Biologists have taken the concept of organism largely for granted. However, advances in the study of chimerism, symbiosis, bacterial‐eukaryote associations, and microbial behavior have prompted a redefinition of organisms as biological entities exhibiting low conflict and high cooperation among their parts. This expanded view identifies organisms in evolutionary time. However, the ecological processes, mechanisms, and traits that drive the formation of organisms remain poorly understood. Recognizing that organismality can be context dependent, we advocate elucidating the ecological contexts under which entities do or do not act as organisms. Here we develop a “contextual organismality” framework and provide examples of entities, such as honey bee colonies, tumors, and bacterial swarms, that can act as organisms under specific life history, resource, or other ecological circumstances. We suggest that context dependence may be a stepping stone to the development of increased organismal unification, as the most integrated biological entities generally show little context dependence. Recognizing that organismality is contextual can identify common patterns and testable hypotheses across different entities. The contextual organismality framework can illuminate timeless as well as pressing issues in biology, including topics as disparate as cancer emergence, genomic conflict, evolution of symbiosis, and the role of the microbiota in impacting host phenotype. Biologists have taken the definition of organism for granted for most of the history of the study of life. However, at a time when biologists have discovered that bacterial‐eukaryote symbiosis is near‐universal, that cancer cells may collaborate to create tumors, and that microbes can communicate and perform collective actions, the concept suddenly seems less clear. Did my genes stuck in my mother affect my brother's development  form a new entity characterized by high cooperation and low conflict among the parts .We argue that a given assemblage of parts will or will not behave as an organism‐like unit, depending upon the specific ecological conditions in time and space in which it occurs. Contextual organismality focuses on studying the traits and mechanisms that are associated with such transitions. For instance, single‐celled microbes in groups, widely regarded as competitors, can show highly cooperative interactions under conditions of starvation, leading to fruiting bodies that allow dispersal and survival of a subset of cells Kaiser . When anDoes the group's cooperation‐conflict level change?When and under what ecological context does it change?What were the important traits that enabled that change?Do different biological groups change under similar circumstances, or are similar traits important?The relevant contexts that determine a group's cooperation‐conflict dynamic vary according to its composition, but can include life stage (development), resource availability, population size, and interactions with other species. Examining different types of biological entities in different contexts reveals exciting new questions that emerge by viewing the phenomenon through the contextual organismality lens:Contextual Organismality in Practice). The answers to these questions can shed light on some of the major challenges in the study of organismality and also generate novel, testable hypotheses to gain further insights into interactions among biological entities.These questions form the basis of investigations guided by the conceptual organismality framework, as we will show in a subsequent section by applying them to biological examples , and the hawkmoth Manduca sexta shifts over developmental time with regard to the relative importance of cooperation versus conflict  due to pollen transport between flowers  in many forms .Thus, a lack of context dependency can be used as an indicator of an organism, that is, a group that preserves high cooperation and low conflict among the parts across widely divergent contexts. This conceptualization of the organism reconciles the fluidity of contextual organismality with organismality approaches that employ fixed definitions of an organism . Potential model systems to test this prediction are interspecific mutualisms that vary in their degree of unification  for the similar responses of cells to starvation across the tree of life.What is an organism?” As with many other fundamental questions, it is unlikely that a single answer will be forthcoming. However, tackling this question has great potential to generate knowledge. An analogy with the question “What is a species?” is appropriate . After over three centuries of study, biology is arguably further away from an answer. Now there are multiple definitions, called species concepts. What has been gained is a breadth and depth in the knowledge of the process of speciation and all the different ways it can happen (De Queiroz It is our hope that the contextual organismality framework will contribute to expanding empirical and synthetic research into one of the fundamental questions of biology: “When is an organism?” to the research agenda. The answer lies in the mechanisms of organismality, which can inform issues as disparate as cancer emergence, the role of the microbiome, and genomic conflict, addressing both pressing and timeless questions in the study of life.Likewise, it is our hope that contextual organismality serves as a useful framework for understanding the process by which organisms emerge. We advocate adding the important question “Associate Editor: R. AzevedoHandling Editor: R. Shaw


Purpose. To assess the effect of three-dimensional (3D) printed personalized moisture chamber spectacles (PMCS) on the periocular humidity. Methods. Facial computed tomography (CT) scanning was conducted on 10 normal subjects. PMCS was designed based on volume rendered CT images and produced using a 3D printer. Periocular humidity of PMCS and commercially available uniformed moisture chamber spectacles (UMCS) were measured for 30 minutes via microhydrometer. Results. The mean ambient humidity was 15.76 ± 1.18%. The mean periocular humidity was 52.14 ± 3.00% in PMCS and 37.67 ± 8.97% in UMCS. The difference was significant (P < 0.001). Additionally, PMCS always demonstrated lower humidity than dew points. Conclusion. PMCS made by 3D printer provides appropriate fitness for the semiclosed humid chamber. PMCS showed higher performance than UMCS. The wearing of PMCS would be an effective method to provide high enough periocular humidity in low humidity environment.  Dry eye is a common ocular disease prompting millions of individuals to seek ophthalmological care . ArtificKorb et al. reported that increased periocular humidity using modified swim goggles resulted in an increase in tear film lipid layer thickness and moderate to total relief of dry eye symptoms . TsubotaUnlike general spectacles, moisture chamber spectacles need to be personalized in shape and size of frame for best performance. The tear retention effectiveness of moisture chamber spectacles is determined by the extent of proper semiclosed chamber formation. The formation of an appropriate chamber depends on how well the spectacles fit the facial contour. There are several commercially available moisture chamber spectacles; however they are single size or minimally adjustable. Previous studies were also performed using spectacles with single or several size variations , 3.Three-dimensional (3D) printing was invented in the 1980s and has shown marked growth in recent years. 3D printing has been advancing in customized product printing and has proved to be effective for medical applications –6. RecenIn this study, personalized moisture chamber spectacles (PMCS) were produced using facial computed tomography (CT) scanning and 3D printing technology. The effect of PMCS on periocular humidity was evaluated and the clinical implication was discussed.This study was approved by the Institutional Review Board of research ethical committees of Aeromedical Center, Republic of Korea Air Force, and conformed to the tenets of the Declaration of Helsinki (ASMC-15-IRB-001). Ten normal subjects who have no history of evidence of ophthalmic disease were recruited.Facial CT scanning for PMCS was conducted on the participants. Facial CT scanning was performed using a 64-channel CT scanner . After conventional CT scan, 3D reconstruction was processed and facial contour was obtained using volume rendering tool, which was incorporated in the workstation .2 and held 0.85 g of distilled water. Silicone part of moisture chamber was constructed on CAD and the mold was printed. Then silicone  was infused into the mold and silicone chamber was formed.The spectacles were designed using computer-aided design (CAD) program . PMCS consisted of mainly 4 parts, that is, frame, side panel, moistened sponge block, and silicone chamber . All parThe present study was designed as cross-over test. The subjects wore a sequence of different moisture chamber spectacles. Commercially available uniformed moisture chamber spectacles (UMCS)  and PMCS were worn naturally for 30 minutes while periocular humidity and temperature were measured. UMCS were worn once again with gentle pressure on the spectacles frame in order to measure periocular humidity with nearly perfect sealing of moisture chamber. The order of the wearing spectacles was randomized. There was an interval of 30 minutes, prior to wearing other spectacles. The microhydrothermometer  was attached to the backside of the spectacles lens . Left eytd = dew point, t = temperature, and RH = relative humidity): t” and ambient temperature for “td.” The calculated RH and measured periocular humidity were compared.The excessively high periocular humidity induces condensation on the spectacles lens and visual blur. The dew point is the temperature at which dew forms. A simple equation for the relationship between relative humidity and the dew point is as follows   and Bonferroni post hoc test were performed for comparisons among measurements. Shapiro-Wilk was used for normality test. Statistical analysis was conducted, using SPSS Statistics Version 21 . All tests were two-tailed and The mean age of participants was ±years. Eight subjects were males and 2 were females. Facial characteristics including interpupillary distance, bitemporal distance, and temple length were described in P < 0.001), (The hydrometer had an accuracy of ±3% RH and a repeatability of ±1% RH. Measurements of periocular humidity over time were shown in  0.001), .P = 0.002). However, the difference of periocular temperature between pressurized wearing of UMCS and natural wearing of PMCS was not significant (P = 0.071), (The thermometer had an accuracy of ±0.5°C and a repeatability of ±0.3°C. Measurements of periocular temperature over time were shown in  0.071), .Measured periocular humidity and calculated humidity level that starts to cause the condensation on the spectacles were shown in The wearing of moisture chamber spectacles can be helpful to improve ocular discomfort associated with dry eye. Based on the definition from the dry eye workshop (DEWS), dry eye disease is defined as multifactorial disease that is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface . The DEWIn this study, PMCS increased absolute value of 36.39% periocular humidity, as compared to the ambient humidity. Moisture chamber spectacles may be particularly helpful to reduce artificial tear use for severe dry eye, involving Sjögren's syndrome or lagophthalmos patients, and ultra-low humidity environmental worker including laboratory employee  or aircrAppropriate fit is the most critical aspect in moisture chamber spectacles. The loose fitness of spectacles reduces relative humidity in the moisture chamber. In the present study, PMCS showed significantly higher periocular humidity than UMCS, as predicted. Additionally, UMCS demonstrated wide individual differences of periocular humidity (standard deviation = 8.96), while PMCS showed narrow individual variations (standard deviation = 3.00). In contrast, tight fitness of spectacles induces excessively high relative humidity, resulting in condensation as well as discomfort. Moisture chamber should be a semiclosed, and not a completely closed space due to condensation. In this study, pressurized wearing of UMCS which formed near completely closed space showed higher mean periocular humidity than condensation humidity. Though the higher periocular humidity provides greater tear retention effect, the spectacles inducing condensation without antifog material are not practical for daily wear. PMCS provided sufficiently high periocular humidity; however the measurements were always slightly lower than the condensation point. These results suggested that personalization of moisture chamber provides appropriate fit.There were some limitations to this study. The present study had small sample size and did not include patient group and clinical evaluation. Further investigation will be needed to determine the clinical effectiveness of PMCS, involving subjective symptoms and objective ocular surface evaluation in patients with dry eye disease with larger sample size.PMCS made by 3D printer provides appropriate fitness to form semiclosed humid chamber. PMCS increased absolute value of 36.39% periocular humidity without condensation as compared to the ambient humidity and demonstrated significantly higher performance than UMCS. The wearing of PMCS would be an effective method to provide highly enough periocular humidity in low humidity environment.


Urothelial carcinoma ranks the ninth among malignant cancers. We conducted this study to identify which patients could benefit more from the treatment of programmed death-1 (PD–1)/programmed death-ligand1 (PD–L1) inhibitors.We performed literature searches, combined data from qualified literature and performed comparative analyses on the effectiveness of anti-PD–1/PD–L1 antibodies in patients with different PD–L1 expression levels.We divided patients into three groups according to the percentages of PD–L1-positive cells, namely the low- PD-L1 (PD-L1 < 1%), the medium-PD-L1 (PD-L1 ≥ 1 and < 5%) and the high–PD–L1 (PD-L1 ≥ 5%) groups. We found that the high-PD-L1 group responded significantly better than other groups , while the latter two groups responded similarly  to both PD–1 and PD–L1 inhibitors. Furthermore, we found that the medium-PD–L1 and high-PD–L1 groups responded similarly to PD-1/ PD-L1 inhibitors , while the low-PD–L1 group responded better to PD-1 inhibitors than PD-L1 inhibitors .Our results suggest that PD–L1 positive patients should be defined as those with ≥ 5% or greaterPD-L1-positive cells. PD-1 antibodies performed better only in the low-group patients, likely because they could block the interactions of PD–1 with both PD–L1 and PD–L2. The incidence of urothelial carcinoma has become the ninth of all malignant tumors and the first in urinary tract cancers . It leadTumor microenvironment plays an important role in tumor immunity . ProgramIt has been shown in many clinical trials that cancer patients with higher expression of PD-L1 received better antitumor effect to PD-1/PD-L1 inhibitors , 11. SimWe retrieved in total 95 articles from public databases using the above-mentioned keywords. After reading abstracts and full texts, we kept six articles for further analysis; in total 828 participants were tested in the six studies. Please consult Figure Three out of the six studies evaluated PD-1 antibodies, of which two evaluated nivolumab , 20 and We first tested whether patients with higher-ratio of PD-L1-positive cells responded better to PD-1/PD-L1 antibodies. Either of 1% and 5% was often used in the literature to classify patients into “positive” and “negative” groups, according to whether there are more than 1% or 5% PD-L1-positive cells in a patient.P = 0.004, OR = 0.54,95%CI:0.39-0.85; see also Figure Of all the studies, 50 of the 345 (14.50%) patients with PD-L1 ≥ 1% level achieved objective responses, while 109 of 483 (22.57%) patients with PD-L1 < 1% level achieved objective response, confirming that patients with higher-ratio of PD-L1-positive cells indeed responded better to PD-1/PD-L1 antibodies  patients with PD-L1 ≥ 5% level achieved objective responses, as compared with 66 objective responses out of 481 (13.72%) patients with PD-L1 < 5% level; therefore patients with higher-ratio of PD-L1-positive cells responded better to PD-1/PD-L1 antibodies . In some studies, patients of the two groups were often combined; again, we found no significant differences in the combined datasets in the responses to PD-1 and PD-L1 antibodies.We next sought to compare the effectiveness of PD-1 and PD-L1 antibodies in patients with different PD-L1-positive cell ratios. In the literature, patients were often grouped into the following subgroups: PD-L1 < 1%, PD-L1 ≥ 1% but < 5%, and PD-L1 ≥ 5%; we referred them as to Low-PD-L1, Medium-PD-L1 and High-PD-L1 groups respectively in the following analysis. As shown in Figure P = 0.046, OR = 1.92, 95%CI: 0.98, 3.89; Fisher’s Exact Test) . However, patients in the Low- and Medium- groups responded similarly to both types of antibodies   Figure . These rIn this study, we performed a meta-analysis on the effectiveness of anti-PD-1/PD-L1 antibodies in urothelial carcinoma patients with different ratios of PD-L1-positive cells. We first confirmed previous findings that patients with higher ratios of PD-L1-positive cells responded significantly better to both PD-1 and PD-L1 antibodies than those with lower ratios of PD-L1-positive cells.We also checked whether patients responded better to one type of antibodies than the other. We found that in most cases, patients had similar ORRs between the two types of antibodies in the Medium-PD-L1 and High-PD-L1 groups. However, in patients with less than 1% PD-L1-positive cells (the Low-group), the objective response ratio was statistically better to PD-1 antibodies than to PD-L1 antibodies. This is likely that PD-1 antibodies are more sensitive to lower ratio of PD-L1-possitive cells, becausePD-1 antibodies block the interaction between PD-1 with PD-L1 and PD-L2, while PD-L1 antibodies only block the interaction between PD-1 with PD-L1 , 23. HowThe definition of PD-L1 “positive” patients is not very clear so far . CutoffsIn conclusion, PD-L1 positive patients of urothelial cancer response more than negative ones, and we suggest 5% or greater as the cutoff to distinguish them. PD-1 antibodies is better than PD-L1 antibodies in PD-L1 < 1% subgroup with urothelial carcinomas patients. Further clinical trials need to be performed to establish the position of immunotherapy in urothelial cancer.trials.gov and American Society of Clinical Oncology (ASCO) meeting abstracts for relevant articles published in English. All the articles were published before July, 2017. We used the following keywords to search for the articles: “PD-1”, “PD-L1” and “urothelial carcinoma”, “urothelial cancer”, “urothelial tumor” combined with “nivolumab”, “atezolizumab”, “pembrolizumab”, “avelumab”, “durvalumab”.We performed a systematic review search of literatures in Pubmed, Embase, clinical The articles must satisfy the following conditions: first, all patients suffering from advanced and metastatic urothelial carcinoma were treated with PD-1/PD-L1 antibodies. Second, certain types of raw data should be available in the original publications in order for us to combine results from multiple studies, including the total numbers of patients treated by PD-1/PD-L1 antibodies, the numbers of patients that responded to the treatment ), and the percentage of PD-L1-positive cells in the tumor microenvironment of these patients. In cases where the exact PD-L1-positive levels were unknown, patients should be grouped into sub-groups according to their rates of PD-L1-positive levels and the grouping criteria should be available.The data extracted from these articles was as below: the first author, the year of the article, the trial name, the antibody name, the different levels of PD-L1 expression and ORR (objective response rate), media progression-free survival (PFS), media overall survival (OS).The outcomes were ORRs and ORs of different PD-L1 levels in urothelial carcinoma patients by different antibodies.https://www.r-project.org) and R Studio (https://www.rstudio.com). Patients were grouped into subgroups according to the percentages of PD-L1-positive cells. The ORR value was calculated for each subgroup. Between-subgroup comparisons were performed by one-tailed Fisher-test using the Fisher. test (matrix) function in R; Odds ratio (OR), its 95% confidence interval (95%CI) and the P-value were recorded for each comparison. It was to consider statistically significant if P < 0.05.All analyses were performed using R 3.4.1 (


The expression of Programmed cell Death Ligand 1 (PD-L1) is observed in many malignant tumors and is associated with poor prognosis including Gastric Cancer (GC). The relationship between PD-L1 expression and prognosis, however, is controversial in GC. This paper purports to use a meta-analysis to investigate the relationship between PD-L1 expression and prognosis in GC. For this study, the following databases were searched for articles published from June 2003 until February 2017: PubMed, EBSCO, Web of Science and Cochrane Library. The baseline information extracted were: authors, year of publication, country where the study was performed, study design, sample size, follow-up time, baseline characteristics of the study population, pathologic data, overall survival (OS). A total of 15 eligible studies covering 3291 patients were selected for a meta-analysis based on specified inclusion and exclusion criteria. The analysis showed that the expression level of PD-L1 was associated with the overall survival in GC . In addition to the above, subgroup analysis showed that GC patients with deeper tumor infiltration, positive lymph-node metastasis, positive venous invasion, Epstein-Barr virus infection positive (EBV+), Microsatellite Instability (MSI) are more likely to expression PD-L1. The results of this meta-analysis suggest that GC patients, specifically EBV+ and MSI, may be prime candidates for PD-1 directed therapy. These findings support anti-PD-L1/PD-1 antibodies as a kind of immunotherapy which is promising for GC. Worldwide, gastric cancer (GC) is the fourth most common malignant disease in males  and the third leading cause of cancer mortality in males , especially in Eastern Asia , Central and Eastern Europe, and South America, and lowest in Northern America and most parts of Africa. DevelopRecently, some clinical trials have indicated that monoclonal antibodies that target PD-1 or its receptor PD-L1 prevent the inhibitory effects of PD-1/PD-L1 pathway and enhance T cell functions, leading to impressive outcomes in patients with cancers–9. HowevThrough a meta-analysis, this review focuses on PD-L1 expression and its association with clinical outcomes in GC. Furthermore, this research attempts to show that the potential of PD-L1 positive patient to obtain optimum treatment benefit, appears promising. It might pinpoint patients most likely to strongly benefit from the inhibition of PD-L1/PD-1 as monotherapy compared to those that may most likely require a different or combinatorial approach in GC.Studies indexed, from June 2003 until February 2017 were systematically searched in the following databases: PubMed, EBSCO, Web of Science and Cochrane Library. The search terms used were: (“stomach neoplasms” OR “gastric cancer” OR “advanced gastric cancer” OR “gastric carcinoma” OR “stomach cancers”) AND (“PD-1” OR “PD-L1” OR “programmed death 1” OR “programmed death ligand 1” OR “programmed cell death ligand 1” OR “programmed death 1 ligand 1” OR“B7-H1”). Additionally, the reference lists of the selected articles were manually reviewed to obtain other potentially relevant articles. Selected publications were all in the English language.From the potentially relevant articles obtained above, those that indicated correlation between prognosis  and PD-1/PD-L1 in GC were selected.For inclusion in this meta-analysis:Articles were limited to those dealing human subjects only.All patients with GC would have been diagnosed by pathological evidence.Expressions of PD-L1 would have been detected by Immunohistochemical (IHC) Assay from gastric carcinoma specimens.All patients had been followed up and results reported.The following criteria were used to exclude irrelevant papers:The literature was not the original article , or a literature duplication.The object of this study was cellular-based or animal-based.The study also covered other malignancies, or the study did not include the analysis of expressions of PD-L1 in subgroups.Data from each included study was independently extracted. The following baseline information was used: authors, year of publication, country where the study was performed, study design, sample size, follow-up time, cut-off criteria for overexpression (the definition of positive PD-L1), baseline characteristics of the study population, pathologic data, overall survival (OS).This meta-analysis was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) 2015 statement–13. The Hazard ratios (HR) including 95% Confidence interval (CI) were used to assess the association between PD-L1 expression level and OS in GC. An observed HR > 1 implied a worse prognostic significance for the group with elevated PD-L1 expression. Conversely, HR < 1 implied a worse prognostic significance for the group with decreased PD-L1 expression. Revman 5.3 Software  was used to evaluate heterogeneity between studies by Cochrane Q-test and P-values. Estimates of HR were weighted and pooled using the Mantel-Haenszel random effect model. The Stata 12.0 Software  was used to evaluate the sensitivity and publication bias of the studies. Publication bias was evaluated by Begg’s and Egger’s test, P < 0.05 was considered statistically significant. Begg’s and Egger’s test of publication bias was not performed on analysis subgroup with less than 5 studies because of low sensitivity of qualitative and quantitative tests.The search strategy originally generated 274 relevant clinical studies in English. Of these, 149 were eliminated because of repetition, 63 were excluded based on evaluation of their title or abstract. The remaining 62 articles were scrutinized by a full-text review. Based on the inclusion and exclusion criteria, 15 studies were selected for this meta-analysis. The detailed search and study selection process is shown in The 15 studies published between 2006 until 2016, included 3291 patients at baseline, with a maximum sample size of 451 and a minimum sample size of 96 participants. The majority of the studies were reported in Asia, 7 in China, 4 in Japan, 3 in South Korea, and 1 in Germany. The defining criterion for PD-L1 positive was that PD-L1 expression was observed in tumor cells by IHC. The discrepancies in the percentage of the PD-L1 positive expression patients were due to the variation in the cut-off values with the maximum ratio at 69.40% and the minimum at 14.32%. 1925(58.49%) patients did not undergo radiation or chemotherapy before surgery in 8 studies, others did not report. The characteristics of the included studies have been shown in A total of 15 studies reported that the expression level of PD-L1 was related to OS. 11 studies indicated that PD-L1 overexpression was associated with poor prognosis of GC, conversely, 3 studies reported that PD-L1 overexpression was associated with better prognosis, and there was no indicated associations 1 study. Analysis showed that the expression level of PD-L1was associated with the OS in GC  .As shown in Begg’s and Egger’s test was used to evaluate the publication bias, respectively. In this meta-analysis, Begg’s and Egger’s test indicated no publication bias among included articles regarding the HR and OS (Begg’s test: P = 0.276 and Egger’s test P = 0.061) . There weff) cells that are produced), the activated Teff cells can’t specifically recognize and bind to cancer cells, T cells therefore cannot kill their target cancer cells. In other words, some molecules, namely factors, may act to modulate activated anti-tumor T cell, such as PD-L1 Increased PD-L1 expression by tumor cells may induce and maintain iTreg cells in the periphery, thereby increasing the suppression of anti-tumor T cell responses and allowing tumor progression[As mentioned by: ‘Regulaeg) cells. PD-L1 iogression.In effect, this research paper tries to evaluate the association between the PD-L1 overexpression and the clinical pathological features. From this meta-analysis, gastric cancer patients with deeper tumor infiltration, positive lymph-node metastasis, positive venous invasion, E-B virus infection positive, MSI are more likely to express PD-L1. The results of subgroup analysis tend to support the idea that patients with positive PD-L1 have unfavorable prognosis.A recent study aiming tet al.[Beyond the differential presence of PD-L1+ cells in EBV+ and EBV- GC, Derks et al. observedTo the best of our knowledge, it is the first meta-analysis to investigate the association between PD-L1 expression and E-B virus infection and MSI in GC. This study shows that the expression level of PD-L1 is higher in EBV+ GC and MSI than in EBV- GC and Microsatellite stability (MSS). The result suggests that specifically EBV+ GC and MSI may be prime candidates for PD-1directed therapy.et al.[et al.[et al.[et al.[The limitations of this study should be stressed on. The heterogeneity among included studies cannot be ignored. We were unable to control for factors such as environmental conditions, racial differences, socioeconomic situation, postoperative treatment, follow-up, all of which are known to influence the OS. Besides, these studies have used a number of monoclonal and polyclonal PD-L1 antibodies for immunohistochemistry and a variety of different scoring schemes/criteria to define positive PD-L1 expression. For example, Eto et al. and Gengl.[et al. reportedl.[et al. and Bogel.[et al. considerl.[et al. to 69%[1l.[et al..Publication bias is another possible reason, wherein studies showing that PD-L1 was not a prognostic factor could be less likely to be published than studies showing that PD-L1 was a prognostic factor. Then the included studies were only English researches, no other languages. Beyond publication bias, the patients covered in this study were strictly from the countries where the studies were made. The conclusion would hence apply to those countries only unless the study is extended to other countries representatively. Perhaps the racial and social factors were also at play here as it has also been observed in other fields other than medicine. Their effects could have been cofounded with those being measured here. More representative samples of patient studies could be needed for stronger conclusion. Also, grey literature was not accounted for in this study as only studies published in the English language were chosen, 46.This meta-analysis was to firstly evaluate the association between the PD-L1 expression and a specific molecular subgroup (EBV+ and MSI) in GC. Furthermore, gastric cancer patient with EBV+ and MSI tend to show PD-L1 expression, which demonstrated that specifically EBV+ GC and MSI may be prime candidates for PD-L1 directed therapy. However, further large-scale and comprehensive researches are needed to support our results and conclusion.S1 Fig(A) sex, (B) age, (C) tumor site, (D) tumor size, (E) tumor differentiation, (F) Lauren Classification, (G) depth of infiltration,(H) lymph-node metastasis, (I) TNM stage, (J) lymphatic invasion, (K) venous invasion, (L) neural invasion, (M) E-B virus infection, (N)MSI-status.(TIF)Click here for additional data file.S2 Fig(A) Asian, (B-1) stages I-IV, (B-2) stages II-III, (B-3) stages I-III, (C) follow-up more than 5 years, (D-1) cut-off value >1%, (D-2) cut-off value >5%, (D-3) cut-off value >10%, (D-4) cut-off value >50%, (E-1) location within tumor cells were cytoplasmic, (E-2) location within tumor cells were cytoplasmic and nuclear, (F) no patients underwent pre-operative chemoradiotherapy, (G) patients underwent postoperative adjuvant Chemotherapy.(TIF)Click here for additional data file.S3 Fig(A) sex, (B) tumor site, (C) tumor size, (D) tumor differentiation, (E) depth of infiltration, (F) lymph-node metastasis, (G) TNM stage, (H) lymphatic invasion, (I) venous invasion.(TIF)Click here for additional data file.S4 Fig(A) sex, (B) tumor site, (C) tumor size, (D) tumor differentiation, (E) depth of infiltration, (F) lymph-node metastasis, (G) TNM stage, (H) lymphatic invasion, (I) venous invasion,(J)OS.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


We exploit this behaviour to present an in-plane dielectric heterostructure with a spatially dependent bandgap, as an initial step towards the creation of diverse lateral junctions with nanoscale resolution.The ability to control the size of the electronic bandgap is an integral part of solid-state technology. Atomically thin two-dimensional crystals offer a new approach for tuning the energies of the electronic states based on the unusual strength of the Coulomb interaction in these materials and its environmental sensitivity. Here, we show that by engineering the surrounding dielectric environment, one can tune the electronic bandgap and the exciton binding energy in monolayers of WS 2 and WSe2 semiconductors via manipulation of the surrounding dielectric environment rather than by modifications of the materials themselves.Electronic bandgap tuning in semiconductors enables key functionalities in solid-state devices. Here, the authors present a strategy to control the bandgap of atomically thin WS The precise and efficient manipulation of electrons in solid-state devices has driven remarkable progress across fields from information processing and communication technology to sensing and renewable energy. The ability to engineer the electronic bandgap is crucial to these applications2 with M=Mo, W and X=S, Se, Te468911131415The emerging class of atomically thin two-dimensional (2D) materials derived from bulk van der Waals crystals offers an alternative route to bandgap engineering. Within the family of 2D materials, much recent research has focused on the semiconducting transition-metal dichalcogenides (TMDCs)—MXImportantly, the Coulomb interactions between charge carriers in atomically thin TMDCs are remarkably strong18192022232425212723242627282930313436372 and WSe2, we achieve tuning of the electronic quasiparticle bandgap, as well as of the exciton binding energy of the two TMDC monolayers by several 100's of meV. We note that graphene is particularly well-suited to demonstrate and explore the concept of dielectric heterostructures. It combines a high dielectric screening with the possibility of adding an arbitrary number of additional layers as thin as only 3 Å. Furthermore, the TMDC/graphene structures have been heavily studied recently in a variety of contexts with potential applications in optoelectronics and photovoltaics394041In this report, we provide direct experimental demonstration of control of the bandgap and exciton binding energy in 2D materials using Coulomb engineering through the modification of the local dielectric environment. By placing layers of graphene and hexagonal boron nitride above and below monolayers (1L) of WS2 partially covered with bilayer (2L) graphene, is presented in 243n=1, 2, 3, …, analogous to the states of the hydrogen atom. (Throughout the rest of the manuscript we omit the term quasiparticle for clarity of presentation.) The difference between the bandgap Egap and the exciton resonance energies defines the respective exciton binding energies. In particular, the energy Δ12 between the exciton ground state (n=1) and the first excited state (n=2) scales with the ground state exciton binding energy EB. Along with the experimentally determined transition energy E1 of the exciton ground state, we can determine the electronic bandgap via Egap=E1+EB.An optical micrograph of a typical sample, 1L WSR/R=(Rsample−Rsubstrate)/Rsubstrate, of the bare 2L graphene, 1L WS2 and the resulting heterostructure at T=70 K are presented in R/R is predominantly determined by the imaginary part of the dielectric function, which is proportional to the optical absorption452 is dominated by the creation of so-called A excitons at the fundamental optical transition in the material, at the K and K′ points of the hexagonal Brillouin zone. In particular, the ground state (n=1) excitonic resonance occurs at 2.089 eV. The first excited state n=2 appears as a smaller spectral feature at 2.245 eV, with an energy separation between the two states of Δ12=156 meV. In addition, the first derivatives of ΔR/R are presented in n=1 state is scaled by factor 0.03 for better comparison. Here, the energies of the peaks correspond to the points of inflection of the asymmetric derivative features, as indicated by dashed lines for the n=2 states. Finally, the shoulder on the low-energy side of the n=1 peak at 2.045 eV arises from charged excitons, indicating slight residual doping in the WS2 material472 response matches our previous observations on uncapped samples supported on fused silica24Typical linear reflectance contrast spectra, Δ2 capped with graphene, the overall reflectance contrast is offset by the graphene reflectance, similar to findings in TMDC/TMDC heterostructures2 exciton resonances to lower energies, where the n=1 transition and the n=2 states are now located at 2.060 eV and 2.167 eV, respectively . In all cases, a decrease in Δ12 separation was observed with increasing dielectric screening of the environment . The bandgap of WSe2 can be thus tuned by more than 100 meV and the largest shift of almost 300 meV is observed for graphene-encapsulated WS2, the structure with the highest dielectric screening. For comparison, the influence of an arbitrary dielectric environment is presented in 2 encapsulated between two thick layers of varying dielectric constants. As we have shown, the change in the bandgap is roughly the same as the change in the binding energy and thus can be as high as 500 meV .In addition to the graphene-capped WS2 and WS2 covered by a bilayer of graphene. The corresponding path is illustrated schematically in 2 exciton n=1 and n=2 resonances, respectively. Each spectral trace corresponds to a different spatial position x on the sample; the bilayer graphene flake covers the WS2 monolayer between 5 and 12 μm on the x axis. Like the data shown in 2 excitons shift to lower energies in the presence of graphene. The peak energies are extracted from the points of inflection of the derivative, indicated by circles in 2 monolayer not being in close contact with graphene . The thickness of the layers was confirmed by optical contrast spectroscopy. The heterostructures were fabricated using well-established polymer-stamp transfer techniques described in refs 2 based samples and ref. 2 samples  calculationsThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.How to cite this article: Raja, A. et al. Coulomb engineering of the bandgap and excitons in two-dimensional materials. Nat. Commun.8, 15251 doi: 10.1038/ncomms15251 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table, Supplementary Notes and Supplementary References


The resolution of conventional terahertz (THz) imaging techniques is limited to about half wavelength, which is not fine enough for applications of biomedical sensing and nondestructive testing. To improve the resolution, a new superlens, constructed by a monolayer graphene sheet combining with a grating voltage gate, are proposed in this paper to achieve deep super-resolution imaging in the THz frequency range. The main idea is based on the Fabry-Perot resonance of graphene edge plasmon waves. By shaping the voltage gate into a radial pattern, magnified images of subwavelength targets can be obtained. With this approach, the finest resolution can achieve up to λ/150. Besides, the superlens can be conveniently tuned to work in a large frequency band ranging from 4.3 THz to 9 THz. The proposal could find potential applications in THz near-field imaging systems. In recent years, terahertz (THz) imaging applications56891319Graphene, emerged as the most promising 2D plasmonic material in the THz to mid-infrared frequency range2427nm is demonstrated, which indicates our proposal could be suitable for biomedical applications.In this paper, a new graphene-based superlens is proposed to break the diffraction limit in the THz frequency range. The superlens is composed by a monolayer graphene sheet and a metallic grating voltage gate. The working mechanism of the superlens relies on Fabry-Perot resonance of graphene edge plasmon waves. The working frequency can be conveniently tuned over a large range by changing the gate voltage. Furthermore, magnified images of subwavelength targets can be obtained if we shape the voltage gate into a radial pattern. A deep resolution of 400 2 substrate. The permittivity of the SiO2 is 3.9. On the back of the substrate, there is a metallic grating voltage gate. Different separations between the graphene and the metallic surface of the grating lead to non-uniform modification of the carrier density, which eventually generates the periodical conductivity along the x direction. This is now a common method used for tuning the conductivity of grapheneσg = σg,r + iσg,i, depends on angular frequency ω, electron relaxation time τ representing the loss mechanism, temperature T, and chemical potential μc. The imaginary part of the conductivity determines the propagation of SPPs waves, the real part of the conductivity represents the losses. When σg,i > 0, a single free-standing layer of graphene effectively behaves as a very thin “noble metal” layer with negative permittivity. Hence, the graphene sheet can surppot a transverse-magnetic (TM) polairized SPPs surface wave. The dispersion of the two dimensional graphene surface plasmon (2DGSP) supported on the sheet can be expressed as32The schematic of the near-field superlens is illustrated in εr1 and εr2 are the relative permittivity of the dielectrics above (here it is the air) and under the graphene sheet, ε0 is the permittivity in vacuum, e is the electron charge, ħ is the Plank’s constant. pq represents the in-plane wave-vector of the plasmon waves.where p, corresponding to the period of the grating. We assume the conductivities of the two adjacent regions (regions 1 and 2) are respectively σg1 and σg2. If the conditions that Im(σg1) > 0 and Im(σg2) < 0 or Im(σg2) ≪ Im(σg1) are fulfilled, Fresnel reflection will occur on the boundary of the two regions. Hence, the surface waves will be confined to a fictitious boundary3336σg1. The width of the ribbons is d, and the duty ratio is 0.5. Below, we will use this equivalent model to present the analysis. Although the microscopic structure of graphene edges modifies the electronic spectrum significantly, the excitation of the edge mode depends only on total charges and total currents. When the separation between the Femi energy and the Dirac point is much larger than the temperature or relaxation energy, the nature of what kind of edge does not affect the calculations reported hereFor the model in λep) and the 2D surface plasmons (λp) differ by a universal numerical factorλep = C0·λp, where C0 is the factor, λp = 2π/Re(qp). The value of C0 increases with the width of the ribbon, but we always have C0 < 1. The physical reason for the smaller wavelength of the edge plasmons compared to the 2D ones is the effective reduction of the Drude weight at the edges, where free carriers exist only on “one side”. The electric field of the edge plasmons decays exponentially away from the edge. For a given ribbon width, high order modes, which are actually the hybrids of 1D edge mode and 2DGSP mode, can be excited at large frequencies. But the 2DGSP mode disappears at low frequencies. Each of the high order modes has a cutoff frequency. Hence, there is always a frequency band for the single fundamental mode (i.e. the 1D edge mode) operation. The narrower the width of the ribbon is, the broader the frequency band will be. In the frequency range we concerned, the condition of single fundamental mode operation is always satisfied by cutting down the width of ribbons to nanometer scale .It has been well demonstrated that the wavelengths of the 1D edge plasmons . These parameters are typical for graphene plasmons observed in THz to infrared frequency range32λep is calculated by 2(DR-DL), DR and DL are respectively the right and the left positions of two adjacent peaks, as illustrated in the inset of d = 0.2 μm, 1 μm and 2 μm are presented in d decreases, the wavelength of the edge mode becomes shorter, which means that the 1D localized mode is more confined to the rims . As we can observe, in the lower frequency range, the gap of the plasmon wavelengths between the edge mode supported on the nanoribbon and the 2DGSP ·m′p) and propagate along each of the nanoribbons, n and m′ are both integers and refers to the n-order harmonic and the m′-th nanoribbon respectively counting from left to right. One of the electric field components on the m-th nanoribbon can be mathematically expressed asFor the graphene nanoribbon array shown in r and Δφ are respectively the reflection coefficient and the phase jump of the reflected surface wave at the boundary, Δφ = tan−1[Im(r)/Re(r)], E0 is related to the coupling strength. As can be observed, regardless of the angle of the oblique incidence (including the evanescent harmonics), the incident plane waves will always be coupled to the graphene edge mode with the same longitudinal wave-vector, i.e. qep. Hence, the phase accumulations of any incident waves are the same at the emergence interface of the graphene nanoribbon array. The scattering waves of subwavelength targets are an integration of all spatial harmonics with corresponding amplitudes and phases. In order to obtain an undistorted image of the targets, the amplitude and the phase at the emergence interface must keep synchronous with the amplitude and phase at the incoming interface. Under this requirement, we can deduce the following equation after considering the boundary condition at both the incoming and the emergence interfacesin which, lg is the length of the nanoribbons. where φ and qep into lg. An analytical method to determine Δφ has been reported in ref. φ is approximately constant during a broadband frequency range. For simplicity here, we can determine Δφ by numerical simulation. When the parameters of the nanoribbons are uc = 0.15 eV and d = 0.2 μm, we get Δφ ≈ 0.54π in the 4 THz-9 THz frequency range. When the working frequency is chosen to be f = 5 THz, we have qep = 46.12k0, in which k0 is the wave-vector in free space. The conductivity of the graphene nanoribbons at 5 THz is σg1 = 0.035 + i0.56 mS. For m = 2, we obtain lg = 0.95 μm.30x-z plane) of two targets are presented. Here, the targets are surface current sources with their directions parallel to z-axis. The x-direction width of the targets is 250 nm. The two targets are separated by 650 nm and located 60 nm away from the incoming interface. The 2D images are detected at the distance of 60 nm away from the emergence interface. As the above theory predicted, the two subwavelength targets are able to be perfectly distinguished in the near-field by the graphene superlens. The x-y plane electric field mapping above the graphene is shown in λ/92 imaging resolution is demonstrated. The near-field 2D images of the two incident targets which are separated 400 nm are illustrated in x-y plane electric field mapping is illustrated in λ/150. Theoretically speaking, this is the ultimate resolution of the designed graphene superlens because the resolution cannot be smaller than the period of the nanoribbon array. To improve the resolution, one can further narrow the width of the nanoribbons.Next, numerical simulation using Comsol is performed to demonstrate the super-resolution imaging of the superlens. In σg1) = 0.56 mS and Im(σg2) = 0.0012 mS. The two targets are separated by 800 nm. As shown in x-y plane electric field mapping in this case is almost the same as y-direction length of the graphene sheet (or nanoribbons) is lg = 1.15 μm, the images of the two targets are disturbed. Hence, we cannot simply regard each of the nanoribbons as a field probe. When lg = 1.53 μm, as shown in the inset of rd order (m = 3) Fabry-Perot resonance is established. The normalized 1D images corresponding to the 2nd, the 3rd and the 4th order Fabry-Perot resonance, plotted along a x-direction line which is 60 nm away from the emergence interface, are also shown in lg = 0.95 μm, 1.53 μm, 2.12 μm, respectively corresponding to m = 2, 3, 4 in Even though all of the above simulations are done on the graphene nanoribbons array, the same results will be obtained if we replace the graphene nanoribbons with a monolayer graphene sheet tuned by a grating voltage gate. At 5 THz, we have Im can be −0.5 mS ~ 0.01 mS. Therefore, the performance of the superlens is not sensitive to σg2. We only need to modulate the value of σg1 for frequency tuning. This is totally different from those hyperbolic-dispersion-based super-resolution imaging mechanisms which need to accurately control σg1 and σg2 simultaneously29Apart from the deep subwavelength resolution, another advantage of our designed graphene superlens is that it can be easily tuned to work over a broadband frequency range. As we have pointed out, what dominates the edge modes propagation on the graphene sheet is qep at different working frequency when lg is fixed. To achieve this, uc must follow a linear relation with the square of f, i.e. uc = α·f2, in which α is a positive constant, known from uc must increase from 0.111 eV to 0.486 eV accordingly, as shown in i0.481 mS to 0.0357 + i1.01 mS. The upper limit of the frequency range is a conservative estimation. If high quality graphene with large carrier density (could be achieved by highly doping) is available, a higher working frequency can be expected.As mentioned above, the principle of realizing super-resolution imaging is to set up the Fabry-Perot resonance. To ensure this, we only need to adjust the gate voltage to get a identical L = [Im(qp)]−1. As shown in μm to 11.66 μm if we tune the frequency from 4.3 THz up to 9 THz. The square dots are the simulated results of the 200 nm nanoribbon. The theoretical and simulated results are in good agreement. The losses decrease when the frequency increases. The minimum propagation length is much larger than the length of the nanoribbons. In nm. The images are detected along a x-direction line which is 60 nm away from the emergence interface. From 4.3 THz to 9 THz, the two peaks corresponding to the two targets can always be distinguished. The image curves at the low (4.4 THz) and the high (8.8 THz) spectral window are also presented in the inset of The lower limit of the frequency range is mainly subjected to the losses. The losses can be characterized by the propagation length of the edge mode, which should refer to the imaginary part of the wave-vector. In the working frequency range, the propagation length can be roughly calculated by r1 and r2 are respectively the inner and the outer radius of the ribbons, θ is the angle of the ribbon. The basic principle is the same as above. But in this case, the plasmon wavelength of edge mode is no longer constant at a given frequency because the width of each ribbon is gradually broadening. Hence, the Fabry-Perot resonance condition now becomesIn practical applications, we always hope magnified images could be obtained so that two objects could be distinguished by conventional imaging system. Hyperlenses which allow magnification of deep subwavelength scale objects have been proposed by using anisotropic materials with hyperbolic dispersionslg = r2 - r1. When the working frequency is 8 THz, the conductivities of the graphene ribbons become σg1 = 0.0357 + i0.898 mS and σg2 = 2.38×10−4 + i5.98×10−3 mS. The magnification factor of image can be calculated by γ = r2/r1. If r1 is fixed, increasing the value of r2 would enlarge the magnification factor. Nevertheless, the value of r2 can only be discrete because the Fabry-Perot resonance condition must be fulfilled. To prove it, we choose r1 = 0.8 μm, θ = 8°. The duty ratio of the gate is 0.5. Two surface sources separated by 800 nm are located 60 nm away from the incoming interface. The simulated electric field mapping at the z = 10 nm plane for the cases of r2 = 3.2 μm and 4.88 μm are respectively shown in z = 200 nm plane is also presented. Because of the evanescent decay of the fields in the z direction, the strongly confined edge modes become 2D “bulk” modes. As shown in in which, The lossless simulations have perfectly demonstrated the mechanism of realizing magnified super-resolution imaging. But in reality, the compromise should be made between the loss and the magnification factor. As we can see in μm). Although in our demonstrations, the length (corresponding to the magnification factor) is not sufficient for compressing the evanescent spatial spectrum of the deep subwavelength targets into propagating spectrum, far-field super resolution imaging could also be predicted if we lower the designed maximum resolution  or mitigate the loss influence. The method of realizing frequency tuning for this superlens is the same as above, therefore, we will not cover it again.Realizing far-field super-resolution imaging requires a relative long graphene ribbon . Even though the demonstrated propagation lengths of the edge plasmon modes are limited, the lengths could be further increased by optimzing the parameters of the graphene and the frequency to reduce loss. We believe it is possible for using the graphene superlens in practical applications. nm. Furthermore, the superlenses can be tuned to work over 4.3 THz ~ 9 THz frequency range. Owing to the strong confinement of the graphene plasmon edge mode, the images are well resolved in both the x and the z directions. It indicates that our proposal may be suitable for 2D super-resolution imaging. The mechanism could be extended to the infrared frequency range. The proposal could find potential applications in THz near-field imaging systems.To conclude, we have introduced a new way to realize deep super-resolution imaging in the THz frequency range by using a monolayer graphene sheet tuned by a grating voltage gate. Magnified images of subwavelength targets also can be obtained by shaping the voltage gate. The finest resolution of the superlenses can reach 400j∥ = σgE∥, where the symbol “∥” means the component is parallel to the surface of the graphene. According to the Kubo formula, the two specific intraband and interband electron transitions contribute to the surface conductivity of a monolayer graphene, which can be calculated from42Numerical simulations are performed by the commercial software, Comsol Multiphysics, which is based on the Finite Element Method (FEM). The direct and iterative solvers are employed. The monolayer graphene sheet or ribbon is characterized by surface currents e is the electron charge, ħ and kB are the Plank’s constant and the Boltzman’s constant, respectively. The chemical potential depends on the carrier density ns which is controlled by the gate voltage. Notice that for relatively small frequencies(ħω/μc ≪ 1), the intraband electron transition dominates the conductivity, hence, In the condition of μc ≫ kBT, a simple semiclassical model for conductivity, which possesses a Drude-like expression, is obtainedin which, The imaging targets in the simulating models are two rectangular surfaces. The width and the length of the two surfaces are 250 nm and 500 nm, respectively. The excitation sources are surface currents added on both of the two rectangular surfaces. The direction of the currents is perpendicular to the graphene. The minimum size of the grid is smaller than 1/6 of the plasmon wavelength.How to cite this article: Tang, H.-H. et al. Tunable Terahertz Deep Subwavelength Imaging Based on a Graphene Monolayer. Sci. Rep.7, 46283; doi: 10.1038/srep46283 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo. F, GF, and A) as well as nearly 600,000 L1 copies rendered immobile by 5′ truncation and the accumulation of internal mutations (Long interspersed element 1 (LINE-1 or L1) is a mobile genetic element active in nearly all mammals . L1 sequutations . The ongutations , and numutations .F, GF, and A subfamily L1 elements, B1 and B2 SINEs, and IAP and ETn long terminal repeat (LTR) elements (mRC-seq) . Using data from mRC-seq reads and PCR validation followed by capillary sequencing, we discerned the complete structures of 17 polymorphic insertions , comprising 11 L1 TF subfamily insertions, two B1 SINEs, and four B2 SINEs. Despite efficient detection of reference IAP and ETn retrotransposons , we identified no polymorphic LTR insertions in our mice. This result may reflect differences in the activity levels of retrotransposon families among inbred mouse strains, revealed by numerous recent studies . Using PCR and capillary sequencing, we validated 11 de novo L1 insertions . All 11 were TF subfamily elements, consistent with previous reports of disease-causing L1 insertions in mice wherein all insertions for which sufficient L1 sequence was present for subfamily distinction were identified as TF elements . Insertion #5 also occurred within an intronic L1mA5 element, and two additional intergenic insertions landed within existing L1 repeats .We regarded insertions detected in one or more offspring but absent from the corresponding parents as potentially de novo , reflecting depletion of mouse L1 3′ termini observed during Illumina sequencing , possibly due to the GC-rich nature of the mouse L1 3′ end sequence , and a previous analysis suggested that TF L1s undergo 5′ truncation less frequently than other L1 elements . Insertions #1 and #2 were identified by mRC-seq in mice SRAB2 and SRAB15, respectively . We did not detect these insertions by PCR genotyping in the somatic tissues of parental mice SRA and SRB; however, insertions #1 and #2 were detected by PCR in both testicles of paternal mouse SRB . Inheritance of each insertion by only 1/20 offspring, coupled with their presence in both testicles of the paternal mouse, suggested germline-restricted mosaicism for insertions #1 and #2 in mouse SRB .Next, we used PCR genotyping to investigate the developmental origin of each de novo L1 insertion . An insertion-specific genomic DNA qPCR assay targeting the 5′ L1-genome junction of insertion #7 revealed its prevalence of ∼11% and ∼4% in the germ cell fraction of the left and right testicle of SRCD14, respectively . As evidenced by our failure to detect L1 insertions #1, #2, and #7 from mesoderm-, ectoderm-, and endoderm-derived tissues of the respective paternal mice, we reasoned that these insertions likely arose in early primordial germ cells (PGCs) during paternal embryonic development, prior to PGC colonization of the genital ridge and the formation of the testes. Thus, we conclude that L1 retrotransposition can occur in early PGCs, resulting in germline-restricted genetic mosaicism and heritable de novo L1 insertions.In an inverse approach to identify germline-restricted mosaic insertions, we performed deep (∼260×) mRC-seq on the germ cell fraction of each testicle of mouse SRCD14. We detected and PCR-validated insertion #7 in both testicles and did not detect it in the somatic tissues of SRCD14 . PCR genotyping revealed bands of varying intensity among the somatic tissues and ovaries of mouse SRE , #6 , and #11  were each identified by mRC-seq in multiple offspring and genotyped as mosaic in the respective maternal tissues. Insertions #8  and #10  were identified by mRC-seq and were confirmed as mosaic in the tissues of mouse SREF17 and mouse F2-145, respectively, but were not transmitted to the limited offspring produced by these animals  . Therefore, we can neither confirm nor rule out the contribution of insertions #8 and #10 to the germ lineage. Taken together and consistent with previous studies . Upon crossing mouse SREF16 to mouse SRCD14, insertion #4 was transmitted to 11/19 offspring . We therefore conclude that mouse SREF16 was heterozygous for insertion #4 and that this insertion arose sufficiently late during germline development of parental mouse SRE or SRF to preclude its detection in bulk gonad tissues, or possibly post-conception in SREF16 at the mature zygote stage.Of the remaining two de novo L1 insertions, insertion #4 likely represented a late germline event. This insertion was identified by mRC-seq in mouse SREF16, one of seven offspring of parental mice SRE and SRF, but was not detected in somatic and germ tissues of SRE or SRF . This rate of transmission suggested either parental heterozygosity or mosaicism with a high degree of germline prevalence. However, we could not detect insertion #9 by nested PCR in the gonads of SRCD11 or SREF20 . The developmental origins of insertion #9 are therefore unclear. We speculate that this event was germline-restricted mosaic in SRCD11 or SREF20, but that the subset of germ cells carrying insertion #9 was depleted by the time the gonads of these animals were harvested, at age 39 and 40 wk , one early embryonic insertion (#5) , and two presumably recent polymorphic L1 TF insertions differentially present/absent among our animals (Poly_L1TF_3 and Poly_ L1TF_4)  carried 3′ transductions, implicating five distinct full-length L1 TF elements present in the C57BL/6J reference genome. Thus, we have identified progenitor elements responsible for de novo heritable L1 insertions.We next sought to identify the progenitor L1 elements responsible for heritable de novo insertions. During transcription of an RC-L1 element, the native L1 polyadenylation signal is occasionally bypassed in favor of a downstream genomic polyadenylation signal, and upon retrotransposition the nascent L1 insertion incorporates a genomic sequence tag, or 3′ transduction, that identifies the progenitor L1 element . Two earSupplemental Fig. S9A; Supplemental Fig. S9B). Given our mouse data indicating each heritable early embryonic L1 insertion was detectable in tissues derived from all three germ layers , we suggest that insertion JH-27 was very likely maternal germline-restricted.Having observed transmission of germline-restricted and somatic/germline mosaic insertions in mouse, consistent with previous studies of human patients , we next2 test with one degree of freedom, two-tailed P-values of 0.49 and 0.67, respectively). Thus, as expected, within a single generation we observe no evidence for positive or negative selection on a de novo L1 insertion. However, future studies tracking the transmission of de novo L1 insertions through many generations may reveal evidence for selection on particular insertions, perhaps dependent on their genomic locations and functional consequences.We uncovered 11 de novo L1 insertions among 85 mouse genomes, providing an estimate of one new insertion per eight mice (11/85 = 0.13 or ∼1/8). This figure is consistent with but more conservative than previous estimations that a new L1 insertion may arise in every two to three mice  and is mWe established for the first time that heritable endogenous L1 retrotransposition events arise in early PGCs, before the PIWI/piRNA retrotransposon defense pathway becomes active in male embryonic gonads , resultiSupplemental Tables 1, 5), suggesting that additional mosaic insertions may have fallen below the detection threshold of mRC-seq. Thus, it is possible that early embryonic retrotransposition frequently generates low-level somatic-restricted mosaicism . Thus, early embryonic insertions present at low prevalence in somatic and germ tissues of a parental mouse nevertheless have the potential to contribute substantially to genetic diversity in the next generation, presumably by their fortuitous presence in a subset of germ cells that ultimately give rise to offspring.Consistent with previous studies , we tracosaicism , as wellosaicism C but wasIn contrast, insertions arising during later germline development  may contribute a broad spectrum of genetic diversity to the gamete pool, yet each insertion individually would have a small likelihood of transmission C. IndeedDe novo heritable L1 retrotransposition is a component of the ongoing evolutionary interplay between retroelements and mammalian genomes, the importance of which is exemplified by recent studies demonstrating the exaptation of LTR retrotransposon sequences and protein products for pluripotency maintenance and embryonic development . FurtherAll animal breeding and handling procedures were carried out in compliance with the guidelines set forth by the University of Queensland Animal Ethics Committee. To establish breeding pedigrees, adult wild-type C57BL/6J mice were ordered from the University of Queensland Biological Resources Facility (UQ-BRF), which in turn sources animals from the Animal Resources Center . The UQ-BRF and ARC provided helpful information regarding the source and breeding history of the animals used in this study. The “P-generation” mice used to initiate the breeding pedigrees in this study were no more than 10 generations removed from the Jackson Laboratory C57BL6/J strain.Supplemental Methods.Genomic DNA from animals and tissues of interest was used to construct Illumina libraries for mRC-seq as described in F GF and A, SINEs B1 and B2, and the LTR elements IAP and ETn . Illumina libraries were pooled to achieve a total mass of 1 µg . A detailed description of mRC-seq hybridization can be found in Supplemental Methods.Hybridization reactions were performed as described in Supplemental Fig. S1B; Supplemental Table 1). We performed mRC-seq on the individual somatic tissues and gonads of mice SRA and SRE and the germ cell fraction of the left and right testicles of SRCD14. We also performed 30× whole-genome sequencing on P-generation animals SRE and SRF, and 22× WGS on their F1 offspring SREF15-SREF21, using pooled somatic tissue genomic DNA . Across all DNA sequencing libraries, we detected 92.8%, 97.8%, and 98.7% of recent reference genome L1 , LTR retrotransposon , and SINE  insertions, respectively .We performed mRC-seq on either pooled genomic DNA from somatic tissues representative of the three embryonic germ layers  or DNA extracted from tail tips and ear punches , generating 2×150-bp read pairs and 2×250-bp read pairs. Some libraries were also sequenced on an Illumina MiSeq platform, generating 2×250-bp and 2×300-bp read pairs. WGS libraries were sequenced on an Illumina HiSeq 2500 platform (Macrogen), generating 2×250-bp read pairs. mRC-seq and WGS data were processed as follows: read pairs were first trimmed from their 5′ and 3′ ends to remove any bases with quality <10, then assembled into contigs using FLASH  (A summary of library pooling and sequencing reads per library is included in ng FLASH  and defang FLASH  (parametETn LTR) , using LETn LTR)  (parametETn LTR) . ClusterETn LTR)  or in alhttp://serialbasics.free.fr/Serial_Cloner.html) and the BLAT tool on the UCSC Genome Browser . Oligonucleotide primers were ordered from Integrated DNA Technologies (IDT).Putative insertions were called as potentially de novo if they appeared in one or more offspring from the same pedigree and were absent from the parental mice. In addition, some insertions were readily detected in offspring and were also detected with 1–2 reads upon deep sequencing of individual tissues of maternal mice SRA and SRE. Such insertions were treated as potentially de novo and chimeric in the maternal mouse. Reads were then manually inspected using SerialCloner . The full details of PCR validation can be found in Supplemental Methods.Empty-filled validation PCRs were carried out using primers specific to the 5′ and 3′ genomic sequence flanking putative insertions. Validation PCRs for 5′ and 3′ junctions were carried out using the appropriate flanking genomic primer paired with a primer internal to the L1 sequence; where necessary, hemi-nested and fully nested PCR reactions were carried out using appropriately designed genomic and L1-specific primers  at a ratio of 3 µL to 1 µg plasmid DNA. G418 selection (400 µg/mL) was initiated at 72 h post-transfection and carried out for 10–12 d  at the Translational Research Institute Flow Cytometry Core. The percentage of EGFP positive cells for each L1 reporter construct was used to normalize the G418-resistant colony counts obtained in the retrotransposition assay . Full deCt values were calculated on the LightCycler software using absolute quantification 2nd derivative max. Details of mosaicism analysis qPCRs can be found in Supplemental Methods.Quantitative PCR using genomic DNA as template was carried out using primers and dual-labeled PrimeTime qPCR probes (5′ 6-FAM-ZEN-3′ Iowa Black FQ) from IDT. Control reactions for DNA input were performed using a predesigned PrimeTime qPCR assay for the single-copy mouse gene RPP25 (Mm.PT.58.21641426.g), with a dual-labeled probe (5′ 6-FAM™-ZEN-3′ Iowa Black FQ). Reactions were run on a Roche LightCycler 480 II with the following cycling conditions: 95°C, 5 min, followed by 45 cycles of 95°C for 10 sec, and 57°C for 1 min, then melt curve (0.11°C per sec from 57°C to 95°C). http://www.ebi.ac.uk/ena/) under project accession number PRJEB10299. Sanger trace files from this study have been submitted to the NCBI Trace Archive (http://www.ncbi.nlm.nih.gov/Traces/home/index.cgi) with TI numbers TI2344112704–TI234412736 and TI234412752.mRC-seq and WGS data from this study have been submitted to the European Nucleotide Archive (ENA; J.A.J. is employed by Roche Sequencing Solutions, Inc., and Roche Sequencing Solutions reagents were used in the study.


Ultra-high molecular weight polyethylene (UHMWPE) is the most common bearing material in total joint arthroplasty due to its unique combination of superior mechanical properties and wear resistance over other polymers. A great deal of research in recent decades has focused on further improving its performances, in order to provide durable implants in young and active patients. From “historical”, gamma-air sterilized polyethylenes, to the so-called first and second generation of highly crosslinked materials, a variety of different formulations have progressively appeared in the market. This paper reviews the structure–properties relationship of these materials, with a particular emphasis on the in vitro and in vivo wear performances, through an analysis of the existing literature. Ultra-high molecular weight polyethylene (UHMWPE) has been used as a bearing material in total joint arthroplasty for more than 50 years now. The idea to replace degraded cartilage with a polymer liner dates back to the late 1950s. At that time, Sir John Charnley chose polytetrafluoroethylene (PTFE), a low friction polymer, as the bearing material to replace the natural acetabulum, articulating against a metallic femoral head, for hip replacement. Due to the unacceptably low wear resistance of PTFE though, the first “low friction arthroplasties” dramatically failed after few years of implantation. In 1962, UHMWPE, a similarly low-friction, but much more wear resistant polymer, replaced PTFE in Charnley’s hip arthroplasty, with remarkably better performances. From then on, arthroplasty has known considerable evolution, but UHMWPE remains the gold standard for artificial hips and now other artificial joints, including the knee and shoulder ..42].Similarly, acetabular cups machined from a polyethylene gamma-irradiated in air at doses ranging from 33 to 1000 kGy, remelted and EtO sterilized, demonstrated an 87% wear reduction with increase in the radiation dose from 33 to 95 kGy, when tested for 5 million cycles in a hip simulator; again, the wear decreased to undetectable levels at a radiation dose higher than 200 kGy . In the Kurtz et al.  summarizThe influence of the type of counterface on the wear behavior of crosslinked polyethylene was also investigated. Bistolfi and coworkers  showed tA previous study  also demRegarding the oxidation stability of the two formulations of 1st generation highly crosslinked polyethylenes, McKellop and coworkers compared the oxidation stability and wear resistance of the same set of samples mentioned above , with orMuratoglu and coworkers  tested tIn another study , the samCollier et al. in an extensive study that compared the physical and mechanical properties of commercial, 1st generation crosslinked polyethylenes from six orthopaedic manufacturers , also coMost of the aforementioned studies attributed the differences in the oxidative stability between remelted and annealed crosslinked polyethylenes to that a thermal treatment above the melting temperature (remelting), leads to a complete melting of the polymer crystallites, hereby allowing termination of the radicals trapped in the crystalline phase and thus brings the amount of residual radicals to undetectable levels ,73. ConvSeveral clinical studies have reported superior in vivo wear performance of highly crosslinked PE, compared to conventional polyethylene in hip replacements ,85,86,87A number of retrieval studies have also reported on the chemical, physical and mechanical properties of these highly crosslinked PE following in vivo aging. According to predictions of in vitro studies, oxidation of the highly crosslinked/annealed formulation was reported by a number of authors ,93,94,95On the other hand, failures of some specific designs of highly crosslinked/remelted have also been described in the literature ,98. Rim As already mentioned, crosslinked/remelted polyethylenes did not contain free radicals at the time of implantation and were expected to be stable to oxidation. Unexpectedly, some recent literature studies reported that measurable levels of oxidation were observed in some retrievals ,103,104.Although the clinical significance of such adverse behaviors of the 1st generation highly crosslinked PE is still under debate, their occurrence stimulated the research of alternative polyethylene formulations to overcome the observed drawbacks.The rationale at the basis of a new generation of highly crosslinked polyethylene was to maintain the superior wear resistance demonstrated by the 1st generation crosslinked PE, while also retaining the mechanical properties and fatigue resistance of the uncrosslinked material and ensuring stable properties, thus oxidative stability, over time.As discussed in the previous paragraph, annealing below the melting temperature preserves most of the original UHMWPE microstructure. Highly crosslinked/annealed PE had been demonstrated to retain mechanical properties and fatigue resistance similar to conventional, gamma-nitrogen sterilized (but unoxidized) material; however, the presence of free radicals in the latter leaves the polyethylene exposed to oxidation . DumbletThis 2nd generation crosslinked PE  was terminally sterilized with gas plasma, so as to avoid the introduction of more free radicals with sterilization. Literature studies reported similar or only slightly reduced mechanical and fatigue properties compared to conventional PE ,107,108 An alternative approach to the 2nd generation highly crosslinked polyethylene involves the addition of an anti-oxidant stabilizer, in order to efficiently inhibit the oxidative degradation, without the need for a thermal treatment of the irradiated polyethylene, so as to preserve the original morphology, mechanical properties and fatigue resistance . AlthougTwo methods are currently used to incorporate vitamin E into UHMWPE ,115. OneSeveral studies have investigated the mechanical properties of both formulations of vitamin E stabilized PE. No significant differences in the static mechanical properties, nor in the fatigue crack propagation resistance have been observed when comparing virgin UHMWPE with blends containing up to 5000 ppm of vitamin E ,135. ConIn addition, Vitamin E-stabilized PE has demonstrated superior oxidation stability under a variety of accelerated aging conditions. For example, blends of UHMWPE with 500, 1000 and 5000 ppm of vitamin E, gamma irradiated to doses up to 100 kGy were found to be more oxidatively stable than virgin, unirradiated UHMWPE, following accelerated aging at 90 °C in air . Oral etThe wear resistance of this 2nd generation highly crosslinked formulation has been extensively tested in several in vitro experiments, under a variety of challenging conditions, and has been compared to conventional and 1st generation highly crosslinked formulations before and after both accelerated and shelf-aging. In a POD wear test, highly crosslinked Vitamin E-doped PE exhibited comparable wear rates to 1st generation highly crosslinked PE irradiated to the same dose , while iBellare et al.  comparedSimilarly, while comparing the wear behavior of standard, unirradiated PE to that of two formulations of crosslinked polyethylene, with and without vitamin E (1000 ppm), both irradiated to 70 kGy, Affatato et al.  found thThese observations suggest that, if vitamin E is necessary to avoid oxidation, then the radiation dose must be optimized in order to obtain enough crosslinking density to preserve the wear resistance of unstabilized, crosslinked PE.Given the concerns mentioned above on the use of 1st generation highly crosslinked PE in knee arthroplasty, the vitamin E crosslinked formulations represented an attractive alternative, in particular in this application. For this reason, many experimental studies investigated the performance of vitamin E stabilized PE for use in the knee with promising results. For example, while comparing the wear rate of two designs (cruciate-retaining and posterior-stabilized) of highly crosslinked UHMWPE doped with vitamin E to that of γ-inert–sterilized direct compression-molded UHMWPE, Haider and coworkers  found thIn another knee simulator experiment, UHMWPE blended with 1000 ppm of vitamin E and radiation sterilized with 30 ± 2 kGy, was artificially aged according to ASTM F2003-2 and tested for 5 million cycle without showing structural failures, as conventional PE did under similar conditions . SimilarTeramura and coworkers  evaluateBladen et al.  demonstrTo investigate the effects of particulate debris in vivo, Bichara et al.  comparedIn addition, it has been shown that vitamin E may also exert some antibacterial activity ,147,148.Literature reports on the clinical outcome of vitamin E stabilized PE are still quite limited in number, but their results are overall promising. One recent study  comparinProspective studies at two to three years follow-up demonstrated no significant differences in femoral head penetration rates between vitamin E doped and virgin highly crosslinked liners ,151,152.However, one study reporting head penetration into vitamin E doped highly crosslinked liners at five years demonstrated less wear compared to that previously reported for 1st generation highly crosslinked PE, at the same interval of five years. Further, the study demonstrated that, after settling of the liners in the early period, no significant head penetration occurred from two- to five-year follow-up and the authors remarked that, overall, the wear observed in the study was well below that at which osteolysis becomes a serious concern .Rowell et al.  analyzedSimilarly, Currier et al.  analyzedUHMWPE remains the most commonly used bearing material in total joint arthroplasty due to its long and relatively successful history. However, UHMWPE is a complex material and its mechanical and tribological behavior is strongly dependent on the morphology and chemical alterations induced by processing conditions, such as sterilization using ionizing radiation or crosslinking. The need for increasing the life expectancy of the implants, in order to meet the demand in younger and active patients, has led to a considerable evolution in the manufacturing of the UHMWPE orthopedic devices over the last few decades. From gamma-air sterilized “historical” polyethylenes, to the first and second generation of highly crosslinked materials, the wear resistance, mechanical properties and overall performances of the UHMWPE biomaterials have greatly improved, as has been extensively reported in the literature. Advances in the sterilization techniques have enabled a substantial reduction of the oxidative degradation experienced by “historical” implants, greatly lowering the incidence of wear-induced osteolysis and early mechanical failures. First generation crosslinked PE has further reduced wear, but at the expenses of fatigue resistance and/or oxidative stability, while 2nd generation crosslinked PE has been introduced to overcome the latter drawback and, overall, it has shown promising, yet early results, despite some issues regarding the oxidative stability of the sequentially irradiated and annealed PE.Nevertheless, the clinical outcome of some of the newest formulations is still largely unexplored. It is therefore of paramount importance to ensure a close, continuous monitoring of the clinical performances of the contemporary UHMWPE biomaterials.


Although chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) seem to be opposite entities from a clinical perspective, common initial pathogenic steps have been suggested in both lung diseases. Emphysema is caused by an elastase/anti-elastase imbalance leading to accelerated elastin degradation. Elastinolysis is however, also accelerated in the IPF patients’ lungs. The amino acids desmosine and isodesmosine (DES) are unique to elastin. During the degradation process, elastases liberate DES from elastin fibers. Blood DES levels consequently reflect the rate of systemic elastinolysis and are increased in COPD. This is the first report describing elevated DES levels in IPF patients. We also demonstrated that the age-related increment of DES concentrations is enhanced in IPF. Our current study suggests that elastinolysis is a shared pathogenic step in both COPD and IPF. Further investigation is required to establish the relevance of accelerated elastin degradation in IPF and to determine whether decelerating this process leads to slower progression of lung fibrosis and better survival for patients with IPF. Although chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) seem to be opposite entities from a clinical perspective, they often coexist in the same patient , and shaet al. recently demonstrated that elastin degradation accelerates during ageing in subjects with or without obstructive lung disease, but that this age-related DES increment is amplified in patients with COPD [The elastin precursor tropoelastin is mainly produced perinatally . Tropoelith COPD . WhetherBy analogy to COPD, we hypothesized that pDES levels would be elevated and that the age-related pDES increment would be enhanced in IPF patients compared to controls with no lung disease.pDES levels were measured by liquid chromatography tandem-mass spectrometry in 154 IPF patients from Maastricht , from Nieuwegein  and from Ann Arbor  as previously described . For eacp<0.0005) compared to controls (337±32 ng/L). No significant differences in pDES levels were found between IPF and COPD patients (p=0.2). FVC data at baseline were available from the Nieuwegein and Ann Arbor cohort; no significant correlation was found between pDES levels and %FVC of predicted at baseline (p=0.3). FVC follow-up data were available from the Nieuwegein cohort; a significant correlation was demonstrated between pDES levels and the change in %FVC of predicted per year (p=0.02).A significant correlation was found between age and pDES (p<0.0005). The equation for the pDES regression line in IPF patients was 76 (95% CI -117–270)+6.76 (95% CI 3.75–9.77)*age (ng/L). Significantly higher pDES levels were found in IPF patients 507±8 ng/L) compared to age-adjusted reference values of non-smoking controls , of smoking controls  and of COPD patients  . Based pPrevious research has mainly focussed on pDES as a biomarker of systemic elastin degradation in COPD . Our prepost hoc study in which AAT augmentation therapy resulted in a decrease of pDES levels in AAT deficient COPD patients [We regard deceleration of elastin degradation as a potential attractive novel therapeutic target in COPD. This intriguing new concept was illustrated by a recently published patients . In IPF,patients . Althougpatients . Future In summary, this is the first study describing elevated pDES levels in IPF patients, which were even higher than in COPD patients. We also demonstrated that the age-related increment of pDES concentrations is enhanced in IPF. Additional studies are needed to establish the relevance of accelerated elastin degradation in IPF and to determine whether decelerating this process translates into slower progression of lung fibrosis and better survival for patients with IPF.


Cryptococcus neoformans, nearly all genes are interrupted by small introns. In recent years, genome annotation and genetic analysis have illuminated the major roles these introns play in the biology of this pathogenic yeast. Introns are necessary for gene expression and alternative splicing can regulate gene expression in response to environmental cues. In addition, recent studies have revealed that C. neoformans introns help to prevent transposon dissemination and protect genome integrity. These characteristics of cryptococcal introns are probably not unique to Cryptococcus, and this yeast likely can be considered as a model for intron-related studies in fungi.In  Cryptococcus is now recognised as a major fungal human pathogen responsible for thousands of deaths every year around the world , Cryptococcus gattii exists in several sub-lineages, some of which can infect immunocompetent patients (Cryptococcus lives in the environment (mainly in soil and decaying wood), its biology has been shaped through its interactions with environmental microbiota, as well as seasonal modifications in the physical and chemical soil parameters  and identified two small introns of 52 and 49 nucleotides  was originally reported to be intronless  , becoming a template for dsRNA synthesis and siRNA production  formation in the wake of the RNA polymerase II, thus avoiding the associated accumulation of DNA damage . Bonnet an cells . They fotability . AltogetSummary and future directions - In summary, these studies have revealed introns as key features in the biology of Cryptococcus and, in the maintenance of their genome integrity. They also suggest that their structure have been finely selected throughout evolution. Nevertheless, a number of questions remain to be answered. (1) For instance, the mechanisms regulating intron gain and loss in fungi is still a very active domain of research. A better description of intron structure and position in the different lineages of C. gattii and C. neoformans  Additionally, the role of introns in gene regulation requires further analyses. The current model on Cryptococcus intron retention-dependent regulation of gene expression  Finally, introns have been identified in miscRNAs, but the function, splicing, and evolutionary conservation of this intron subclass have not been studied yet.oformans  will likoformans , a precipression  suggestsentified . In metaentified . In fung