-based rigid docking , the NIBThe COX-2 ligands ,19 were In theory, the R-NiB or any other rescoring methodology can improve the docking yield , becauseDespite this specific example , the abiThe ShaEP scoring can be combined with the original PLANTS scoring in the consensus scoring  Figure A. The R-The normalization and re-weighting of the PLANTS and ShaEP scoring A are perThe cyclooxygenase 2 (COX-2) benchmarking example shows that the negative image-based NIB;  1] scre scre1] sThe better performance of the cavity-based rigid docking routine over the flexible docking suggests that the shape complementarity is a crucial factor for the COX-2 inhibitor binding, at least with the benchmark test sets. That is not to say that other docking algorithms or PLANTS cannot outperform the NIB methodology in virtual screening experiments on a case-by-case basis . Both thThe NIB model generation is a straightforward process when the target protein’s ligand-binding site is a well-defined cavity . The chaImportantly, the COX-2 example shows that the rigid docking  is not oPrior to this study, the ligand 3D conformer generation had been tested thoroughly for the purpose of assessing its effects on the ligand-based screening ,32; howeThe effectiveness of the outputted 3D conformers differed between the software in the NIB screening. Their rigid docking performance is undoubtedly case-specific, and one should not draw too far-reaching conclusions based on the COX-2 benchmarking only. For example, the single low-energy conformers worked well with COX-2 ; Table 2The negative image-based rescoring R-NiB;  producesn = 10) to have enough explicit solutions to re-rank and improve the docking performance utilizing the cavity’s shape/charge information. The NIB screening , althouThe test sets, containing both active ligands ligs in  and decoOBABEL. Open Babel Package or OBABEL 2.4.0  was usedMAESTRO. The 3D conversion of the ligands (SMILES-to-MOL2) was performed using LIGPREP in MAESTRO. The protonation was set to match pH 7.4, and potential tautomers were created. The ligand 3D conformer generation was performed with CONFGEN in MAESTRO using an OPLS3  force field .MARVIN. MOLCONVERT 17.6.0 in MARVIN (ChemAxon) was used for the ligand 3D conversion (SMILES-to-MOL2). CXCALC 17.6.0 in INSTANT JCHEM (ChemAxon) was used to protonate and create the potential tautomers at pH 7.4 and generate 3D conformers for the ligands. The number of conformers was scaled from 1 to 64 according to the rotatable bond number calculated with MayaChemTools . The parRDKit. RDKit open-sourced cheminformatics was used in the ligand 3D conversion (SMILES-to-MOL2) and conformer generation. The protonation at pH 7.4 was prepared using MARVIN, and partial charges were added using OBABEL with the MMFF94 force field .PLANTS. The flexible docking poses , which wThe ligand preparation was performed using five 3D conformer generation routines  to studyThe X-ray crystal structure of COX-2 with the bound inhibitor celecoxib (PDB: 3LN1) ,39. Thiswww.medchem.fi/panther). A few of the previous default settings were amended for this study . The fundamental input options and updates to the packing method selection in the model generation with PANTHER  center coordinate(s) for generating the NIB model. The centroid should be within a protein cavity that can accommodate the envisioned ligand binding (Center(s) (-cent) option designates the user-defined  option is a key determinant of how the pocket is filled, together with the selected centroid C.Keep (-keep)/Do not fill (-nofill) option is used to define a list of ligand residues with which the NIB model should not overlap. The area taken by these user-defined residues is excluded from the resulting NIB model. By default, some commonplace cofactors  and water molecules (HOH and WAT) are listed for this option, but the list can be edited to include or exclude any residues of the input PDB file.Multibox (-mbox) option makes it possible to generate a custom NIB model utilizing multiple close-by centroids. This option is especially useful when a single centroid is not enough to build a model that mimics the irregular shape of the target protein’s cavity. For example, the negative image of a peptide-binding site or groove at the protein surface can be built by picking several neighboring centroids and utilizing them together with the multibox option A.Basic multipoint (-bmp) option can be used to generate the pocket center based on any kind of residue included in the input PDB file  option can be used to restrict the volume that the NIB model occupies based on a bound ligand residue in the cavity of the input PDB file D,E. WhenPacking method (-pack) option, which is by default face-centered cubic (FCC), adds the filler atoms of the NIB model. The FCC has been altered to correspond the correct lattice in the updated version of PANTHER . In addiThe input files and NIB models used in the benchmarking example  are given = 10) outputted by the molecular docking software PLANTS [The NIB screening  and the e PLANTS  are not e PLANTS .By combining the ShaEP-based R-NiB scoring with the PLANTS-based molecular docking scoring, it is possible to improve the flexible docking yield further . This inx-axis logarithmic) and to calculate the early enrichment and area under curve (AUC) values in This study described the practical steps and software settings to be used during the negative image-based NIB;  screenin


