BioLuminescence Imaging (BLI), we have now the opportunity to spatiotemporal visualize cell behaviors, among which proliferation, apoptosis, migration and immune responses, in any body district in living animal in a time frame process. We provide here a review of the available models to visualized cancer and cancer-associated events in living animals by BLI and as they have been successful in identifying new stages of early tumor progression, new interactions between different tissues and new therapeutic responsiveness.Preclinical animal models are valuable tools to improve treatments of malignant diseases, being an intermediate step of experimentation between cell culture and human clinical trials. Among different animal models frequently used in cancer research are mouse and, more recently, zebrafish models. Indeed, most of the cellular pathways are highly conserved between human, mouse and zebrafish, thus rendering these models very attractive. Recently, several transgenic reporter mice and zebrafishes have been generated in which the luciferase reporter gene are placed under the control of a promoter whose activity is strictly related to specific cancer cellular processes. Other mouse models have been generated by the cDNA luciferase knockin in the locus of a gene whose expression/activity has increased in cancer. Using  In vivo bioluminescence imaging, BLI, represents an interesting current and future new approach to molecular imaging. It allows imaging of internally generated light linked to specific physiological and/or pathological cellular processes in living small animals. This non-invasive technique, allows quantification in the same animal of spatial and temporal progression of the process of interest and identification of animal-to-animal variations  luciferase, a heat-unstable enzyme with a half-life of approximately 2h thus useful to study dynamics of biological processes. Animals do not produce the substrate for the light producing process, the luciferin, thus giving an excellent signal-to-noise ratio, as there is virtually no background in the animal tissues. Upon intraperitoneal (i.p.) injection the luciferin distributes throughout the mouse rapidly and it passes across blood-tissue barriers including placenta , in which an NF-Y–dependent promoter controls luciferase expression. The model stems from a decade of research to identify an NF-Y-dependent cyclin B2 minimal promoter driving luciferase expression in all phases of the cell cycle. In these mice luciferase is expressed in each proliferating cell. Using BLI in these mice we visualize areas of physiological cell proliferation as well as regeneration processes in response to injury demonstrating a role of NF-Y activity on hepatocyte proliferation during liver regeneration. MITO-Luc reporter mice would be useful to study the involvement of genes in cell proliferation and aberrant proliferation in disease pathogenesis. They will be also useful to study the effect of new anti/pro-proliferative drugs and assessment of their efficacy both on target and non-target tissues. We have demonstrated that MITO-Luc mouse model is suitable to characterize the effects of small molecules acting on proliferation of bone marrow (BM) and spleen cells in time course experiments in intact animals (Progression through each phase of the cell cycle is governed by cyclin/cyclin-dependent kinases (Cdks) complexes. We and others have contributed to demonstrate that the transcription of a plethora of cyclins and cdks, responsible for the cell cycle progression, is driven by the transcription factor NF-Y . Based o animals .Cyclin-Cdk complexes are negatively regulated by Cdk-inhibitors (CKIs) among which p16INK4a and p21Cip1. Apart from their capacity to arrest the cell cycle they have been shown to participate in an increasing number of cellular processes. p16INK4a limits cell-cycle progression and promotes cellular senescence in response to stress such as oncogene activation, while p21Cip1 is mainly involved in growth arrest, quiescence and induction of differentiation.In 2006 Guaven and colleagues described a mouse model useful to visualize the transcriptional control of ATM, a gene whose mutations lead to a pleiotropic phenotype, including a predisposition to develop malignancies. In these animals the luciferase gene is under the control of the murine Atm promoter. Using this tool the authors demonstrated for the first time that ionizing radiation and heat stress induce ATM promoter in different mouse tissues .cis-regulatory elements  promoter. In the same year a transgenic mouse expressing luciferase under control of AFP promoter has been described . Both moelopment .With the purpose to monitor pancreatic T-cell tumors non-invasively by BLI, Zumsteg and colleagues in the 2010 developed mice in which the expression of a bicistronic mRNA coding for large T antigen and luciferase occurs specifically in β-cells of Langerhans islets. In these mice the authors were able to illuminate tumor progression as well as lymph node and liver metastases .G12D/+;Pdx-1-Cre (KC) and LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice, in which the Cre-recombinase, transcribed by the pancreas-specific Pdx-1 promoter, leads to the expression of oncogenic mutant for of Kras alone or in combination with a mutant p53 protein, respectively  are the LSL-Krasectively , 2005. Opearance . From a To track responses to small molecules, Zhong and collegues developed and HPV-positive oral tumor mouse model in which HPV oncogenes E6, E7, mutant kras as well as a luciferase reporter (iHPV-Luc) are conditionally express in the epithelial cells upon a tamoxifen-regulated Cre recombinase system. In these transgenic mice tamoxifen treatment resulted in oral tumor development the development of which can be easily monitored by BLI .To achieve bioluminescence in B-cell lymphoma Scotto and colleagues have developed a transgenic mouse carrying CherryLuciferase fusion protein targeted to one allele of the CD19 locus crossed with GEMM of an aggressive lymphoma due to a chromosomal translocation of the c-myc gene .in vivo  luciferase transgene. In these mice it is possible to visualize cells that have undergone Cre-mediated recombination in alive animals. The mice have been crossed with transgenic mice in which a conditional oncogenic Kras expressed in lung epithelial cells induces non-small cell lung cancer and the authors demonstrate that, in this contest, longitudinal non-invasive visualization of lung tumorigenesis by BLI is feasible .An interest GEMM is represented by the Vegfr3 mouse model, where an EGFP-luciferase fusion protein, is expressed under transcriptional control of the endogenous Vegfr3 promoter. In these mice Martinez-Corral and colleagues were able to track tumor-induced lymphangiogenesis in the context of papillomas induced by dimethylbenzanthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) . SimilarAmong different transgenic models developed to spatio-temporally image inflammation in living animals, the most used is the one generated by Few studies visualize inflammation processes related to cancer. One interest study by Rauch and colleague have employed a mouse model of spontaneous lymphoma in which inflammation associated to malignant transformation is coupled with light emission. They crossed transgenic mice in which luciferase transcription is regulated by HTLV-1 LTR promoter with mice expressing Tax oncogene under the human granzyme B promoter in activated T cells and natural killer cells. These mice develop leukemia and lymphoma and the authors were able to discover an already unknown inflammation step preceding tumorigenesis . Using tin vivo the immune response to antigens relatively few transgenic animal models have been produced. To study adoptive T-cell therapy efficacy in the presence of large solid tumors a transgenic mouse models that allow monitoring T-cell activation in response to cancer has been generated. In this model renilla luciferase is under control of a constitutive promoter while firefly luciferase transcription is driven by the NFAT promoter thus allowing the concomitant analysis of T-cell migration and NFAT-dependent activation. The T-cells isolated from these mice are useful for longitudinal studies have been employed to study the kinetics of T-cell activation upon immune reconstitution of myelo ablated hosts in the presence of two tumor models (+CD4+ regulatory T cells (Tregs). These cells play a major role in maintaining self-tolerance and limiting immune responses to pathogens. In these mice the promoter of Foxp3 gene transcribes luciferase and the diphtheria toxin receptor. By BLI the authors demonstrated that Tregs were mainly located in lymphoid organs. Treg depletion upon diphtheria toxin treatment was also monitored by BLI, and longitudinal studies showed that the Treg compartment was recovered to its original size due to homeostatic proliferation of the remaining Tregs  cells establish a systemic macro-environment that supports outgrowth of otherwise-indolent disseminated tumors. LBCs secrete cytokines that activate BMCs . This caBLI reporter animals are going to revolutionize the way biological processes, among which cancer, can be studied. Signals are generated with a fast kinetics, enabling the visualization of molecular events in real time, not only on the target organs but in all body districts allowing the visualization of the communicative reprogramming occurring in the entire animal. The non-invasive nature of the model allows investigations of physiological events from embryos to adult animals, as well as longitudinal studies of disease progression and/or drug response. The ability to monitor individual animals throughout longitudinal studies without sacrificing them significantly reduce the number of animals required in each experiment. It will also be possible to detect even sligth changes due to variability among individuals. Although they are already giving important results on basic and applied cancer research, we can anticipate that in the future they will be increasingly used in translational cancer research, finding application among the others in drug discovery development and toxicology.IM, LdL, AG, and GP conceived and designed the review. GP wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evodia rutaecarpa (Juss.), has been reported to affect many physiological functions. Topoisomerase inhibitors have been developed in a variety of clinical applications. In the present study, we report the topoisomerase I (TopI) inhibitory activity of EVO, which may have properties that lead to improved therapeutic benefits. EVO is able to inhibit supercoiled plasmid DNA relaxation catalyzed by TopI. Upon treatment 0~10 μM EVO TopI was depleted in MCF-7 breast cancer cells in a concentration-dependent and time-dependent manner in 0~120 min. A K-SDS precipitation assay was performed to measure the extent of Top I-trapped chromosomal DNA. The ability of EVO to cause the formation of a TopI-DNA complex increased in a concentration-dependent manner, in that the DNA trapped increased by 24.2% in cells treated with 30 μM. The results suggest that EVO inhibits TopI by stabilizing the enzyme and DNA covalent complex.Evodiamine (EVO), an alkaloidal compound isolated from  The religation step consists of transesterification involving a nucleophilic attack by the hydroxyl oxygen at the 5′ end of the broken strand. The equilibrium constant of the breakage and closure reactions is close to unity, and the reaction is reversible.DNA topoisomerase enzymes regulate the topological state of DNA that is crucial for initiation and elongation during DNA synthesis. Topoisomerase I (TopI) produces a single strand break in DNA, allowing relaxation of DNA during its replication. The single strand break is then religated, thus restoring the DNA double strands. The enzymatic mechanism involves two sequential transesterification reactions . In the Some TopI- and TopII-targeted drugs are reported to stabilize the covalent topoisomerase-DNA complex, thereby preventing religation . The TopThe inhibitor-trapped TopI-cleavable complex triggers replication fork arrest and breakage to generate a DNA break that is responsible for the G2/M arrest and activation of DNA damage signals including nuclear factor κB activation, p53 upregulation, Chk1 phosphorylation, and ATM/ATR activation . TopoisoEvodia rutaecarpa (Juss.), has been reported to possess many physiological functions including vasorelaxation, antiobesity  assay to test the cytotoxicity of reagents and cell viability was described previously . MCF-7 cEVO is reported to have various pharmacological effects, but this is the first demonstration of EVO as a TopI inhibitor. EVO acts by stabilizing the covalent complex between TopI and DNA during the procedure of TopI-catalyzed DNA strand breakage and religation, which results in a barrier to DNA replication and transcription.


Multiblock copolymers constitute a basis for an emerging class of nanomaterials that combine various functional properties with durability and enhanced mechanical characteristics. Our mini-review addresses synthetic approaches to the design of multiblock copolymers from unsaturated monomers and polymers using olefin metathesis reactions and other ways of chemical modification across double C=C bonds. The main techniques, actively developed during the last decade and discussed here, are the coupling of end-functionalized blocks, sequential ring-opening metathesis polymerization, and cross metathesis between unsaturated polymers, or macromolecular cross metathesis. The last topic attracts special interest due to its relative simplicity and broad opportunities to tailor the structure and hence the properties of the copolymer products. Whenever possible, we analyze the structure–property relations for multiblock copolymers and point to their possible practical applications. Nowadays, olefin metathesis has become a well-established field of organic and polymer chemistry. The discovery of metallocarbene initiators that are capable of catalyzing metathesis polymerization in a living fashion turned it into a powerful tool of polymer design . Hundred4910In this mini-review we consider the approaches to multiblock copolymer syntheses via olefin metathesis reactions developed mainly over the past ten years. The following sections address the achievements and perspectives of three main techniques used for this purpose, namely, sequential ring-opening metathesis polymerization, coupling of end-functionalized blocks, and macromolecular cross metathesis.Ð = Mw/Mn = 1.0–1.19) and enables control over the block sequence and length in the copolymer chains (sequence-controlled multiblock copolymers). However, in practice this method is restricted to copolymers with a limited number of blocks, such as tetrablocks or pentablocks . A polymers  64]..n-octylfThe ADMET technique was used not only for the synthesis of polymer telechelics but also for their assembling into multiblock copolymers. A simple one-pot way for the preparation of random multiblock copolymers was proposed in reference . A mixtu2 with a norbornenyl-containing initiator (An approach utilizing macromonomers or macrocycles was used for the synthesis of multiblock copolymers with random or sequence-controlled structure . The ROM6nitiator  68]. Th. Th2 witVarious linear and star-shaped (triarm) ABA and ABCBA amphiphilic multiblock copolymers containing acetal-protected sugars (APS) were prepared by the coupling of an end-functionalized ROMP copolymer of norbornene (NB) and APS-substituted NB with poly(ethylene glycol) (PEG) . Ring-opThe MCO was obtained by ring-closing metathesis and contained easily cleavable ester linkages. It gave the possibility to сut the multiblock copolymer into pieces under alkaline conditions in order to obtain telechelic polyoctenylene with carboxyl end groups. The last reaction represents an example of the so-called sacrificial synthesis, another effective approach to telechelics .Hiff and Kilbinger generated cleavable ABAB pentablock and ABABABA heptablock metathesis copolymers via the sequential ROMP of seven-membered cyclic acetals  and N-substituted NB dicarboximide derivatives . The sub1 or a pyridine-containing end-terminator to yield pyridine-functionalized telechelic polymers 2. On this basis, supramolecular copolymers with alternating blocks were constructed using AgBF4 to remove Cl from the pincer complex and generate a cationic Pd ligand, which can coordinate with pyridyl ligands in a new pincer complex.Supramolecular multiblock copolymers with the possibility to introduce stimuli-responsive functionalities were obtained using a bimetallic ruthenium initiator . The iniA range of Zr(IV) and Hf(IV)-based bisamido complexes can catalyze both ROMP and addition (AP or vinyl) (co)polymerization of NB –75. The E-stereoregular multiblock copolymers and heterotelechelic polymers were successfully synthesized by the cross metathesis between different ROMP-derived poly(3-substituted cyclooctenes),  . Th. ThE-stectenes),  87]. Th. ThE-stectenes),  88–93].–93.E-steJ mol−1) ,96 is thJ mol−1) 91,93. ObJ mol−1) .MCM is an interchain cross reaction characterized by reshuffling of monomer units in the macromolecular backbones via break up and formation of new double bonds according to the olefin metathesis mechanism. In the beginning, an exchange of chain segments between the parent homopolymers results in the formation of diblock copolymers. Then random multiblock copolymers are formed , their aTg values are observed which get closer and finally merge into one, when long sequences of chemically identical units stemming from the parent homopolymers are exhausted.Therefore, the copolymer chain structure can be controlled by altering the reaction time, molar ratio of the starting polymers, catalyst type and concentration, as well as solvent type and initial polymer concentration –8993. It891H NMR spectroscopy was implemented to track the evolution of the chain structure in the course of MCM between polybutadiene (PBD) or polyisoprene (PI) and olefin-containing polyesters or polyurethane, as well as changes in the chain stereospecificity during the reaction between 3-substituted PCOEs [13C NMR [91L was calculated from an integral ratio of homo  and heterodyad (A–B) signals in the NMR spectra:ed PCOEs –86. Cros[13C NMR –9193. ThLA = [I(CA–A)+I(CA–B)]/I(CA–B); LB = [I(CB–B)+I(CB–A)]/I(CB–A); where I(CA–A) and I(CB–B) are the peak intensities of the initial homodyads, A–A and B–B, and I(CA–B) and I(CB–A) are the peak intensities related to the alternating dyads.The average block lengths decreased with the conversion, reaction time, and catalyst concentration and asymptotically approached the value of 2, characteristic of a completely random (Bernoullian) equimolar copolymer. Thus, a proper choice of the MCM conditions enables one to obtain copolymers with a controllable average block length ranging from the initial homopolymer length to a few monomer units.Important data on the kinetics of MCM between PNB and PCOE mediated by Gr1 were obtained by combining in situ NMR studies of the Ru-carbene transformations and ex situ NMR monitoring of the dyad composition evolution . It was It takes about a day for the interchain exchange between the homopolymers with carbene-functionalized end groups to yield a statistical NB–COE copolymer and during this process its molar mass remains almost unchanged. The slowest elementary reaction, which controls the overall kinetics, is the interaction between [Ru]=PCOE carbenes and C=C bonds in PNB chains (An increase of the PNB concentration in the mixture results in a growth of the copolymer degree of blockiness . This fecis/trans-isomerization of double bonds in the MCM process were obtained . It. It3Si-ss formed –88. Besis formed  via douboic acid . It was trans-octenylene blocks. Compared with the pure PCOE or its equimolar blend with PNB, the NB–COE copolymers form considerably smaller crystallites (Copolymer crystallinity can be studied in detail by combining WAXD and DSC methods, including recently emerged technique of thermal fractionation by successive self-nucleation and annealing ,102–103.tallites  92]..trans-ocIt is rather clear nowadays that the olefin-metathesis reaction is a versatile tool for the synthesis of multiblock copolymers with diverse chemical structures. Due to the rapid progress in the catalyst design for living polymerization, sequential ROMP has become a well-established method of obtaining copolymers with sequence-defined structures. However, in many aspects, this technique remains laborious and even cumbersome. Most publications report on the multiblock copolymers synthesis by the coupling of premade individual blocks. A key point here is related to advances in the development of synthetic approaches for fabricating symmetric and asymmetric telechelics and monochelics, macromonomers and macrocycles based on different olefin-metathesis techniques like CTA, ADMET, etc. A subsequent assembling of macroblocks into copolymers can be carried out by combining olefin metathesis with other reactions such as ATRP, RAFT, click-reaction, and so on, which permit to gain certain control over the final copolymer structures. The most recent approach to the multiblock copolymer synthesis implements the macromolecular cross-metathesis reaction, which is still poorly studied. For this method, the simplicity of realization is counterweighted by inability of precise control over block sequences and considerable drop in the average molecular mass of reacting polymers as a result of their interaction with metathesis catalysts. Nevertheless, the average block lengths can be easily tailored and the resulting copolymers reveal the ability to self-assemble into ordered structures, enhanced mechanical properties, and nontrivial crystalline and thermal characteristics. Recent kinetic studies with the use of in situ and ex situ NMR have shed some light on the regularities of the macromolecular cross-metathesis reaction, which appeared to be somewhat opposite to the notions about metathesis copolymerization. Perspectives of the entire field under review are related to the elaboration of novel post-modification methods for obtaining new functionalities and enhancing various characteristics of multiblock copolymers. In our opinion, further development of the olefin metathesis methods for the multiblock copolymer synthesis will be directed by the search for new properties and possible applications.


Screening programs for lung cancer aim to allow diagnosis at the early stage, and therefore the decline in mortality rates. Thus, the aim of this retrospective cohort study was to the comparison of screened and non-screened lung cancer in terms of lung cancer characteristics, overdiagnosis and survival rate. A retrospective study in which 2883 patients with 2883 lung cancer diagnosed according to the hospital-based lung cancer register database between 2007 and 2017. A comparison was performed in term of clinical characteristics and outcomes of lung cancer between the screened and non-screening patient groups. 2883 subjects were identified (93 screened and 2790 non-screened). Screened group patients were younger , and were more likely to be female than non-screened group . The screened group showed significantly better outcomes in overall mortality than the non-screened group . In a Cox proportional hazard model, lung cancer in the screened group proved to be an independent prognostic factor in lung cancer subjects. Our findings point to the improved survival outcome in the screened group and might underline the benefit of low-dose computed tomography (LDCT) screening program in Asian populations with the high prevalence of non–smoking-related lung cancer. Further study aimed at the LDCT mass screening program targeting at light smokers and non-smoker outside of existing screening criteria is warranted. In Taiwan, lung cancer is the leading cause of cancer-related deaths and among both men and women over the last decade. 5884 males and 3348 females died of lung cancer in 2015, accounting for the most common cancer-related mortality rate among 10 most common cancers according to the Taiwan Cancer Registry annual report.1. Around 70~80% of patients with lung cancer diagnosed have stage III or IV disease at presentation in Taiwan, which generally lead to poor prognosis with the high mortality rate2. Therefore it is crucial that screening and diagnosis of lung cancer at early stage because when found early, lung cancer is highly curable3.The National Lung Screening Trial (NLST) demonstrated that lung cancer screening using low dose computed tomography (LDCT) resulted in a significant reduction in lung cancer-specific overall mortality rate in comparison with chest radiography in high-risk heavy smokers5. In addition, the average PM2.5 levels in Taiwan were much higher than the levels in European cities according to the European Study of Cohorts for Air pollution Effects (ESCAPE), especially in Kaohsiung city6. Therefore, genetic factors and environmental pollution may play an important role in the development of non-smoking related lung cancer. Therefore, the LDCT screening selection criteria should be modified due to regional genetic and environmental differences. In Kaohsiung Veterans General Hospital , self-paid LDCT for lung cancer screening has been conducted for population-based mass screening aged 40~80 years since 2007 in a population with the high prevalence of non-smoking-related lung cancer7. However, all screening programs involve the trade-offs between potential benefits and potential harms. There are still many unresolved problems about the benefits, harms and the cost of the mass screening, included light smokers and non-smokers11.Previous studies have demonstrated that family history and gender are two important factors associated with non-smoking related lung cancerThus, the aim of this retrospective cohort study was to a comparison of screened and non-screened lung cancer in terms of lung cancer characteristics, overdiagnosis and survival rate.Between January 2007 and September 2017, 2883 patients were diagnosed with lung cancer at Kaohsiung Veterans General Hospital. The focus in this present study is on clinical characteristics and outcomes of lung cancer between screened and non-screening patient groups. The institutional review board approved this retrospective study, and thus informed consent was waived. All research was performed in accordance with the relevant guidelines and regulations and all Institutional Review Board requirements.12. Data regarding curative surgical treatment and targeted therapy use were also recorded.Patients were classified two groups: Group 1 (screened group): patients who had no clinical symptoms and incidentally detected lung cancers by self-paid LDCT exam aged 40–80 years. Group 2 (non-screened group): patients with the clinical scenario of lung cancer-related symptoms or abnormal finding (highly suspicion of lung cancer) in the chest radiograph were further diagnosed as lung cancer. Patients were assigned to screened and non-screened subgroups based on the stringent definition. Our database included the following clinical characters: age, sex, tumor size, histopathologic type, adenocarcinoma spectrum classification, lung cancer clinical stage, death, survival time, mortality rate, smoking habit, betel nut and alcohol drinking habit. The histological diagnosis was described according to the World Health Organization classification. All the patients were staged according to the 7th edition of the TNM staging system published in 2009All statistical analyses were performed with SPSS 17.0 for Windows , STATA 13.0 package  and MedCalc 13.2.2.0 . Differences in continuous variables between 2 groups were compared by the independent Student t test. Categorical variables were summarized as frequencies and percentages and compared using the chi-square test to examine differences in demographic characteristics. Fisher’s exact test was used to analyze when the smallest expected value is less than 5. Overall survival time was defined as the period from the date of lung cancer diagnosed until the date of death from any cause or until the date of the last follow-up at which point the data were censored. Overall survival was estimated using the Kaplan-Meier method. A log rank test was used to compare prognosis between screened and non-screening groups. Cox regression analysis was performed to calculate and compare the survival rate. A multivariate analysis was used to estimate the hazard ratios (HRs) based on the Cox regression model, adjusted for age, gender, smoking, alcohol, betel nut, tumor size, curative surgery, target therapy, histology and screened or not. A p value of 0.05 was regarded as significant.The study protocol was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (VGHKS19-CT2–09). The need for informed consent was waived by the institutional review board due to the minimal risk retrospective study.During the study period, 2883 lung cancer patients were analyzed according to the hospital-based cancer registry dataset. The screened group consisted of 93 subjects, and the non-screened group consisted of 2790 subjects. The general characteristics of the lung cancer subjects between the two groups are summarized in Table The mean age in the screened group was significantly younger than that in the non-screened group . The screened patients had a significantly greater proportion of female gender than the non-screened group . Patients in the non-screening group had the significantly higher proportion of smoking habit (p < 0.0001), surgery (p = 0.0001) and target therapy (p = 0.001) than the screened group. There were no significant differences between the two groups in terms of alcohol drinking and betel nut habits. For survival interval analysis, the mean survival date in the screened group was significantly higher than that in the non-screened group .The comparison of the difference in histopathology and clinical lung cancer stage distribution between the two groups are summarized in Table Stage I was the most frequent stage in the patients with screened group. In comparison, patient in the non-screened group presented with stage IV in most cases.in situ, the screened patients had a significantly greater proportion of carcinoma in situ than the non-screened group .For carcinoma in situ or its precursor lesions.For lung cancer histopathology distribution, a significant difference in histopathology distribution was seen between screened and non-screened groups. With regard to histological subtype, adenocarcinoma was significantly more prevalent in the patients with screened group . In contrast to that, the subjects with non-screened group have a significantly greater proportion of the patients with small cell carcinoma  and squamous cell carcinoma . With regard to histological subtype of adenocarcinoma spectrum, a significant difference in histological subtype of adenocarcinoma spectrum was seen between the screened and non-screened groups. Invasive pulmonary adenocarcinoma was the most frequent histological subtype in the patients with non-screened group. In comparison, the patients in screened group presented with higher portion of minimally invasive adenocarcinoma, adenocarcinoma Table P < 0.0001 for all). The mean survival time for the screened group was 860.31 ± 502.20 days (median 793 days), and the mean survival for the non-screened group was 574.94 ± 558.48 days (median 404 days). After exclusion of AAH or AAH/AIS patients, the screened group still showed significantly better outcomes in overall mortality than the non-screened group shown in Supplement Tables Patients in the screened group had a lower overall mortality than that in the non-screened group. One-, and Five-year mortality rates increased significantly from the screened group to the non-screened group P < 0.000 for all.Survival analysis for lung cancer using Cox regression model for multivariate effects, the hazard ratio for lung cancer mortality was determined adjusting for age, gender, smoking, alcohol, betel nut and screened status Table . MultivaThe focus of the present study is to compare between screened and non-screened lung cancer in terms of lung cancer characteristics, overdiagnosis and survival rate. In this study, we demonstrated four major findings. The first one is that we observed that adenocarcinoma was significantly more frequent in patients in the screened group. The second finding is that stage I was significantly more frequent in patients in the screened group. Third, favorable overall survival was significantly superior in the screened group and lead to a reduction of 68.31% compared with non-screened group.in situ lesions in the screened group and lead to an increase of 14.86% compared with the non-screened group in regard to overdiagnosis.Fourth, there was an obvious increase of carcinoma 1. In this present study, we found a significant decrease in the overall mortality rate of 68.31% in the screened group compared with the non-screened group. Our present result is similar to the findings of this previous study1. Further, in accordance with our results, lung cancer subjects in the screened group also had smaller primary tumor size and lower clinical stage at the time of initial diagnosis. These finding were associated with the lower mortality rate, longer survival periods, and an increased surgical treatment rate. However, there are also a number of important differences between the NLST population and our screened group. Our screening population found a higher proportion of non-smoking women with lung cancer diagnosed. These findings also support that there appears to be an increasing trend of non-smokers related lung cancer in Asian population such as China, Japan, Korea and Taiwan in recent years7. Currently most of the society recommends that lung cancer screening targeted in adults age 55 to 80 year who have a 30 pack-year smoking history and currently smoke or have quitted within the past 15 years14.In 2011, the NLST reported a reduction in lung cancer mortality of 20% in subjects who underwent annual LDCT exam compared with annual chest x-ray exam during the time period of 5 years15. Recent research has demonstrated that lung cancer screening using LDCT resulted in a 43% decrease in all-cause death in comparison with chest radiography in a population-based cohort including non-smoker and light smoker in Hitachi city, Japan16. Therefore, the effectiveness of lung cancer screening program specifically targeting at non-smoking Asian population with the high prevalence of non-smoking related lung cancer should be further investigated.The present results also support previous studies concerning broadly expanding inclusion criteria to include never-smokers in lung cancer screening program in Asian population with the high prevalence of non-smoking related lung cancer18. In this study, we attempted to estimate the proportion of carcinoma in situ or the proportion of early adenocarcinoma spectrum lesions to assess the extent of overdiagnosis. For carcinoma in situ, the screened patients had a significantly greater proportion of carcinoma in situ than the non-screened group (15% vs. 0.14% p < 0.0001). For adenocarcinoma lesions, the screened patients had a significantly greater proportion of precursor lesions of adenocarcinoma spectrum  than the non-screened group (23.65% vs. 0.19% p < 0.0001). To estimate the percentage of overdiagnosis in NLST trial, Patz et al. estimated that more than 18% of all lung cancers detected by LDCT in the NLST seem to be indolent17. Our findings also support the same results.In this study, we assessed the factors affecting the prognosis of lung cancer between the screened and non-screened groups. In the multivariable Cox regression model, lung cancer in the screened group is the most important factor responsible for improved survival prognosis. Therefore, it is crucial that widely implementation of LDCT lung cancer screening program in Asian population including non-smoker and light smoker based on the lung cancer risk prediction model. However, overdiagnosis is always an important concern in cancer screening because it potentially leads to overtreatment and potential harmful treatments20. Recent evidences have demonstrated that a strong correlation between radiologic features and adenocarcinoma spectrum lesions22. In addition to the rapid development of computer image processing and radiomics technology, recent studies have also demonstrated that use of CT radiomic texture analysis such as tumor size, morphology, attenuation, entropy and volumetric doubling time analysis could facilitate more accurate differentiation between pre-invasive lesions and invasive pulmonary adenocarcinomas29. Our previous studies also have shown that novel subclassification of subsolid nodules has the high positive predictive value to rule in invasive pulmonary adenocarcinoma, which manifesting as part-solid nodules24. Incorporation of these CT radiomic features can help further refine the management of subsolid nodules in the Asian lung cancer screening program with the high prevalence of non-smoking related lung cancer30. The goal of lung cancer screening program is to reduce mortality rate from lung cancer death and avoid overdiagnosis/overmangement. Thus, we should always be aware of overdiagnosis as a counterpart of lung cancer screening program in Asia. Because of more indolent growth for preinvasive lesions which manifesting as pure groundglass nodules, the combination strategies of shared decision making and watchful waiting with annual or biannual follow-up for a period of at least 5 years a would be a safe option to allow early detection of disease progression and to avoid the potential harms of overdiagnosis and overtreatment32. These strategies could improve the efficacy of lung cancer screening programs in Asian populations with the high prevalence of non–smoking-related lung cancer25.There is even a more obvious trend because of the higher prevalence of non-smoking lung adenocarcinoma spectrum lesions in Asia34.The main strength of this study is the hospital-based lung cancer registry database, which mainly reflect the entire lung cancer population in a hospital-based cohort. Therefore, the large hospital-based cohort could result in sufficient power to detect small differences in patient outcomes and prognosis. Several previous studies investigated the difference between screened and symptomatic group for surgically diagnosed lung cancer subjects, which can’t represent the entire distribution of lung cancer in the population30.This study also has its limitations. First, this study is a retrospective cohort study design. Therefore, the retrospective aspect may introduce selection bias and information bias, which could bias this study’s results. Second, it is uncertain that the current study results could be widely generalized to other hospitals in Asia. Thus further large multicenter studies are needed to investigate the effect of implementation of mass lung cancer screening program at target-risk population based on lung cancer-risk prediction model to establish the generalizability in AsiaIn summary, a very high proportion of non-smoking related lung adenocarcinoma is detected at the early stage in patient underwent mass lung cancer screening program, which lead to significantly improved survival outcome. These results suggest that broadly expanding inclusion criteria to include never-smokers in lung cancer screening program in Asian population may be crucial in reducing mortality rate and improving survival outcome. To maximize the cost-effectiveness and health benefits of LDCT screening and reduce the potential risk in over-diagnosis and over-management among non-smokers, further prospective-cohort studies should address the effectiveness of implementation of LDCT lung cancer screening program targeting at high-risk population based on lung cancer prediction risk model.Supplement Table 1&2


Exercise therapy is recommended for knee osteoarthritis (OA), but the underlying mechanisms of pain relief are not fully understood. The purpose of this study was to explore the effects of exercise on muscle perfusion assessed by dynamic contrast enhanced MRI (DCE-MRI) and its association with changes in pain in patients with knee OA.ClinicalTrials.gov: NCT01545258) performed at an outpatient clinic at a public hospital in Denmark. We compared 12 weeks of supervised exercise therapy 3 times per week (ET) with a no attention control group (CG). Analyses of covariance (ANCOVA) were used to assess group mean differences in changes from baseline to week 12 in knee muscle perfusion quantified by DCE-MRI, patient-reported pain and function using the Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire, knee extensor and flexor muscle strength tests, and the six-minute walking test (6MWT). Spearman’s correlation coefficients were used to determine the correlation between changes in DCE-MRI variables, KOOS, muscle strength, and 6MWT. The potential effect mediation of the DCE-MRI perfusion variables was investigated in a post-hoc mediation analysis.Exploratory outcome analyses of a randomised controlled study with per-protocol analyses  adhered to the protocol and had complete DCE-MRI data. At follow-up, there were significant group differences in muscle perfusion changes and clinically relevant group differences in KOOS pain changes  in favor of ET. There were no significant between-group differences on muscle strength and function. The changes in pain and muscle perfusion were significantly correlated . The mediation analyses were generally not statistically significant.Of 60 participants randomised with knee osteoarthritis, 33 (ET, The pain-reducing effects of a 12-week exercise program are associated with changes in knee muscle perfusion quantified by DCE-MRI in individuals with knee OA, but whether the effects are mediated by muscle perfusion changes remains unclear.NCT01545258, first posted March 6, 2012.ClinicalTrials.gov:  Exercise therapy is recommended as first line treatment of knee osteoarthritis (OA) based on extensive research evidence on beneficial effects of exercise on pain and function –4. HowevThe beneficial effects of exercise on knee OA pain and function may be caused by increased muscle strength, increased knee range of motion, or improvements in proprioception , 6. AlsoMuscles are the primary tissue affected by most exercise types; thus, any physiological changes caused by exercise are expected to be reflected in the muscles. Indeed, exercise causes physiological changes in the muscles, such as increased capillarisation or angiogenesis –11. We hDynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) is a method to quantify tissue perfusion through temporal variations of the MRI signal intensity following intravenous injection of a contrast agent . Such quThe purposes of this study were to explore 1) the effects of a 12-week therapeutic exercise program on muscle perfusion in the peri-articular knee muscles assessed by DCE-MRI and 2) the association between changes in muscle perfusion and changes in pain, physical function and performance in patients with knee OA. We hypothesized that exercise would increase muscle perfusion in parallel with improved pain, physical function and performance when compared with a no-attention control intervention.www.ClinicalTrials.gov: NCT01545258).This is an exploratory sub-study of a previously published randomised controlled parallel-group-trial investigating the effects of a 12-week exercise program on the pain sensitivity in patients with knee OA focusing on the per-protocol population  performe2. Exclusion criteria included participation in exercise therapy within the previous 3 months, having inflammatory and autoimmune diseases, and lower extremity joint replacement.A comprehensive description of the recruitment process and inclusion/exclusion criteria is published elsewhere . BrieflyThe sample size of the main trial was based on group differences in the primary outcome, pressure-pain threshold, and was calculated to 60 participants .After baseline assessments, participants were randomly assigned (1:1 stratified by gender) to exercise therapy (ET) or to a control group (CG). A computer-generated list of random numbers was used and concealed from the researchers enrolling and assessing participants. Participants were aware of their group allocation; outcome assessors and data analysts were kept blinded to the allocation .The ET group was offered facility-based, functional and individualised exercise therapy supervised by a trained physiotherapist 3 times weekly for 12 weeks. A full description of the.exercise program is available in  and in tMRI of the most symptomatic knee at baseline was performed on a 3 T Siemens Verio®  system using a 15-channel dedicated send/receive coil. In addition to a standard clinical MRI protocol, a sagittal DCE T1 VIBE sequence was performed with a temporal resolution of 9 s and 30 repetitions. During the third repetition an intravenous injection of 0.1 ml/kg body weight Gadolinium (Gd) contrast agent  was administered (2 ml/s). The total scanning time was 30 min with the DCE-MRI sequence performed after 20 min. The full MRI protocol is available in the Additional file The investigator analysing the DCE-MRI images (EB) was blinded to group allocation and clinical data and supervised by experienced radiologists (MB and RR). Dedicated software  was used for all DCE-MRI analyses , 21: mottrans, was calculated as described in Riis et al. 2016 [As previously described in detail , 21, heuOutcomes were measured at baseline and after the 12 weeks of intervention. For the purpose of this exploratory outcome analysis, we examined changes from baseline to follow-up, looking at the outcomes: muscle perfusion from DCE-MRI, patient-reported pain and function using the Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire (0(worst)–100(best) , physicaDCE-MRI derived perfusion variables are listed and explained in Table n = 10) in which manual segmentations were repeated after a minimum of 4 weeks. The intra-observer reliability of the volume (cm3) of the Total Muscle VOI was ICC = 0.99 (95% CI: 0.98 to 0.99). ICC of the muscle perfusion variables was ≥0.99 . Due to low reproducibility of the Ktrans it was not included in the further analysis. Details of reproducibility of the DCE-MRI variables are presented in the Additional file Intra- and inter observer reproducibility of the DCE-MRI analyses were assessed for the Total VOI on a random subsample n = 0 in whicThe reliability of KOOS , muscle As pre-specified in the main trial protocol, the study focuses on individuals who adhere to the protocol. The per-protocol population was defined as participants with 12 or fewer incidences of non-attendance at exercise sessions in the ET group, and no exercise in the CG. The present exploratory analyses aim at investigating the effects of exercise on muscle perfusion in the peri-articular knee muscles; thus, only individuals included in the per-protocol population with complete DCE-MRI data sets at baseline and follow-up were included in the present analyses , analyses of covariance (ANCOVA) was used with change from baseline at follow-up as dependent variable, and baseline value of the dependent variable, treatment group, and gender (due to stratification by gender when randomising) as covariates . For senTo explore whether changes from baseline to follow-up in the muscle perfusion variables were associated with changes in pain and function (KOOS) and physical performance measures (purpose 2), we assessed the rank correlation (Spearman’s rho) of changes from baseline between the DCE-MRI muscle perfusion variables, KOOS subscales, muscle strength, and 6MWT across the entire population.Furthermore, we investigated the potential effect mediation of changes in the DCE-MRI perfusion variables for changes in KOOS pain in a post-hoc mediation analysis using a model-based approach with nonparametric bootstrap for variance estimation. The mediation models were adjusted for the same variables as the primary analysis (i.e. baseline KOOS pain and gender). We estimated an average causal mediation effect , i.e. thn = 16; CG, n = 17). In the DCE-MRI per-protocol population group, imbalances were present in age, weight and BMI at baseline, with the ET group being older and less overweight. Details of study flow are given in Fig. n = 15; CG, n = 12).Of the 60 included participants in the main trial, 31 were randomised to ET and 29 to the CG. The present analyses involved participants who adhered to the protocol with complete DCE-MRI data: 33 participants constituted the DCE-MRI per-protocol population  and Initial Rate of Enhancement Index (IRExNvoxel%) were statistically significantly different in favor of ET, showing similar patterns as for the Total Muscle and Flexor VOIs, with a relative increase in the ET group compared with the CG. The results are summarized in Table There was a statistically significant and clinically relevant group difference (10.7 KOOS points (95%CI 3.3 to 18.1)) in changes from baseline in KOOS pain in favor of ET similar to the per-protocol population in the main study [No statistically significant group differences were observed in changes from baseline to follow-up in the muscle strength variables and in 6MWT Table .The results were robust to sensitivity analysis, with only slight changes in the estimates . Further, changes in KOOS pain were positively correlated with changes in IRE Composite score (IRExNvoxel) and ME Composite score (MExNvoxel) in the Flexor VOI . This indicates that pain reduction was associated with increase in the degree and area of perfusion of the peri-articular muscles  (Nvoxel%) and Maximal Enhancement Index (MExNvoxel%) in the Total and Flexor VOIs . Changes from baseline in isometric knee extensor muscle strength were the only muscle strength variable which correlated significantly with changes in the muscle perfusion variables . No other statistically significant associations were found between changes in muscle perfusion variables and changes in the variables of physical performance   there was no statistically significant mediation of the pain-reducing effect of exercise in patients with knee OA, except for three muscle perfusion variables in the Flexor VOI. These were initial Rate of Enhancement Composite Score (IRExNvoxel), Maximal Enhancement Composite Score (MExNvoxel) and Maximal Enhancement index (MExNvoxel%) Table , mediatiOur results suggest that exercise therapy sustain peri-articular knee muscle perfusion compared to a no attention CG, as the participants in the ET exhibited a constant level of muscle perfusion in contrast to decreased perfusion in the CG over the 12-week intervention period. Besides the differences in muscle perfusion, exercise had a clinically relevant effect on self-reported pain. Further, across the population, changes in muscle perfusion were associated with changes in self-reported pain but not with physical function and performance. Although the mediation analyses overall were not statistically significant, all effects were in the expected direction. Altogether, these results indicate that muscle perfusion may be part of the underlying mechanisms of the pain-relieving effects of exercise. The present results extend our previous cross-sectional observation that more widespread muscle perfusion associates with less pain [The current study does not provide insight into the underlying mechanism of changed muscle perfusion. Exercise causes structural adaptations in the muscles, such as increased capillarisation or angiogenesis as a physiological response –11. BasePain experience is affected by psychological variables, such as depression and quality of life , 36, andAs exercise therapy cannot be blinded to the provider or to the patient, the observed clinical effects may be facilitated by effects associated with attention and study participation . HoweverIncreased perfusion of intra-articular structures (e.g. synovium and the infra-patellar fat pad) are conventionally interpreted as signs of inflammation, and studies have repeatedly shown that intra-articular perfusion associates with a higher degree of pain –17, 27. www.koos.nu). This increases the relevance of our findings. In contrast, the MCIDs in the muscle perfusion variables have not been established, which encumbers interpretation of our results. However, the observed differences in muscle perfusion variables were all beyond the smallest detectable changes, and the reliability of the image analyses was satisfactory, which altogether strengthens our confidence in the results.We assessed knee OA pain using the KOOS questionnaire and found clinically relevant group differences that exceed the minimal clinically important difference (MCID) of 8–10 KOOS points as suggested by the developers of the questionnaire  and performance-based tests are recommended and frequently used , 42, 43.The use of a rigorous randomised study design with pre-specified methods using standardised and validated image analyses with high reproducibility are major strengths of our study. On the other hand, the limited sample size and the many variables and statistical tests performed increase the chances for multiplicity. The fact that the study was designed and powered for another outcome are limitations and calls for replication in larger studies specifically designed for DCE-MRI. Nevertheless, we could detect clinically meaningful important signals associated with the effects of exercise, a universally recommended treatment for knee OA. Another important aspect to consider is the per protocol design of the study. This was done to investigate the underlying mechanisms of exercise and thus it is necessary to focus on the participants who had complete DCE-MRI assessments at both baseline and follow-up and who had adhered to the protocol, i.e. received the allocated intervention (exercise vs control). This is fundamentally different from intention to treat study designs and therefore this study cannot be used to inform clinical practice. An important limitation is that out of 14 potential mediation analyses  we only analysed mediation of effects on KOOS pain as this was the only outcome with significant group differences. This analysis did not support that changes in muscle perfusion mediate pain improvements.The pain-reducing effects of a 12-week exercise program are associated with changes in muscle perfusion quantified by DCE-MRI in individuals with knee OA, but whether the effects are mediated by muscle perfusion changes remains unclear.Additional file 1: Supplementary information.


Fetal adenocarcinoma of the lung is a rare lung neoplasm that accounts for only 0.5% of all primary lung cancers. Because of its rarity, effective treatments for the management of the tumor are poorly understood. We herein report a case of adenocarcinoma with fetal features of the lung with invasion of the right superior sulcus that was treated with neoadjuvant chemoradiotherapy followed by surgical resection.A 54-year-old man was referred to a medical institution due to right inner forearm pain. On computed tomography of the chest, a 56-mm mass with invasion of right superior sulcus was discovered. Bronchoscopic biopsy revealed non-small cell lung carcinoma. We performed concurrent chemotherapy (2 cycles of cisplatin and vinorelbine) and thoracic radiation therapy (40 Gy in 20 fractions). As the result of extreme tumor reduction after neoadjuvant chemoradiotherapy, we could perform right upper lobectomy by complete video-assisted thoracoscopic surgery. Since no viable cancer cells were detected from the pathological examination of the resected tissue, the specimen obtained by bronchoscopic biopsy was reexamined by immunohistochemistry. The analysis supported a pathologic diagnosis of adenocarcinoma with fetal features.We experienced a case of adenocarcinoma with fetal features of the lung in which the patient showed a complete response to neoadjuvant chemoradiotherapy. In addition, the tumor invading the right superior sulcus was completely resected by video-assisted thoracoscopic lobectomy. Neoadjuvant chemoradiotherapy followed by surgery may be also an effective treatment for advanced-stage high-grade fetal adenocarcinoma of the lung, similarly to other subtypes of advanced-stage primary lung cancer. Fetal adenocarcinoma of the lung (FLAC), which is classified into low-grade (L-FLAC) and high-grade (H-FLAC), is an extremely rare subtype of pulmonary tumor occurring with a relative incidence of 0.5% among all lung cancers . BecauseA 54-year-old Japanese man with a 30 pack-year smoking history was referred to a medical institution due to pain and paresthesia from the ulnar surface of the right forearm to the small finger of the right hand in the distribution of the C8 and T1 dermatomes that had persisted for approximately 2 months. He had no remarkable medical history. The patient’s laboratory data revealed an elevation carcinoembryonic antigen (CEA) level of 8.4 ng/ml. A chest X-ray showed a tumoral shadow at the right apex of the upper-lung field.  was performed. Chest CT after chemoradiotherapy indicated remarkable reduction of the tumor  invading the superior sulcus that showed a complete response to neoadjuvant chemoradiotherapy, which was followed by right upper lobectomy under complete VATS. This report suggests that neoadjuvant chemoradiotherapy may be effective for H-FLAC. We diagnosed the tumor in this case as adenocarcinoma with fetal features based on new criteria for adenocarcinoma in small biopsies and cytology of the 2015 WHO classification of lung tumors . CollectThis report also suggests that superior sulcus tumors may be successfully resected by complete VATS lobectomy in cases in which neoadjuvant chemoradiotherapy showed a high degree of efficacy. Preoperative concurrent chemoradiotherapy is recommended as the standard treatment for resectable non-small cell lung cancer of the superior sulcus . HoweverWe reported an extremely rare case of adenocarcinoma with fetal features (high grade) invading the right superior sulcus, which showed a complete response to neoadjuvant chemoradiotherapy and which was successfully resected by complete VATS. However, the role of neoadjuvant chemoradiotherapy for H-FLAC is not well established. Although the complete resection of superior sulcus tumor without bony thorax defects by VATS alone may be possible after a good response to induction therapy, the optimal procedure remains to be established. This case provides and suggests a useful approach to the management of advanced-stage H-FLAC. More cases are needed to clarify the effects of this treatment method.


BackgroundThe genetic etiology of colorectal cancer (CRC) is the occurrence of mutation in the genes involved in signal transduction pathways including that of cellular responses to endoplasmic reticulum (ER) stress. This study examines alterations of pre-messenger ribonucleic acid (pre-mRNA) splicing in X-box binding protein (XBP) transcripts related to the ER stress pathway in CRC.Materials and methodsIn this study, samples were deparaffinized and underwent RNA extraction. A total of 30 synthesized complementary deoxyribonucleic acid (cDNA) templates from the extracted RNAs related to tumor and non-tumor CRC samples, collected over three years and containing pathological data, were subjected to semi-quantitative reverse transcriptase polymerase chain reaction (sqRT-PCR). These cDNA templates were amplified in reaction tubes with specific primers for both spliced and non-spliced isoforms of XBP. Results with P< .05 were considered statistically significant.ResultsMicroscopic assessment represented lymphocyte-rich effusion in tumor samples. sqRT-PCR electrophoresis results showed spliced and non-spliced forms of XBP messenger RNA in the studied samples. In addition, our data showed there were more than 7.8 times the total number of spliced variants in the marginal tumor samples than in the tumor tissue samples (P<.05).ConclusionAlterations of expression in genes involved in stress signaling pathways in cancer have been identified previously. Our results showed an inverse relationship between XBP splicing and CRC tumor tissue, possibly lead to the inactivation of apoptosis in the downstream response to ER stress. However, we propose that the remaining genes in this pathway should undergo gene expression analysis using a greater number of samples. Colorectal cancer (CRC) is the third most common cancer, with an incidence of 1.36 million people per year worldwide. The genetic causes of this heterogeneous disease are the occurrence of changes in pathway genes including tumor protein p53 (TP53), transforming growth factor beta 1 (TGFB), Janus kinase/ signal transducer and activator of transcription (JAK / STAT), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), and Wnt family member 1 (WNT1) . One patOne of the major functions of the ER network is to coordinate between synthesis and secretion of proteins. The first step of maturation and folding of the secretory and membrane proteins occurs in the ER. Changes in homeostasis lead to the accumulation of unfolded or inappropriately folding proteins, ER stress, and activation of the ER stress response pathway. Depending on the duration and amount of ER stress, the pathway can either send survival signals by activating anti-apoptotic pathways or trigger death signals by inducing programmed cell death . Some caATF6 is activated after dissociation from HSPA5 and digested with peptidase in the Golgi apparatus. This portion is transferred into the nucleus as a transcription factor, leading to the expression of some chaperone proteins and X-box Binding Protein (XBP). XBP is transferred into the cytoplasm and spliced by IRE1. The spliced form is also transferred as a transcription factor to the nucleus. If the stress persists, the genes involved in apoptosis are activated. A study of the detailed processes of each of the mentioned components in this signaling pathway is important to understand its molecular mechanisms. XBP is essential for the survival of a tumor under hypoxic conditions . XBP mesThe ER stress response pathway in CRC is very important, and the mechanisms of this pathway in CRC are poorly understood; therefore, we assessed XBP transcript splicing in CRC tumor samples compared to non-tumor or marginal tumor samples. The results of this study may clarify the paradoxical mechanisms of this signaling pathway in CRC.Clinical samplesA total of 30 paraffin-embedded tissue blocks from tissue samples were collected from the pathology department of hospitals in Arak, Iran over the course of three years. Tumor and non-tumor samples of these blocks were confirmed using pathological examination. From each tissue block, one slide was stained with hematoxylin and eosin.Ribonucleic acid extractionThe collected samples underwent deparaffinization and rehydration, then were digested with Proteinase K. These experiments were performed by adding xylene to tissue then washing with various dilutions of ethanol. After digestion with Proteinase K, RNA extraction was performed according to its protocol  and the samples were treated with DNaseI .Complementary deoxyribonucleic acid synthesisA two-step cDNA synthesis protocol was performed with the help of Random Hexamer primer and Reverse Transcriptase Enzyme (Cinagen-PR911658) in reaction tubes at a volume of 20 μL according to the optimized protocol for cDNA. The cDNAs were transferred to a -20°C freezer.Semi-quantitative reverse transcriptase polymerase chain reaction sqRT-PCR was optimized by synthesizing cDNA as templates (2 μl) and 2X MasterMix  with specific primers 10 pmol) for amplification of spliced and non-spliced variants of XBP with a touchdown program  in a thermocycler machine. Used primers amplified a 152 base pair (bp) (non-spliced isoform) or a 126 bp fragment (spliced isoform). The products were loaded on 1.3% agarose gel  which was stained with Safe Stain . The bands were analyzed using the Gel Doc system . To confirm the amplified products, some of the PCR products were sent to Macrogen (South Korea) for sequencing on an Applied Biosystems . The sequence of these primers [ pmol forStatistical analysisStatistical analysis was performed using MedCalc and GraphPad Prism software version 7.0 (unpaired t-test).Bioinformatics analysis determined the XBP gene contains five exons that code several types of transcripts. The type I variant is a longer transcript, although it produces a shorter protein isoform. The type II variant in the coding sequence is shorter than variant 1, resulting in a spliced-type with a C-terminal domain longer than the non-spliced isoform  acts as a transcription factor in the pathway responding to ER stress.Extracted RNA from tissue samples Figure  had concFigure Additionally, we used another set of primers to amplify the spliced variant of XBP again. The 62 bp band confirmed the spliced products Figure . Also, tThe data showed there were more than 7.8 times the total number of spliced variants in the marginal tumor samples than in the tumor tissue samples P<.05)  as regulators of signaling pathways in various types of cancer , it is sWe found an inverse relationship between XBP transcription and CRC tumor tissue, possibly due to the inactivation of apoptosis in the downstream response to ER stress. However, the remaining genes in this pathway should undergo gene expression analysis using a greater number of samples.


Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5′RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19–26 years) and middle-age (45–58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen. A number of previously published studies suggests that the function of adaptive immunity is impaired in aged individuals , 2. The Functional defects of antibody-mediated vaccine-induced immunity in elderly adults are manifested both in the hampered generation of a primary response and in decreased effect of booster vaccination: low production of vaccine-specific antibodies, low affinity and opsonic capacity of generated antibodies, reduced vaccine longevity , 17–23. Recent advances in high-throughput sequencing (HTS) allow performing a targeted readout of hundreds of thousands of B-cell receptor (BCR) heavy chain (IGH) sequences from samples of interest –30, provThe HTS approach was also employed for studies of influenza , 27, tetThere is also an increasing number of data characterizing changes in the antibody repertoires with respect to vaccine immune stimulus and age. We have found three HTS-based studies of BCR repertoires aimed at revealing the age-related differences in vaccine response, all tracking the changes upon influenza vaccine challenge , 32, 43.Wu et al.  analyzedJiang et al.  analyzedde Bourcy et al.  analyzedAll these data have highlighted the importance of B cell repertoire dynamics consideration in vaccine studies in the elderly adults, but were limited to tracking the response to a common pathogen with a substantial exposure history. Here we focused on investigation of the age-related differences in BCR repertoire structure of the plasma B cells responding to the live yellow fever (YF) virus vaccine in young (19–26 years old) versus middle-age (45–58 years old) individuals as a model of response to a previously unencountered pathogen.We utilized our protocol based on 5′-RACE with unique molecular identifiers (UMI) that allows nearly error-free, full-length (FR1-FR4 plus IgD/IgM/IgG/IgE/IgA isotypes identification) sequencing of IGH variable region repertoires , with mihighCD38high plasma B cells from peripheral blood samples obtained from healthy volunteers 9 days after their first vaccination with live YF vaccine. In this time frame, the concentration of plasma B cells in peripheral blood increases dramatically and mainly represents the cells that respond specifically to the vaccine antigens ;Profiles of newly acquired and potentially pre-existing somatic hypermutations.We reveal a number of antibody repertoire features that were distinct between young and middle-age individuals, highlighting age-related differences in humoral immune response directed against newly encountered antigens, that are already detectable by the age of 50.n = 10, Table high and collected directly into RLT buffer (Qiagen) for storage and RNA extraction. The numbers of sorted plasma cells per sample are shown in Table This study was approved by the local ethical committee and conducted in accordance with the Declaration of Helsinki. All donors were informed of the final use of their blood and signed an informed consent document. The cohort of healthy donors  composition of NDN regions of two sequences is highly similar the sequences are considered to originate from one ancestor sequence and connected by an edge on the lineage tree.An edge connects a pair of clonotypes that are likely to come from a pair of cells, one of which is a hypermutated [bears a B-cell receptor with mutation(s)] sub-variant of another. The direction of edge shows which of the clonotypes is a parent one and which is a child one. As we use full-length immunoglobulin sequencing data, one can infer edge direction using parsimony principle: parent clonotype mutations should be a subset of child mutations.in-silico mixing Raji hypermutating cell line repertoire and PBMC IGH samples. CDR3 hypermutations were obtained using Smith-Waterman alignment for each pair of connected nodes with different CDR3 sequences. Parsimony principle was applied to remove improbable nodes and infer the direction of edges. In order to eliminate duplicate paths and form a tree structure we next removed all incoming edges except the one with lowest number of mutations. To normalize samples for accurate comparison, we extracted 10,000 randomly sampled IGH cDNA molecules from each dataset.The similarity is computed by summing the information content of each K-mer , thus K-mers containing many non-template bases are scored the most. The similarity score threshold for drawing an edge was selected according a benchmark performed by Averaged CDR3 physicochemical properties of repertoires accounting for the clonotypes size were computed using custom R script. To estimate the energy of the interaction between CDR3 and a random epitope, we used Miyazawa-Jernigan statistical potential , that isThe selection strength was estimated using BASELINe framework  which cohttp://www.imgt.org/IMGTeducation/Aide-memoire/_UK/aminoacids/IMGTclasses.html) for the young versus middle-age individuals , and included CDR3 length, added N nucleotides, and physicochemical characteristics for the five amino acid residues located in the middle of CDR3 . The laThe analysis revealed several features that significantly differed between the responding plasma cell IGH repertoires of the two age groups but not between the isotypes Figure . Of noteWe have not detected prominent differences in IGHV gene segment usage as well as in isotype usage between plasma cell IGH repertoires of young and middle-age individuals vaccinated with YF Figures . The lisNote that potential biases in isotype and IGHV gene segment usage are corrected by UMI-based analysis, since sequencing reads that cover the same cDNA molecule (irrespectively to the efficiency of amplification of each particular isotype or gene segment) are clustered together, each cDNA molecule to a single UMI-labeled group of sequencing reads.The analysis of antibody repertoires can be extended by grouping IGH clonotypes into clonal lineages (trees) that share a common ancestor  and reprThis analysis revealed that basic graph characteristics, such as the Gini inequality coefficient, and number of singletons (clones including only one clonotype) were significantly different between young and middle-age YF-vaccinated donors Figure . The GinNote that the trees may include impossible lineage relations such as IgA to IgM isotype conversion. This reflects the fact that our analysis is limited by sampling depth and particular time point. We do not observe the whole pre-history of hypermutation process, thus additional unseen IGH sequence variants that can resolve this ambiguity may exist, e.g., unseen IgM sequence variant parent to observed IgM and IgA variants.Bulk load of somatic hypermutations per clonotype obtained without using the information on the trees structure was comparable between young and middle-age plasma cell IGH repertoires Figure . We contIn order to estimate the burden of somatic hypermutations that could earlier accumulate within memory B cells clones currently participating in immune response, we analyzed the frequency of hypermutations within the identified roots of the trees—the IGH sequence variants that were closets to germline within each tree. This analysis also has not revealed significant differences between the young and middle-age individuals in a load of root somatic hypermutations, potentially pre-existing within responding IGH clones Figure .To test for the intrinsic differences in the structure of hypermutations, we estimated the average “selection strength” that drived the accumulation of somatic hypermutations in young versus middle-age individual repertoires using BASELINe framework , which iFinally, we focused on the newly generated mutations that are directly observed (identified in the edges of the trees), i.e., hypermutations that are supported by both observed “parent” and “child” clonotypes in the dataset. A dedicated tree building algorithm allowed us to infer the set of currently ongoing hypermutations on the entire length of the immunoglobulin sequence Figure . Note thMiddle-age individuals had higher total numbers of the newly acquired unique somatic hypermutations Figure , while tOur comparative analysis of immune response to a novel pathogen, performed using immune repertoire sequencing and modeled by a live YF vaccine, revealed several differences between the two age groups indicating that humoral adaptive response already undergoes significant changes by the age of 50.First, physicochemical properties of the hypervariable IGH CDR3 region that are linked to antigen recognition , 59 chanSecond, the analysis of clonal lineages suggests that young individuals produce a more diverse IGH repertoire implying higher efficiency of the adaptive immune response . Middle-Third, responding clonal lineages of the middle-age individuals hypermutated more intensely Figure  but lessOf note, it was earlier demonstrated that AID levels and intensity of hypermutation decrease with aging, but only after the age of 60 , 69, 70—We have not obtained data on serum titers of YF-specific antibodies due to technical unavailability of samples. In general, it is known that the serum titers are similar for these age groups , 72, andFurther studies involving a larger set of novel antigens and comprehensive longitudinal tracking of the response, as well as studies of vaccinations with vaccine boost, can shed more light on the fine properties of age-related changes in B cell response.DS and GS performed cell sorting. AD, AO, ML, and MS analyzed the data. AO, MS, and DC prepared the figures. ML, MT, EM, and OK worked on samples, library preparation, and sequencing. OB and DC designed and managed the entire study. MS and DC wrote the manuscript. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A 35-year-old woman underwent a caesarean at 39 weeks and 6 days of her pregnancy. The operation was done without complications. Over the next days, the patient complained about abdominal bloating and nausea. Clinical examination noted a decrease in peristalsis. The biological analysis showed an inflammatory syndrome with a C-Reactive Protein (CRP) level of 143 mg/L and hyperneutrophilia up to 13930/µl. Abdominal contrast-enhanced Computed Tomography (CT) was performed and revealed distension (with air-fluid levels) of the stomach, the small bowel and both ascending and transverse colon Figure . The patOgilvie’s syndrome is also called acute colonic pseudo obstruction (ACPO) and is a clinical condition with symptoms, signs and radiographic appearance of acute large bowel obstruction, mostly right and transverse, without a mechanical cause. ACPO may be linked with a defect of the parasympathetic innervation of the left colon causing a loss of its peristalsis, which would lead to upstream dilation and hyperpressure within the right colon. In young women, caesarean section is the most common surgical procedure associated with the onset of this syndrome that occurs post-operatively at an average of 4–5 days. The clinical features of ACPO include lower incomplete intestinal obstruction and mostly abdominal distension . Plain a


The main objectives of this four-arms RCT are (1) to investigate the synergetic effects of a group-based multidomain training program that combines cognitive video-game training with physical exercise, in comparison to those produced by cognitive training combined with physical control activity, physical training combined with cognitive control activity, or a combination of both control activities; (2) to investigate in a memory-based task switching task whether event Related Potential (ERP) latencies of the P2 component are shorter, and N2 and P3b components are enhanced after training; and (3) to find out whether possible enhancements persist after a 3-month period without training. One hundred and twenty participants will be randomly assigned to one of the four combinations of cognitive training and physical exercise. The cognitive component will be either video-game training  or video games not specifically designed to train cognition . The physical exercise component will either emphasize endurance, strength, and music-movement coordination  or stretching, toning and relaxation . This RCT will investigate the short and long-term effects of combined multi-domain training compared to cognitive training and physical training alone, on executive control and memory functions of healthy older adults, in comparison with the performance of an active control group. This trial is an ongoing project started in 2018. Trial registration: Clinicaltrials.gov ID: NCT03823183;


Dietary protein profoundly influences organismal traits ultimately affecting healthspan. While intracellular signalling downstream of altered amino acid supply is undoubtedly important, peptide hormones have emerged as critical factors determining systemic responses to variations in protein intake. Here the regulation and role of certain peptides hormones in such responses to altered dietary protein intake is reviewed. This isGastrin is involved in the acute regulation of protein digestion . In addiGhrelin is secreted by the stomach, mainly by the gastric fundus in response to lack of food . The levSecretin is the first discovered hormone by the seminal work of Bayliss and Starling . It is sCholecystokinin (CCK) is secreted by the I-cells of the duodenal and jejunal mucosa  in respoGlucagon-like peptide 1 (GLP1) is derives from the proglucagon gene, and is secreted mainly by the L-cells of the distal small intestine and colon in response to food intake . GLP1, mGlucagon like peptide 2 (GLP2) also derives from the proglucagon gene, and is typically co-secreted with GLP1 . GLP2 maGlucose-dependent insulinotropic polypeptide  is released from intestinal K-cells predominantly in the duodenum in response to the presence of nutrients in the intestinal lumen . GIP is Peptide tyrosine-tyrosine (PYY) is released from the intestinal L cells from the distal parts of the GI tract including the ileum and colon  in respoFibroblast growth factor 19  is secreted from the enterocytes of the small intestine in response to meal feeding and coordinates postprandial nutrient homeostasis . The maiInsulin is the most well studied postprandial hormone. It is secreted by the beta-cells of the pancreatic islets in response to meal feeding . It actsGlucagon (GCG) is typically known as a fasting hormone , but is Amylin (a.k.a. amyloid polypeptide) is an anorexigenic hormone secreted by the pancreatic islet beta-cells  in respoPancreatic polypeptide (PP) is secreted by the F-cells of the pancreatic islets  in a bipSomatostatin  is secreted by the delta-cells of the pancreas . In geneFibroblast growth factor 21 (FGF21), an hepatocyte/liver-derived peptide hormone, has emerged as a potent regulator of systemic response to altered nutrient supply . OriginaInsulin-like growth factor 1 (IGF1) is a liver derived hormone and part of the growth hormone-IGF axis. Whether altered dietary protein affects IGF1 is currently controversial with some studies showing a positive relationship ,120,121,Leptin is an adipose tissue derived peptide hormone with levels correlating with whole-body fatness in multiple mammalian species. Studies of both human and mouse leptin receptor mutants have revealed an integral role in body weight regulation , althougAdiponectin is secreted from adipose tissue in a fashion inversely related to fatness  and reguAside from several intracellular signalling cascades, peptide hormones play a major role in mediating the systemic metabolic response to alterations of dietary protein intake. In particular, both glucagon and PYY are mediators of the response to heightened protein intake modulating liver amino acid metabolism and hypophagia, respectively, while FGF21 has emerged as a bone-fide endocrine signal of low protein intake affecting both the altered feeding and systemic metabolic response . Future


Neisseria [N. gonorrhoeae and N. meningitidis]. PEA decoration of lipid A is mediated by the enzyme EptA (formerly termed LptA) and promotes resistance to innate defense systems, induces the pro-inflammatory response and can influence the in vivo fitness of bacteria during infection. These important biological properties have driven efforts dealing with the biochemistry and structural biology of EptA that will facilitate the development of potential inhibitors that block PEA addition to lipid A.Bacteria cause disease by two general mechanisms: the action of their toxins on host cells and induction of a pro-inflammatory response that can lead to a deleterious cytokine/chemokine response  often seen in bacteremia/septicemia. These major mechanisms may overlap due to the action of surface structures that can have direct and indirect actions on phagocytic or non-phagocytic cells. In this respect, the lipid A (endotoxin) component of lipopolysaccharide (LPS) possessed by Gram-negative bacteria has been the subject of literally thousands of studies over the past century that clearly identified it as a key virulence factor in endotoxic shock. In addition to its capacity to modulate inflammatory responses, endotoxin can also modulate bacterial susceptibility to host antimicrobials, such as the host defense peptides termed cationic antimicrobial peptides. This review concentrates on the phosphoethanolamine (PEA) decoration of lipid A in the pathogenic species of the genus  Neisseria gonorrhoeae (GC) and N. meningitidis (MC) to decorate their lipid A with phosphoethanolamine (PEA) has profound implications for their ability to survive host-derived antimicrobials and influence the host’s pro-inflammatory response during infection. In the past decade a number of studies have been reported that advance our knowledge on the molecular mechanisms of this lipid A modification.The capacity of N. gonorrhoeae (GC) that threaten the efficacy of currently used antibiotics. Accordingly, this review is concerned with bringing together results from molecular and structural studies that have focused attention on the enzyme EptA that is responsible for PEA decoration of lipid A in the context of biological studies that support this modification as a virulence factor.We propose that this strategy would render bacteria susceptible to innate host defenses and reduce the potentially damaging action of the pro-inflammatory response during infection. We posit that EptA inhibitors would serve as adjunctive therapeutics to counteract multidrug-resistant strains of N. gonorrhoeae causes the sexually transmitted infection termed gonorrhea with a world-wide yearly estimate of >78 million infections  syndromes meningitis and/or fulminant septicemia seem to have appeared much later than gonorrhea in the evolution of Antibiotic resistance expressed by GC and MC has considerable implications for treatment options affecting severe complications and control of the disease in the community , has been shown to be required for multiple aspects of GC and MC pathogenesis including colonization, inflammation and survival in neutrophils, and we propose this enzyme has exciting potential as a target for development of anti-virulence compounds.Neisseria spp.  and 3-deoxy-D-manno-2-octulosonic acid (KDO) attached to a lipid A moiety embedded in the outer membrane  which mimics human glycosphingolipids such as the human I erythrocyte antigen (k antigen (N-acetyl-glucosamine (GlcNAc) residue -3-hydroxydodecanoic acid [12:0 (3-OH)] and the amino groups at positions 2 and 2′ being substituted with (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O (12:0)-14:0]. The β1-6 linked di-glucosamine backbone of lipid A can be variably substituted at the 1 and 4′ positions with phosphate, di-phosphate, O-phosphorylethanolamine, and O-pyro-phosphorylethanolamine , and nfnB, which encodes a putative nitroreductase . Expres-4 (4 logs greater than spontaneous mutation) a single T insertion occurs due to slipped-strand mispairing resulting in premature truncation of EptA (62 amino acid protein).EptA expression is governed at the translational level by two means: phase variation of the open reading frame and protein stabilization by oxidoreductases. Translation of full-length EptA is subject to high frequency mutation due to a phase variable poly-T track in the 5′ end coding sequence . In the N. gonorrhoeae and MC contain a cohort of three oxidoreductases  which are oxidized at the active site with a disulphide bond by membrane bound DsbB. The oxidoreductase rapidly and irreversibly donates the disulphide bond to thiol groups on substrate proteins resulting in the release of an oxidized substrate carrying a disulphide bond and a reduced oxidoreductase. DsbA1 and DsbA2 are paralogs, with DsbA2 being encoded on a genetic island that is distributed in MC but absent in GC . EptA is an integral membrane protein consisting of an N-terminal transmembrane (TM) domain and a C-terminal soluble periplasmic-facing domain. The solved crystal structure of the soluble domain showed it adopts a hydrolase-type fold with a bound Zn2+ ion at the enzyme active site near to the catalytic nucleophile, Thr280 .EptA catalyzes the transfer of PEA from phosphatidylethanolamine (PtdE) to lipid A at 1 and/or 4′ head group positions  oriented approximately parallel to one another and spanning the inner membrane in a previously uncharacterized fold. A patch of positively charged residues  at the cytoplasmic end of TMH5 are likely to provide interactions with the phospholipid head groups at the inner membrane surface. The bridging helix (residues 194–208) has a highly unusual configuration in the structure and is linked to the soluble domain by an extended coiled region (residues 210–231). There is a large interface between the soluble and membrane domains which is conserved in all lipid A PEA transferases suggesting this region has a conserved function. Intrinsic tryptophan fluorescence experiments and molecular dynamics simulations has led to the proposal that the protein is highly dynamic in the membrane and support a model that the enzyme carries out catalysis through a “ping pong” kinetic mechanism. In this model, the first step requires EptA to bind PtdE using the small periplasmic helices (PH2 and PH2′) located between TM3 and TMH4. PEA is cleaved from PtdE and transferred to the Thr280 residue to form an PEA-enzyme intermediate. The soluble domain then rotates way from the TM domain, resulting in an open conformation capable of accepting the larger lipid A into the binding site pocket and completing the transfer of the PEA from the enzyme intermediate to the 1 and 4′ positions of lipid A.The membrane domain contains five transmembrane helices  that suppresses the growth of most Gram-negative bacteria but not Neisseria spp. which are resistant to this antibiotic. This unfortunately prevents the detection of MC or GC with “phase-off” eptA in pathology diagnostic laboratories as “phase-on” eptA confers colistin resistance.se sites . EptA muse sites . Removalse sites . Althougse model . No studThe complement pathway is an important arm of the innate immune system. Complement protects against infection and under physiological conditions, its activation is tightly controlled by soluble and membrane-associated complement inhibitors. Both GC and MC have sophisticated mechanisms for evading complement-mediated killing during colonization and invasive disease , 2016. TEvasion of complement-mediated killing is commonly associated with escaping the ability of normal human serum to the kill the organism once it enters the bloodstream. In this case, the role of complement resistance by MC during invasive disease is substantial in enabling bacteremia . Female eptA mutant induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Consistent with these tests, analysis of the chemokine/cytokine responses of female mice with lower genital tract infection showed that the parent GC strain possessing lipid A with PEA decorations elicited a robust pro-inflammatory response that was significantly dampened in mice infected with the GC eptA null mutant; while markers of the anti-inflammatory response were not significantly different in mice infected with either strain.While the endotoxins of MC and GC have an identical lipid A, most information on the structure and functional relationships that are necessary for eliciting the pro-inflammatory response has been conducted with meningococcal lipid A . Both eneptA mutant had a substantial fitness defect in vivo compared to the wild-type strain during a competitive, mixed infection. etpA mutants.The binding of lipid A to the TLR4/MD-2 complex on macrophages and neutrophils results in cellular activation, the release of cytokines, chemokines, reactive oxygen species (ROS), and nitric oxide which will kill phagocytosed bacteria. In the female mouse model of lower genital tract infection, eptA mutants from MC and GC are exquisitely sensitive to killing by CAMPs and are rapidly killed by neutrophils as a result  and non-oxidative mechanisms  to kill the bacteria . Neutropa result . Interesa result  and addia result .Neisseria spp. This feature stimulates the pro-inflammatory responses during IMD and gonorrhea but also provides a protective role against clearance by innate immune cells such as neutrophils and macrophages that are attracted to the site of infection. In both instances, the engulfment of the bacteria leads to a frustrated innate immune response that results in chronic inflammation and in the case of GC infected neutrophils become vehicles for transmission between hosts.The decoration of lipid A with PEA in GC and MC is an essential pathogenesis factor that distinguishes these pathogens from most commensal Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, etc.), rendering them resistant to colistin therapy (Neisseria sp. will be prove to be a beneficial approach to the development of novel therapies. Therapeutics to boost the bactericidal activity of phagocytic cells are currently in development (We propose that inhibition of EptA will improve killing and clearance of these pathogens by neutrophils thus improving clearance of infection from mucosal surfaces and providing a mechanism that curtails transmission of GC in neutrophils. In addition, EptA enzymes are found in many gram-negative pathogens (e.g.,  therapy . Detaile therapy  or inhibelopment , which iCK and WS wrote the drafts of the manuscript with editorial changes and input by KN, AA, and AV. AA and KN prepared the figures and performed experiments described in the text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.