N-bromosuccinimide or N-chlorosuccinimide was performed. This procedure provides a convenient route to synthetically useful mono-halogenated heteroarene intermediates such as halo-indoles, -aza-indoles, -indazoles and -aza-indazoles. The mild conditions employed and simple protocol provides an advantage over traditional halodecarboxylation procedures that require expensive and toxic metal catalysts, basic conditions, time-consuming intermediate isolation and elevated reaction temperatures.The halodecarboxylation of heteroarene carboxylic acids by treatment with  Halodecarboxylation of the Barton ester results in the formation of both the desired halide and the undesired 2--pyridine side-product, limiting the atom efficiency of the reaction  and N-chlorosuccinimide (NCS).With the above in mind, current methods of halodecarboxylation suffer from the use of highly toxic and expensive metals employed in stoichiometric amounts, requires the use of corrosive elemental halide sources or suffer over halogenation. It is, therefore, desirable to establish a more reliable and economical method for mono-halodecarboxylation of heteroarene carboxylic acids to furnish synthetically useful haloheteroarenes. Presently, we describe a simple base-free method for the regiospecific mono-halodecarboxylation of substituted and unsubstituted heteroarene carboxylic acids under mild and metal-free conditions at room temperature with 2.1 with NBS. The reaction proceeded with two equivalents of NBS in N,N-dimethylformamide (DMF) at room temperature affording product 2 in 35% yield . Examples of indazole acids bearing electron withdrawing (products 3–5) and electron donating substituents (products 8 and 9) underwent decarboxylative bromination in excellent yields under the reaction conditions. However, when the electron donating methoxy group was moved to the 4, 6 and 7 positions on the indazole , no product could be detected. The use of N-methyindazole to afford compound 13 appeared to be unsuccessful, with no halodecarboxylated product observed by LCMS—it appears as though the presence of the free indazole N–H is a requirement for decarboxylation when using the current set of reaction conditions. Halodecarboxylation to afford the 5-amino indazole analogue (product 7) also proved unsuccessful. Initial mechanistic investigations reveal that the reaction possibly proceeds through a non-radical pathway, as the reaction continued to proceed when performed in the absence of light. However, detailed mechanistic investigations are continuing within our laboratory, and so a clearer insight into the lack of reactivity of some substrates will be detailed in due course.With the halodecarboxylation procedure in hand, the indazole carboxylic acid scope was investigated . A varieN-iodosuccinimide (NIS) as halide sources  have been reported in the literature to be problematic to isolate [15 and 16 in reasonable yields.To demonstrate the versatility of the halodecarboxylation procedure for heteroarenes, further reactions with NBS were investigated . Althoug isolate ,27, unde17 and 18 in good yield, implying that more electron deficient systems undergo halodecarboxylation efficiently under the current conditions. Attempts to further expand this methodology to simple benzoic acids have currently proved unsuccessful, although this remains an active area of research within our group.Aza-indole and aza-indazole acids underwent halodecarboxylation smoothly to furnish the corresponding haloheteroarenes 3.In summary, a mild and efficient protocol for the mono-halodecarboxylation of heteroarene carboxylic acids has been developed. It is noteworthy that this halodecarboxylation is metal-free and displays significant advantages over traditional methods of halodecarboxylation that commonly require harsh reaction conditions, toxic reagents or suffer over bromination. The proposed method extends the substrate scope of traditional halodecarboxylations to heteroarene substrates. This methodology provides a novel and direct route to haloheteroarene building blocks that can be used in transition metal-catalysed cross-coupling reactions to construct molecules of scientific importance. Investigations into the site-selective halodecarboxylation of other heteroarene acid precursors are currently underway in our laboratory.


Tool wear is a significant issue for the application of micro end mills. This can be significantly improved by coating materials on tool surfaces. This paper investigates the effects of different coating materials on tool wear in the micro milling of Ti-6Al-4V. A series of cutting experiments were conducted. The tool wear and workpiece surface morphology were investigated by analyzing the wear of the end flank surface and the total cutting edge. It was found that, without coating, serious tool wear and breakage occurred easily during milling. However, AlTiN-based and AlCrN-based coatings could highly reduce cutting edge chipping and flank wear. Specifically, The AlCrN-based coated mill presented less fracture resistance. For TiN coated micro end mill, only slight cutting edge chipping occurred. Compared with other types of tools, the AlTiN-based coated micro end mill could maximize tool life, bringing about an integrated cutting edges with the smallest surface roughness. In short, the AlTiN-based coating material is recommended for the micro end mill in the machining of Ti-6Al-4V. Micro end milling has many advantages in high-accuracy and high-efficiency machining and is capable of machining small complex structures with various materials at micro- and meso-scales compared with other micro machining methods ,2. Howev2O3 + ZrN coated micro end mills is superior, and delamination is the principal wear mechanism of TiCN, TiSiN and Alumina (Al2O3) coated micro end mills in laser assisted micro-milling of hardened steel. Kang et al. [7N3)-based cermet micro end mill is better than WC micro end mill, and the wear mechanisms of the Ti(C7N3)-based cermet micro end mill are adhesion wear, micro chipping and diffusion wear in machining of aluminum alloy 2024. Torres et al. [Many studies have shown that optimizing coating materials improves the cutting performance of micro tools. Ucun et al.  investigg et al.  reportedg et al.  reportedg et al.  reportedg et al.  reporteds et al.  found thAs for traditionally coated cutting tools, An et al.  reportedThis paper aims to investigate the effects of different coating materials on cutting performance in the micro milling of Ti-6Al-4V. A series of cutting performance experiments were conducted on Ti-6Al-4V using micro end mills coated in different materials. To clarify the tool wear mechanisms, end flank wear length, the total cutting edge length reduction and workpiece surface morphology were also investigated.® ALNOVA), AlTiN-based (BALINIT® LATUMA) and TiN (BALINIT® BALINIT A) , respectively, and their characteristics are listed in In the machining of difficult-to-cut material like titanium alloy Ti-6Al-4V, the micro end mill is prone to serious wear, with the cutting edge damage occurring early, thus significantly reducing the cutting performance. Coating the micro end mill is an effective way to solve this problem. This is because the coating material can increase the hardness and chemical stability of a surface and reduce the friction coefficient between the tool and workpiece. Micro end mills with two flutes and a diameter of 0.5 mm were used in the experiments. Surface morphologies and energy spectrum analyses of the coated mills’ flank faces are shown in The cutting experiments were conducted on a five-axis machining center . Ti-6Al-4V was selected as the workpiece material. The cutting parameters of the experiments are listed in The surface morphologies of micro end mill end teeth flank faces with different coatings after a cutting length of 640 mm are shown in The flank face wear morphologies of differently coated micro end mills after a cutting length of 640 mm are shown in To evaluate the effects of differently coated micro end mills on cutting performance, the end teeth flank wear length and total cutting edge length reduction were investigated. The corresponding results are shown in xAlyN coated cutting tools may have sufficient potential to become a machining alternative compared to the AlTiN coating. However, the affixed CrxAlyN coating has a poor machining performance due to its brittle structure or high coefficient of friction [In micro milling, the cutting edges of tools suffer cyclic flexural and compressive stress, thus the stress concentrates in the cutting edges. Dislocation behavior occurs in coating material, resulting in micro crack nucleation. Under the action of alternating stress, the initiation of micro cracks begins, and then the micro cracks propagate, resulting in cutting edge chipping. The cutting edge chipping causes the reduction of the total cutting edge length, leading to a decrease in microgroove width. Moreover, with an increase in cutting length, the cutting edge radius becomes larger due to micro chipping of the cutting edge, and then plowing behavior prevails. Under high pressure, workpiece material tends to adhere to the surface of the micro end mill in the contact zone, and the workpiece material welds to the tool surface forming an adhesion layer. A built-up edge and built-up layer are also generated as the result of the high cutting pressure and the high chemical affinity with Ti-6Al-4V. The coatings are pulled off and dragged by the adjacent Ti-6Al-4V, and the dislocation, plastic deformation and breakage of the coatings occur at the cutting contact. Moreover, the contact of micro milling is at the microscale, and adsorption energy becomes the key factor dominating tool adhesion at the microscale. The formation mechanism of a built-up edge and built-up layer depends on both tool and workpiece materials . The harfriction . Thus, cfriction .The surface morphologies of microgrooves machined with differently coated micro end mills are shown in The surface morphologies of microgroove bottoms are shown in (1)The AlTiN-based and AlCrN-based coatings can lead to the reduction of cutting edge chipping and tool wear length compared to an uncoated micro end mill.(2)Compared to uncoated, AlCrN-based and TiN coated tools, the AlTiN-based coated micro end mill has the longest service life. Its cutting edge remains intact after a longer cutting distance. Moreover, the surface roughness of the machined Ti-6Al-4V is the lowest for this coating when compared with the other three types of coating. Thus, the AlTiN-based coated material is suitable for the micro end mill in the machining of Ti-6Al-4V.(3)The tool failure modes of all coated micro end mills are cutting chipping at cutting edge and coating delamination. The dominant wear modes are severe adhesive wear and abrasion wear on the tool flank face and rake face.This paper investigates the effects of coating materials on the cutting performance of micro end mills. A series of cutting experiments were conducted on Ti-6Al-4V with different materials. The end flank wear length and the total cutting edge length reduction were also investigated. Based on this work, the following conclusions were obtained: