Background: COVID-19 is a serious and potentially deadly disease. Early diagnosis of infected individuals will play an important role in stopping its further escalation. The present gold standard for sampling is the nasopharyngeal swab method. However, several recent papers suggested that saliva-based testing is a promising alternative that could simplify and accelerate COVID-19 diagnosis.Objectives: Our aim was to conduct a meta-analysis on the reliability and consistency of SARS-CoV-2 viral RNA detection in saliva specimens.Methods: We have reported our meta-analysis according to the Cochrane Handbook. We searched the Cochrane Library, Embase, Pubmed, Scopus, Web of Science and clinical trial registries for eligible studies published between 1 January and 25 April 2020. The number of positive tests and the total number of tests conducted were collected as raw data. The proportion of positive tests in the pooled data were calculated by score confidence-interval estimation with the Freeman–Tukey transformation. Heterogeneity was assessed using the I2 measure and the χ2-test.Results: The systematic search revealed 96 records after removal of duplicates. Twenty-six records were included for qualitative analysis and 5 records for quantitative synthesis. We found 91% (CI 80–99%) sensitivity for saliva tests and 98% (CI 89–100%) sensitivity for nasopharyngeal swab (NPS) tests in previously confirmed COVID-19 patients, with moderate heterogeneity among the studies. Additionally, we identified 18 registered, ongoing clinical trials of saliva-based tests for detection of the virus.Conclusion: Saliva tests offer a promising alternative to NPS for COVID-19 diagnosis. However, further diagnostic accuracy studies are needed to improve their specificity and sensitivity. COVID-19, caused by the SARS-CoV-2 virus, is a serious and potentially deadly disease. Globally, as of 5 May 2020, there have been 3,489,053 confirmed cases of COVID-19 reported to WHO, including 241,559 deaths , is the gold standard for detection of SARS-CoV-2 infection , thus exOne candidate for non-invasive specimen collection is saliva. The saliva secreted by salivary glands contains water, electrolytes, mucus, and digestive and protective proteins –9. But wSince early January 2020, several papers have been published on the possible use of saliva as a specimen for detecting SARS-CoV-2 in the diagnosis of COVID-19. Until now there has been no systematic review or meta-analysis of this topic. Our aim, therefore, was to conduct a meta-analysis, thus overcoming the limitations of the small sample sizes in individual studies, in order to estimate the diagnostic sensitivity of saliva-based detection of the virus. We also aimed to summarize the study protocols that have been registered in clinical trial registries to investigate saliva-based COVID-19 diagnosis in the future.https://osf.io/3ajy7).The reporting of our meta-analysis follows the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . The PRIStudies eligible according to our inclusion criteria did not present sufficient raw data to complete 2 × 2 contingency tables. True positive, true negative, false positive and false negative values were not generally available, thus sensitivity and specificity could not be separately calculated. Instead, positive event rates were pooled for statistical analysis. Details of the analysis are described in section Summary Measures and Synthesis of Results.We included records if they met the following eligibility criteria: (1) records published in scientific journals or clinical trial registries; (2) patients diagnosed with COVID-19; (3) index test: saliva specimens with PCR diagnostics for detecting SARS-CoV-2; (4) reference standard (comparator test): NPS specimens with PCR diagnostics for detecting SARS-CoV-2; (5) records written in English or available in English translation. Exclusion criteria: (1) publications with no primary results such as reviews, guidelines and recommendations; (2) publications dated before 1 January and after 25 April, 2020; (3) gray and black literature.Systematic searches for records published in English after 1 January 2020 were performed in five major literature databases  and also in five clinical trial registers . The last update of our systematic search was performed on 25 April 2020. Cited and citing papers of the relevant studies were screened for further eligible studies.The following key words were applied to each database to identify eligible records: (COVID 19 OR COVID19 OR Wuhan virus OR Wuhan coronavirus OR coronavirus OR 2019 nCoV OR 2019nCoV OR 2019-nCoV OR SARS CoV-2 OR SARS-CoV-2 OR NCP OR novel coronavirus pneumonia OR 2019 novel coronavirus OR new coronavirus) AND .We used EndNote X9.3.3 reference manger to organize records. After removal of duplicates, two authors (A.H. and I.M.) independently screened the records for eligibility based on the titles and abstracts. Papers included at this stage were further appraised by reading the full text. Any disagreement between reviewers was resolved by consulting a third reviewer (L.M.C.).Using a preconstructed, standardized data extraction form, two authors (A.H. and I.M.) independently collected data from the included records. From primary studies the following information was extracted : first aAre saliva specimens reliable for detecting SARS-CoV-2 in COVID-19 patients confirmed by nasopharyngeal swab testing?We evaluated the potential for bias, the quality of reporting and the applicability of the studies using the QUADAS-2 tool  , which iWe used the preconstructed form available on the QUADAS-2 web page of the University of Bristol .In the synthesis of quantitative data we included patient-based data from consecutive case series. Case reports from single participants were excluded.The sensitivities of the saliva and NPS tests were assessed in patients who had previously been confirmed to be infected, having had both a positive NPS test and well-defined clinical symptoms on admission to the hospital. Extracted data were limited to test results from subsequent occasions when both saliva and NPS samples were collected concurrently. Therefore, the sensitivity of the NPS test is based on the matching NPS tests when saliva tests were also performed.The sensitivity of the saliva test in the patient-based pooled data was calculated using the methods recommended by the working group of the Cochrane Collaboration. Because some of the sensitivity values are close to or equal to 1, the score confidence interval estimation  was applI2 measure and the χ2-test, where p < 0.1 is taken to indicate significant heterogeneity. I2 values of 25, 50, and 75% were identified as low, moderate and high estimates, respectively .–18.14–18In the qualitative synthesis we also included another consecutive case series . But in In the individual studies included in the quantitative synthesis, the sensitivity of the saliva test among COVID-19 infected patients ranged from 78%  to 100% Pooled event rates (positive and negative test results) from saliva specimens show that the sensitivity of the saliva test was 91% (CI 80–99%) among COVID-19 patients diagnosed in the recruitment period . By defiI2-test  indicating the contribution of confounding factors. On the other hand, we found a low level of heterogeneity among the NPS test results .We assessed our pooled results for inconsistency using the I2-test . In the Interestingly some of the data suggest that NPS tests may occasionally be negative when the corresponding saliva test gives a positive result. Azzi et al. reported that two patients showed positive saliva tests while their NPS tests were negative , and a c10 units higher in the NPS specimens than in the saliva specimens  for clinical trial protocols that are planned to evaluate saliva specimens for COVID-19 diagnosis. By using the same keywords as for the studies already completed, we found 18 registered clinical trials on planned or ongoing clinical studies. All of them appeared in the ClinicalTrials.gov registry . This new saliva-based diagnostic collection method, which RUCDR has developed in partnership with Spectrum Solutions and Accurate Diagnostic Labs (ADL), claims to allow an easier and therefore broader screening of the population compared with the current method using nose and throat swabs. Another accelerated EUA for the “Curative-Korva SARS-Cov-2 Assay,” which was specifically designed for use with oral fluid samples, was also approved to permit the testing of oral fluids, i.e., saliva (https://www.fda.gov/media/137088/download). Nasopharyngeal swabs, oropharyngeal swabs and nasal swabs can also be used with the Curative-Korva SARS-CoV-2 Assay, but their performance with this assay has not yet been assessed (https://www.fda.gov/media/137088/download). These two saliva-based, FDA-approved assays are now in use to test for COVID-19 infection, in spite of the fact that no independent, scientific analysis has yet established their effectiveness. Our present work is the first integrative meta-analysis study to review the existing multi-study evidence for validity of the saliva-based approach.In April 2020 the Food and Drug Administration (FDA) granted emergency use authorization (EUA) to Rutgers' RUCDR Infinite Biologics and its collaborators for a new specimen collection approach that utilizes saliva as the primary test biomaterial for the SARS-CoV-2 coronavirus, the first such approval granted by the federal agency  and 98% (CI 89–100%) for saliva and for NPS samples, respectively, based the pooled event rates among COVID-19 patients. Clearly the two confidence intervals overlap, suggesting that the outcomes of the saliva tests and NPS tests are not very different. There appears to be a slight tendency for NPS tests to be more sensitive but this is not statistically significant. On the other hand, one study reported the opposite tendency with the virus detectable in the saliva but not the NPS sample on a significant number of occasions . AlthougAt present only two studies have considered the specificity of the saliva tests. In one of those tests only one saliva sample was found to be positive among 50 apparently healthy individuals who were PCR-negative for the NPS test . In the For optimal saliva-based testing at least three conditions have to be improved by standardization and validation . (1) A sNot surprisingly the studies included in our analysis used different sampling methods to collect saliva. This may have had a significant effect on the sensitivity of the saliva test. Azzi et al. used a simple drooling technique to collect saliva and they resuspended the collected specimens in 2 ml of PBS . In contOther factors, such as the type of transport medium, the temperature during transportation, and the time passed between specimen collection and RNA extraction, may also affect the outcome of the tests . UnfortuIt is likely that a simple drooling technique, with no specific target volume and no extra stimulation of saliva secretion, will provide the greatest sensitivity if the viral RNA in whole saliva derives mainly from sources other than the secretions of the salivary glands. Drooling is a well-established saliva collection method that is generally recommended for analytical purposes . Due to In the present meta-analysis the overall sensitivity of the saliva (index) test is assessed by comparison with the NPS (reference standard) test using patient-based pooled data. This simple comparison does not allow us to address any of the more complex questions that arise from the widely varying presentation of different COVID-19 patients. For example, are there significant differences in the sensitivities of the two sampling methods according to the primary location of the infected cells? Are there higher viral loads in the saliva, and is there therefore a higher saliva test sensitivity, in COVID-19 patients who only present with a loss of taste sensation or who are asymptomatic? Are saliva tests more or less sensitive than NPS tests in patients whose infection is mainly localized to the respiratory tract? Correlation studies comparing saliva and NPS viral loads in patients categorized by the nature and severity of their symptoms should be very informative. Time series data on the relative viral loads in the saliva and NPS specimens may be useful in predicting the progression of the disease and in guiding treatment. But, as discussed above, these studies will require careful optimization and standardization, particularly of the saliva collection protocol.ClinicalTrials.gov registry (The need for reliable, non-invasive and easy-to-perform tests for COVID-19 has focused special attention on saliva in the last few months. Between 1 January and 25 April 2020, 18 clinical trials involving saliva specimens have started according to the registry . Among tA limitation of the present work is the relatively small number of studies and small sample sizes available regarding this topic. Despite the large number of records found in the systematic search of the literature, only 6 studies could be included. Although intensive research is in progress regarding COVID-19, there are still only a handful articles fulfilling our eligibility criteria. The limited amount of reported data makes it difficult to perform comprehensive analyses and to thoroughly investigate the causes behind certain trends in the results. Another issue that hinders in-depth analysis is the lack of methodological homogeneity, and the inadequate reporting of methods and outcome parameters. A significant limitation is the lack of false-positive data, based on an independent reference, that would be required for 2 × 2 contingency tables to allow estimation of the test specificities. Thus, the more rigorous statistical methodologies specially developed for meta-analysis of diagnostic test accuracy could not be used in this work.All studies except two , 21 inveIn the present meta-analysis we provide evidence that saliva tests are a promising alternative to nasopharyngeal swab tests for COVID-19 diagnosis. Optimized and validated saliva assays offer the possibility of reliable self-collection of samples for COVID-19 testing in the future. However, there are many open questions to be answered before the precise specificity and sensitivity of the saliva-based tests can be determined and appropriate standardized procedures introduced into clinical practice.Publicly available datasets were analyzed in this study. This data can be found here: The data that support the findings of this study are available from the corresponding author, upon reasonable request.LC, SK, ÁN, ZL, ZS, PH, MS, and GV devised the project, the main conceptual ideas and planned the research. LC, SK, NF, ZS, and GV worked out the methodology. IM, AH, and LC performed the data collection, literature search, study selection, and data extraction. LC, IM, and AH also organized and maintained research data for analysis. NF performed analytic calculations and applied statistical models for synthetizing data. ZS and SK also aided the research by interpretation of raw and synthetized data. NF visualized synthetized data into forest plots. LC and GV worked on summarizing results into figures and tables. SK and GV were responsible for managing and coordinating the research activity. PH and GV took leadership responsibility for the research activity, provided resources, and acquired financial support for the research project. ZS, PH, MS, and GV validated reproducibility of the results. LC, NF, IM, AH, and GV wrote the manuscript with input from all authors. SK, ÁN, ZL, ZS, PH, MS, and GV extensively reviewed the work and further edited the manuscript. All authors contributed to the article and approved the submitted version.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.


To the editor,We all know that Coronavirus disease 2019 (COVID-19) has been declared a pandemic on March 11th, 2020. Such dramatic scenario deeply impacted on the healthcare systems worldwide.If telemedicine allows for the remote provision of healthcare by means of electronic communication tools in case of medical conditions, surgical indications could be not deferrable   , 2 .Major surgical societies have been prompted in publishing position papers and guidelines for best surgical practice. Among these, the European Association of Urology (EAU) Robotic Urology Section (ERUS) recently published its Guidelines on dealing with robotic surgery in the COVID-19 era   .Such guidelines include behavioral good clinical practice rules aimed to maximize the safety and the protection against COVID-19 for both patients and healthcare professionals involved in the robotic surgical activity. We followed the principles included in the ERUS guidelines either for pure-laparoscopic or robot-assisted procedures performed at our Institution since the beginning of the COVID-19 crisis. All patients with indication to surgery received preoperative health screening, with none of them reporting symptoms suggestive for COVID-19. Procedures were performed in a dedicated operative room. All the necessary protection tools and general recommendations to reduce the transmission of the disease were adequately followed   . SelectAt the end of a three-weeks period, the teams involved in the operative room setting  were screened with a COVID-19 IgM/IgG rapid test lateral flow immunoassay, nowadays validated for the rapid diagnosis of COVID-19   .VivaDiagTM COVID-19 IgM/IgG was performed according to the manufacturer’s instruction   . After To date, real-time polymerase chain reaction in respiratory samples is the gold standard method for diagnosing COVID-19   . NevertThis is why we adopted VivaDiagTM COVID-19 IgM/IgG test. Although sensitivity has been published to be low   , 5 , spAt a price running to 10 euros per person screened, we believe it could represent a value-for-money passport for immunity of health-care professionals.


Sustained attention encompasses a cascade of fundamental functions. The human ability to implement a sustained attention task is supported by brain networks that dynamically formed and dissolved through oscillatory synchronization. The decrement of vigilance induced by prolonged task engagement affects sustained attention. However, little is known about which stage or combinations are affected by vigilance decrement. Here, we applied an analysis framework composed of weighted phase lag index (wPLI) and tensor component analysis (TCA) to an EEG dataset collected during 80 min sustained attention task to examine the electrophysiological basis of such effect. We aimed to characterize the phase-coupling networks to untangle different phases involved in sustained attention and study how they are modulated by vigilance decrement. We computed the time–frequency domain wPLI from each block and subject and constructed a fourth-order tensor, containing the time, frequency, functional connectivity (FC), and blocks × subjects. This tensor was subjected to the TCA to identify the interacted and low-dimensional components representing the frequency-specific dynamic FC (fdFC). We extracted four types of neuromakers during a sustained attention task, namely the pre-stimulus alpha right-lateralized parieto-occipital FC, the post-stimulus theta fronto-parieto-occipital FC, delta fronto-parieto-occipital FC, and beta right/left sensorimotor FCs. All these fdFCs were impaired by vigilance decrement. These fdFCs, except for the beta left sensorimotor network, were restored by rewards, although the restoration by reward in the beta right sensorimotor network was transient. These findings provide implications for dissociable effects of vigilance decrement on sustained attention by utilizing the tensor-based framework.The online version of this article (10.1007/s10548-020-00795-0) contains supplementary material, which is available to authorized users. Human attentional resources are not limitless. Sustaining attention on stimuli for a prolonged duration results in task performance declines and mental fatigue increases. This effect is known as time-on-task effect or vigilance decrement  and fMRI have uncovered that the fronto-parietal attention network decreases during prolonged sustained attention task engagement  platform, which is a free, open platform to support our research and enable collaboration. The contributors of this dataset have reported a different set of results in a prior study  collected during a modified version of sustained attention task   and vertical (below and above the left eye) EOGs.The preprocessing was conducted in MATLAB with the EEGLAB toolbox  and principal component analysis (PCA)) for extracting interested brain activities . We selected the desirable TCA components that both consistent with prior knowledge with a visual inspection and closely related to the behavioral measurements. The component which only meets the criterion of prior knowledge or only meets the criterion of significant correlations were not selected, as shown in Figs. S1–3.In order to examine the changes of fdFC caused by vigilance decrement, the elements in feature factor were subjected to the one-way analysis of variances (ANOVAs) with the within-subject factor block (first 60 min without reward) and results were corrected by the Greenhouse–Geisser. We also conducted the pair-wise comparisons between blocks 6 and 1 in the correct rejections condition and between blocks 3 and 1 in the hits and misses conditions to directly reveal the differences of fdFC between low and high vigilance state (without considering variations in different blocks). We performed the pair-wise comparisons between blocks 7 and 6 in the correct rejections condition and between blocks 4 and 3 in the hits and misses conditions to indicate the modulations of fdFC produced by motivation. When the effects of reward on specific component were detected, we ran pair-wise comparisons between blocks 7 and 1 in the correct rejections condition and between blocks 4 and 1 in the hits and misses conditions to quantitatively investigate the improvement of fdFC by motivation relative to that in the high vigilance state. In case of significant differences between blocks 7 and 1, we further ran the comparisons between blocks 8 and 1 to explore the continuous effect of motivation on fdFC. Both paired-sample t-test and Kruskal–Wallis test were applied to pair-wise comparisons. When the representations of the specific component followed the Gaussian distribution measured by the Jarque–Bera test, we used the paired-sample t-test, otherwise the Kruskal–Wallis test.After testing the distribution of behavioral data and representations of component with Jarque–Bera test, the Pearson correlation (following the Gaussian distribution) or Spearman rank correlation (non-parametric test) were used for correlation analysis between behavioral measurements  and the features of TCA components.The type I errors should be controlled during multiple comparisons when more than one TCA component was selected and multiple pair-wise comparisons were conducted. The p-values from selected components were corrected by the false discovery rate (FDR) to control the false discoveries  the pre-stimulus alpha right-lateralized parieto-occipital FC, (II) the post-stimulus theta fronto-parieto-occipital FC, (III) the post-stimulus delta fronto-parieto-occipital FC, (IV) the post-stimulus beta right sensorimotor FC, and (V) the post-stimulus beta left sensorimotor FC, as shown in Fig. The component as shown in row I of Fig. The component in Row II of Fig. The component  the pre-stimulus alpha right-lateralized parieto-occipital FC and (II) the post-stimulus theta fronto-parieto-occipital FC, as shown in Fig. The component in row I of Fig. Row II of Fig. By applying the analysis framework composed of wPLI and TCA to the high-temporal resolution EEG collected during a sustained attention task over 80 min, we examined how a cascade of fundamental functions was reflected by the fdFC and which stages or a combination of stages were affected by vigilance decrement and motivation. In tandem, we performed the analysis framework in the correct rejections, hits, and misses conditions to explore the distinctive involvement of functions in different conditions. Following the main steps of the framework Fig. , we firsIn the present study, the time window of the alpha right-lateralized parieto-occipital FC ranged from − 1000 to 0 ms. The theta fronto-parieto-occipital FC peaked around 280–320 ms and the delta fronto-parieto-occipital FC peaked around 460–490 ms across emerged conditions. The beta (peaked at approximately 20 Hz) right and left sensorimotor FCs peaked around 670 and 740 ms, respectively. These fdFCs emerged in the temporal order of the alpha parieto-occipital, theta fronto-parieto-occipital, delta fronto-parieto-occipital, and beta right/left FCs. When these results are interpreted in the context of the roles suggested for the fdFCs, a series of fundamental functions underlying sustained attention can be tracked.A previous study has demonstrated that the event-related desynchronization within the alpha band in the occipital brain regions is associated with the anticipatory attention for a forthcoming stimulus  and detecting target (the hits condition), but not in error responses of detecting target (the misses condition). As referred above, the delta FC is related to working memory. It is likely that the inability to detect targets is owing to a failure of the target-related working memory process. The working memory encompasses subprocesses of information encoding, maintenance, or retrieval  of a sustained attention task lead to vigilance decrement, impairing all types of fdFCs. Participants were motivated with an extra monetary reward in the last 20 min interval. Four types of fdFCs, except for the left sensorimotor FC, were restored by reward, although the recovery of the right sensorimotor FC was transient. The right sensorimotor FC increased only in the first 10 min and then fell down to the low vigilance level in the last 10 min. The restoration of fdFCs by motivation appears to inconsistent with the overload theories, which states that cognitive resources are limited and vigilance decrement is determined only by resource depletion. The partial and transient improvement by motivation might agree with the motivational control and energetical costs theoretical model, where participants evaluate the costs and benefits and also assess their energetic resources to decide to expend or reserve their efforts.There are still three caveats in explaining the results. First of all, the modulation effect of reward on the pre-stimulus alpha parieto-occipital FC is detected in the correct rejections and hits conditions, but not in the misses condition. It is possible that the differences are from the robustness of motivation itself or the instability of preparation of attention in the misses condition. Secondly, the number of trials cannot be equalized in the correction rejections and hits/miss conditions because of the experimental design. This imbalance might result in some differences in the TCA components. Finally, the task-related component selection is critical for the application of the tensor-based framework. Not only with the behavioral data association analyses but also more robustness and efficient methods should be developed for TCA component selection to provide comprehensive consideration.We apply an analysis framework composed of wPLI and TCA to a long period of sustained attention EEG dataset and derive a cascade of fdFCs involved in a sustained attention task. The pre-stimulus alpha parieto-occipital FC, post-stimulus theta fronto-parieto-occipital FC, delta fronto-parieto-occipital FC, and beta right/left sensorimotor FCs are derived, corresponding to different functions in sustained attention. We successfully detect the modulations of fdFCs affected by vigilance decrement and motivation. All these fdFCs are impaired by vigilance decrement. Especially, the pre-stimulus alpha FC parieto-occipital drifts rightward with time-on-task. The impairments of fdFCs are partially restored by motivation. The post-stimulus beta left sensorimotor network is not modulated by rewards. The right sensorimotor FC is more associated with motivation than the left sensorimotor FC, although the effect of improvement by motivation on the right sensorimotor FC is transient. Our results lay the ground for the hybrid model that vigilance decrement is determined by motivational control and energetical costs. The analysis framework provides feasibility for identifying dynamic organizations of frequency-specific FC in cognitive tasks.Supplementary file 1 (DOCX 19053 kb)Below is the link to the electronic supplementary material.


Immune related long non-coding RNAs (IR-lncRs) plentiful in immune cells and immune microenvironment (IME) are potential in evaluating prognosis and assessing the effects of immunotherapy. A completed and meaningful IR-lncRs analysis based on abundant pRCC gene samples from The Cancer Genome Atlas (TCGA) will provide insight in this field.Background: Papillary renal cell carcinoma (pRCC) was the 2Results: 17 IR-lncRs were selected by Pearson correlation analysis of immune score and the lncRNA expression level, and 5 sIRlncRs were significantly correlated with the OS of pRCC patients. 4 sIRlncRs  with the most remarkable prognostic values were identified to establish the IRRS model and the OS of the low-risk group was longer than that in the high-risk group. The IRRS was certified as an independent prognosis factor and correlated with the OS. The high-risk group and low-risk group showed significantly different distributions and immune status through PCA and GSEA. In addition, we further found the expression levels of SNHG16 was remarkably enhanced in female patients with more advanced T-stages, but ADAMTS9-AS1 showed the opposite results.Conclusion: The IRRS model based on the identified 4 sIRlncRs showed the significant values on forecasting prognoses of pRCC patients, with the longer OS in the low-risk group.Methods: We integrated the expression profiles of LncRNA and overall survival (OS) in the 322 pRCC patients based on the TCGA dataset. The immune scores calculated on account of the expression level of immune-related genes were used to verify the most relevant IR-lncRs. Survival-related IR-lncRs (sIRlncRs) were estimated by COX regression analysis in pRCC patients. The high-risk group and low-risk group were identified by the median immune-related risk score (IRRS) model established by the screened sIRlncRs. Functional annotation was displayed by gene set enrichment analysis (GSEA) and principal component analysis (PCA), and the immune composition and purity of the tumor were evaluated through microenvironment cell count records. The expression levels of sIRlncRs of pRCC samples were verified by real-time quantitative PCR. In 2004e of RCC . Characte of RCC . Given tWith the deeper understanding of the crucial roles of immune and stromal cells on tumor biological progress, a growing body of researchers were motivated to discover immunotherapy of pRCC, and increasing immunotherapy drugs obtained rewarding treatment effects –10. HoweWith the increasingly clear cognition of the crucial role of genetics and epigenetics on tumor pathological features, biological behaviors, therapy strategies and prognosis, more pRCC-related immune genes and genetic modification approaches were identified to be involved in the diagnosis process and prognosis prediction , 14 LongHowever, the potential of certain genetic markers in immune microenviroment (IME) especially for iIR-lncRs on prognosis forecast have yet to be adequately elucidated , 19. TheTo give an insight into the clinical potency of IME and IR-lncRs on prognosis evaluation of pRCC, we extracted a class of IR-lncRs in IME predicting poor prognosis in pRCC patients, together with their clinicopathologic signatures, we further calculated their correlations with OS. The results establish a more personalized precision predicting model of pRCC, and provide the guiding light for making clinical decision.Transcriptome data and clinical data of patients with pRCC were fetched from TCGA database. Next, transcriptome data was processed to convert the data ensembl ID into gene names. Following that, transcriptome data were divided into lncRNA and mRNA. From the Immune system process M13664 and Immune response M19817 of Molecular Signatures Database, we identified 331 pRCC IRGs, of which 17 lncRNAs were validated to be the IR-lncRs by correlation analysis.Based on COX Regression model, we then identified 5 IR-lncRs which were associated with prognosis (sIRlncRs), such as AP001267.3, SNHG16, AC021054.1, AC026471.3 and ADAMTS9-AS1. The relationships between these sIRlncRs and prognosis were clearly illustrated in the forest map .The top 4 sIRlncRs  among the 5 sIRlncRs were included to establish the risk evaluating model, by which the pRCC samples were divided into the high-risk group and the low-risk group based on the intermediate risk score . The morTo investigate the relevance of the sIRlncRs and clinicopathological features of pRCC, we analyzed the correlation between the risk score and the clinical and demographic characteristics, such as age, gender, stage, T-stage, N-stage and M-stage. Under the IRRS, the scores of older patients , female We also analyzed the relationships between the compositions of the IRRS and the aforementioned tumor characteristics which all illustrated in We further found the expression levels of AP001267.3 and ADAMTS9-AS1 were gradually decreased in the more advanced stage, T-stage, M-stage and N-stage, while the expression levels of AC026471.3 and SNHG16 were enhanced –6D. DeteWe then conducted the independent risk analysis, the results showed age, stage, N-stage, M-stage and risk score were significantly correlated with OS in univariate analysis (P<0.05). But in the multivariate analysis, M-stage and risk score showed more remarkable correlations with OS . The ROCWe employed the PCA to detect the different distribution patterns between the low-risk group and the high-risk group by the immune gene sets and the genome-wide expression sets. In the IRGs set, the low-risk group and the high-risk group were observably separated with the lower immune scores in the low-risk group . While wIn order to verify whether the immune genome accurately reflects the status of the tumor immune microenvironment, we analyzed the relationships between the sIRlncRs and immune cell infiltration –9F. We fWe previously explored the roles of SNHG16 and ADAMTS9-AS1 on predicting prognosis by bioinformatics methods . To furtThe significance of tumor immunization activities on tumorigenesis and development and the individual variation at the genetic level attracted a growing body of researchers pay attention to discover the implications of differential genes which were potential for distinguishing pRCC patients responding heterogeneously and predicting prognosis, thus increasing miRNAs and lncRNAs are being identified to be labelled on the immune correlation –23 HowevTo detect the roles of lncRNAs in assessing prognosis, Seema Khadirnaikar established a lncRNA immune prognostic signature score model based on seven lncRNAs, and demonstrated a negative correlation between the score and survival of patients with ccRCC . To elucIn the present study, 322 pRCC patients were enrolled in a genome-wide analysis for lncRNAs, combining with 311 IRGs screened in Molecular Signatures Database v4.0 , and 17 IR-lncRs were identified eventually. We further detected the relation between the prognosis of pRCC patients and the expression levels of the 17 IR-lncRs, of which 5 sIRlncRs indicated the significant correlation with OS. Utilizing multivariate Cox and risk score model, we further identified 4 IR-lncRs to establish a risk evaluating model which was available to distinguish pRCC patients into the high-risk group and low-risk group with obviously differences of OS. Due to the molecular heterogeneity, the accuracy and sensitivity of the present clinical risk factors in predicting the survival of pRCC patients remain unsatisfying, we further validated the predicting value of the three sIRlncRs by multivariate analysis. We found the 4 sIRlncRs were independent of traditional risk factors and molecular characteristics. According to the IRGs set, the low-risk group and the high-risk group tended to be divided into two parts, with the low-risk group having lower immune scores than the high-risk group. GSEA was employed to further verify the functional annotation, and we found the more abundant immune-related responses and processes in the high-risk group. Therefore, immune-related scores are bound up with the immune status of pRCC, with higher scores indicating the poor prognosis. Besides, we found only B cell showed the most prominent relationship with sIRlncRs, which is probably due to the remarkable effects of macrophage polarization on the tumorigenesis, progression, prognosis and tumor behaviors of pRCC. The results motivated us to further discover the underlying functions and mechanisms in future studies.Furthermore, SNHG16 and ADAMTS9-AS1 were further verified in 22 pRCC samples, and the significant correlations between the two sIRlncRs and T stages and genders were detected. We found female patients obtained the higher expression levels of SNHG16 and the lower expression levels of ADAMTS9-AS1 than male. It is probably due to the transcription factors which affect genes expression levels are different between male and female. Some sex-biased genes have transcription factors with different expression levels between different genders in the promoter region where genes can be activated. Besides, sex chromosomes and hormones control the differences between male and female, and these differences could eventually affect transcription factors . These fIn the analyzing process, the large numbers of pRCC patients were enrolled to further enhance the reliability and persuasion in guiding clinical strategic decisions. Besides, some specific IR-lncRs with remarkable differences under variable risk factors have been further corroborated to be implicated in the progression and prognosis of pRCC. More importantly, these IR-lncRs as the molecular bioindicators, showed significant potency in forecasting and evaluating the OS.Although we elucidated the roles of sIRlncRs on forecasting prognosis and verified the expression levels of SNHG16 and ADAMTS9-AS in tumor tissues, some limitations remain to be further discussed. Firstly, we didn’t combine with the detection of proteomics, metabolomics and immunogenomics. Then, the practical application values of these sIRlncRs have yet to be adequately elucidated and need to be further wide verification. Thirdly, except for SNHG16 and ADAMTS9-AS, other sIRlncRs included in the IRRS model are also needed to be explored.In conclusion, we comprehensively analyzed and verified the effects of IR-lncRs on forecasting clinical prognosis of pRCC. The results will contribute to develop a reliable and referable risk evaluating model and provide new insight into the immune-related researches and treatment strategies.Informed consent forms have been signed by all patients before this study. The research protocol has been approved by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University and is based on the ethical principles of medical research involving human subjects in the Helsinki Declaration.A total of 22 clinical samples of pRCC and corresponding adjacent tissues were collected from patients who received treatment in the First Affiliated Hospital of Chongqing Medical University from October 2018 to December 2019. The collected tissue samples were immediately frozen in liquid nitrogen and then stored at –80°C until RNA extraction.https://portal.gdc.cancer.gov/), that contained data from 32 non-tumor tissues and 289 pRCC tissues. The clinical data about these patients was also downloaded and extracted (the OS of patients≤30 days were excluded because these patients probably died for unpredictable factors such as infection and hemorrhage). Raw data was collected to do further analyses. These data were currently updated in November 11, 2019. RNA-seq results were combined into a matrix file by a merge script in the Perl language (http://www.perl.org/). The Ensembl IDs of genes was converted into a matrix of gene symbols by the Ensembl database (http://asia.ensembl.org/index.html).Transcriptome RNA-sequencing of pRCC samples were downloaded from the The Cancer Genome Atlas (TCGA) data portal (http://www.broadinstitute.org/gsea/msigdb/index.jsp) was used to explore the immune-related gene participating in the immune process. Immune related gene was used to establish the immune score of pRCC gene by GSEA. The correlation between the immune score and the expression of lncRNA in pRCC patients was analyzed by Pearson correlation analysis. IR-lncRs was identified by a criterion of |r|>0.8 and P<0.001.The Molecular Signatures Database v4.0 . Univariate COX analysis was used to screen sIRlncRs (p<0.01). Hazard ratio (HR) was used to specified sIRlncRs into protective and deleterious portion. These sIRlncRs were selected for follow-up study.To verify the reliability, sIRlncRs were submitted for the multivariate analysis, while the integrated sIRlncRs were still used as an independent prognostic indicator to develop the IRRS (p<0.05) . In ordeThe survival ROC curve was employed to verify the prognostic performance through the survival ROC package of the R software. The survival time, survival status and risk scores of patients with pRCC were used to predict the prognosis over a 5-year period, then the ROC curve was drawn and the value of AUC was calculated. Abscissa was the false positive rate and ordinate represented the true positive rate. Principal component analysis (PCA) was displayed to demonstrate the expression levels of pRCC samples and gene set enrichment analysis (GSEA) was used to detect the different functional phenotypes between the low-risk group and high-risk group. The tumor infiltrating immunocytes were evaluated through the TIMMER database. The levels of immune infiltration in pRCC patients were downloaded, and the relationship between IRRS and immune cell infiltration was calculated.−ΔCt method. The primer sequences are shown in Triazole (Invitrogen) was used to extract total RNA from tissues and cell lines under various experimental conditions according to the manufacturer's instructions. cDNA Synthesis Kit  combining with RNA (1μg) was utilized to reverse transcribed cDNA. The reaction steps were as follows: 37°C for 15min, 85°C for 5s, and quantitative polymerase chain reaction (qPCR) was performed on an ABI 7500 real-time PCR system (Applied Biosystems) using SYBR-Green method (TaKaRa). The reaction cycle conditions were performed . Relative expression level of lncRNAs normalized to β-actin was calculated by the 2P<0.05 was considered significantly statistical difference.Univariate Cox regression analysis and Pearson correlation analysis were used to identify the target IR-lncRs. Kaplan-Meier curve was used to evaluate the OS between low-risk group and high-risk group. Univariate and multivariate Cox regression analysis were used to verify the independent prognostic factors for pRCC patients. All statistical analysis was conducted using SPSS21.0 software  and GraphPad Prism5 . Varieties in clinical parameters were determined using independent t-tests. Supplementary Figure 1


Several studies have shown enhanced performance in change detection tasks when spatial cues indicating the probe’s location are presented after the memory array has disappeared  compared with spatial cues that are presented simultaneously with the test array . This retro-cue benefit led some authors to propose the existence of two different stores of visual short-term memory: a weak but high-capacity store (fragile memory (FM)) linked to the effect of retro-cues and a robust but low-capacity store (working memory (WM)) linked to the effect of post-cues. The former is thought to be an attention-free system, whereas the latter would strictly depend on selective attention. Nonetheless, this dissociation is under debate, and several authors do not consider retro-cues as a proxy to measure the existence of an independent memory system . We approached this controversial issue by altering the attention-related functions in the right superior parietal lobule (SPL) by transcranial magnetic stimulation (TMS), whose effects were mediated by the integrity of the right superior longitudinal fasciculus (SLF). Specifically, we asked whether TMS on the SPL affected the performance of retro cues vs. post-cues to a similar extent. The results showed that TMS on the SPL, mediated by right SLF-III integrity, produced a modulation of the retro-cue benefit, namely a memory capacity decrease in the post-cues but not in the retro-cues. These findings have strong implications for the debate on the existence of independent stages of visual short-term memory and for the growing literature showing a key role of the SLF for explaining the variability of TMS effects across participants. Classical theories of visual short-term memory (VSTM) usually distinguish between two main systems: working memory, which is a low-capacity but durable memory system that lasts seconds to minutes and is robust enough to prevent visual interference,  and visuIn typical visual working memory (WM) paradigms, such as change detection tasks , particiOne of the interpretations of the so-called retro-cue benefit is that it results from a fragile but large-capacity visual short-term memory store ), which would be an intermediate system between the iconic and visual working memory (WM) ,6,7. MorThe multiple store theory postulates a differential impact of the attention selection mechanisms in the two memory stores. More specifically, this theory proposes that FM traces are formed without the need for selective attention, while visual WM traces actually depend on selective attention to be created. Retro-cues would be just a tool to report, but not to build, the fragile memory representations. This assumption has been defended by Vandenbroucke et al. , who manEven though the multiple store theory conceptualizes retro- and post-cue trials as two different tools to gauge two independent memory systems—the formation of which is considered independent of and dependent on attentional processes, respectively—this dissociation is not unanimously accepted. Many studies  from the performance in post-cue trials . We applied online TMS to the right SPL to interfere with attentional orienting while measuring participants’ memory capacities in a change detection task with retro-cue vs. post-cue trials. TMS pulses were applied online, either to the right SPL or to an active control site (the vertex). The right SPL was selected, as this area has consistently been involved in the endogenous control of attentional orienting toward both external  and internal (WM) objects . PreviouTo specifically understand how and whether the temporal modulation of the attentional function produced by SPL stimulation affects the encoding or maintenance phases, TMS pulses were delivered either before or after the memory array presentation (see magnetic resonance imaging (MRI) and TMS protocol and We hypothesized that if FM traces were formed and maintained independently of focal attention, then the disruption of the activity of the right SPL should only affect the memory content in the post-cue trials, when endogenous attention—at encoding and maintenance—is supposed to be necessary to protect the memory traces from interference, leaving the performance unaffected in the retro-cue trials. Furthermore, TMS modulation of the right SPL attentional function is specifically expected from participants with reduced integrity of the right SLF , who should be more prone to TMS effects. Data will be analyzed and discussed, taking into account both the multiple store theory approach  by compaA total of 17 right-handed, healthy volunteers  took part in the study. The sample size was established on the basis of the previous literature, employing a similar sample size and obtaining reliable effects ,7,19,48 The experiment was run on a computer with a 1 GHz Pentium III processor. The stimuli were presented and the data were acquired by E-prime software . The stihttp://www.axilumrobotics.com/en/, accessed on 30 June 2019) and asked to maintain their gaze on the central cross during all of the trial. At the beginning of each trial, six placeholders and a central red fixation cross that turned black after 500 ms were presented. After 1000 ms, the six letters constituting the memory array were then presented for 250 ms within the placeholders. The participants were required to remember as many letters as possible. We tested the participants’ memory for letters instead of line orientations to minimize the grouping or chunking effects [The behavioral procedure see  was simi effects , increas effects  and consSince previous research suggested that training participants to use a retro-cue was necessary to guarantee a proper measure of fragile memory , particiStructural T1-weighted anatomical magnetic resonance (MR) scans of all participants were acquired at the Mind, Brain and Behavior Research Center (CIMCYC) at the University of Granada. We used a 3-T Siemens magnetization-prepared rapid gradient echo  = 2530 ms, echo time (TE) = 3.5 ms, slice thickness = 1 mm, and field of view (FOV) = 256 mm). Prior to the TMS experimental session, we determined the hotspot for the first dorsal interosseous (FDI), defined as the optimum site over the primary motor cortex (M1) which evoked the highest contralateral motor evoked potentials (MEPs) in the relaxed FDI. Afterward, we calculated the individual resting motor threshold (rMT), which was the minimum stimulus intensity eliciting MEPs >50 μV in five out of ten consecutive trials ,52,53. EIn each trial, we applied a burst of four TMS pulses at 8 Hz either during the encoding phase , and 125 ms after the onset of the memory array) or during the maintenance phase at 0, 125, 250 and 375 ms after the onset of the first blank retention interval see . This prhttp://www.axilumrobotics.com/en/, accessed on 30 June 2019) and a TMS neuronavigation system , which allowed us to estimate and track in real time the relative position, orientation and tilting of the coil on the sectional and 3D reconstructions of the participants’ MRIs with a precision of 5 mm. The TMS robot warranted the precise stimulation of the selected brain regions (the right SPL and the vertex) by automatically readjusting the position of the coil if a movement larger than 5 mm was detected.The scalp coordinates of the stimulation sites were localized by using the native space of each participant’s T1-weighted anatomical MRI scans. The TMS coil was controlled by a robotic arm . The cod z = 78 ), which d z = 78 ,60,61,623) resolution and coverage of the whole head with a posterior–anterior phase of acquisition (echo time = 88 ms and repetition time = 8400 ms). At each slice location, 6 images were acquired with no diffusion gradient applied and 60 diffusion-weighted images, in which gradient directions were uniformly distributed in space. The diffusion weighting was equal to a b-value of 1500 s/mm2. In each slice, diffusion-weighted data were simultaneously registered and corrected for motion and geometrical distortion, adjusting the gradient accordingly . Then, individual dissections of the tracts were carried out with TrackVis software . Both branches (SLF-I and SLF-III) were isolated using a multiple region of interest (ROI) approach. Parietal and frontal ROIs  and parietal and frontal ROIs  were delineated around the white matter for both the SLF-I and the SLF-III, respectively. A no-part ROI in the temporal white matter was also used to exclude streamlines of the arcuate fasciculus projecting to the temporal lobe [DTI analyses were performed following the previously reported procedure . A totaral lobe ,64. Finaral lobe ) was extral lobe .The behavioral performance was analyzed by calculating the memory capacity K index of each participant by using Cowan’s K formula : K = [, which constitute a statistically powerful method that takes into account both fixed factors (the variables of interest) and random effects (variation that is not explained by the variables of interest). In particular, one of the advantages of the linear mixed model is that it resolves the issue of independence among repeated measures by controlling for individual variation among the participants . In otheThus, we constructed linear mixed models using the lmer function of the lme4 R package, version 1.1–21 , with pap values of the significant fixed effects were provided by the ANOVA function of the lmerTest R package, version 3.1–0 [To determine the best structure for the random and fixed components, we followed a well-known procedure . Specifion 3.1–0 , using ton 3.1–0 . Because1,90 = 7.10, p = 0.009, η2p = 0.072). To follow up on this four-way interaction, we first split the data by the stimulation time window. As can be observed in 1,38 = 0.06, p = 0.80, η2p = 0.002)    , while performance in the retro-cue trials remained unaffected (p = 0.56). It should be noted that the analysis did not show any association with the right SLF-I. 1,90 = 7.10).The analysis of the memory capacity showed a final model with significant four-way interaction between the cue type, stimulation site, stimulation time window and right SLF-III integrity  . To follow up on this three-way interaction, we split the data by the stimulation time window. The analysis showed that the interaction between the stimulation site and the right SLF-III integrity was not significant when the stimulation was applied at the maintenance phase . A significant interaction between the stimulation site and the right SLF-III integrity was instead found when TMS was applied during the encoding phase . Bonferroni-corrected comparisons showed that vertex stimulation during encoding did not produce any modulation of the retro-cue benefit, depending on the right SLF-III integrity (p = 0.1), while SPL stimulation significantly increased the retro-cue benefits of those participants with lower right SLF-III integrities (p = 0.004)  B. This rIn the present study, we used a causal approach to investigate whether and how endogenous top-down spatial attention might dissociate performance in retro-cue trials (supposedly gauging FM) from performance in post-cue trials (supposedly gauging WM). With this aim, while participants performed a change detection task measuring both types of short-term memories, we disrupted their attention with online TMS, applied to the right SPL (in comparison with a control area) during both the encoding and maintenance phases. We found that applying TMS over the right SPL during the encoding phase caused a decrease of the memory capacity in the post-cue condition, especially for those participants with lower right SLF-III integrities, while memory capacity in the retro-cue trials was unaffected. These findings are in good agreement with the previous literature showing that TMS effects are dramatically modulated by interindividual variability in white matter connectivity ,39,40,73In general, the present findings fit well with the multiple store theory , which pHowever, if the results are observed from a different perspective by looking at the individual differences between the retro-cue and post-cue trials (the retro-cue benefit), another explanation that does not necessarily imply the existence of fragile memory as a system independent from spatial attention and WM is possible. As was previously mentioned, TMS over the right SPL led to a decrease in memory capacity in the post-cue condition associated with the right SLF-III integrity, along with an increase of the retro-cue benefit. Thus, a possible alternative interpretation of this result is that the reduction of the memory capacity in the post-cue trials produced by TMS stimulation during encoding was compensated by the action of the retro-cues. Previous research has indeed suggested that retro-cues boost the accessibility of short-term memory representations to levels higher than those acquired after encoding  by at leIt could be speculated that post-cue trial performance likely depends on the initial distribution of the attentional resources at the encoding phase. Since the participants did not know which item would be probed until the presentation of the test array in the post-cue trials, attention in this condition should not be focused on any specific location but distributed rather homogeneously across the different placeholders. The previous literature has suggested that the attentional weight associated with memory array items and generated by spatial selection criteria are computed in the PPC ,85,86,87Finally, the present findings show no TMS effect when the SPL was stimulated at the maintenance phase. Apparently, this result is in contrast with the attentional rehearsal theory , which sIn conclusion, by a causal approach with TMS we have shown that online SPL stimulation during encoding reduced the memory capacity in post-cue trials depending on the right SLF-III integrity: the more reduced the right SLF-III integrity, the more decreased the memory capacity in post-cue trials, leaving the performance in retro-cue trials unaffected. This pattern of results further demonstrated that the memory benefit of retrospectively attending to internal representations of previously observed stimuli (the retro-cue benefit) can be altered by TMS over the right SPL at encoding, depending on the individual structural characteristics of long-range white matter connections. No modulations were observed when SPL was stimulated during memory maintenance, thus suggesting that the attention role of SPL is more related to the encoding phase. The present findings provide new information for the better comprehension of the interplay between spatial attention and VSTM, suggesting an important role of SPL during encoding and highlighting the necessity of considering individual differences in the structural characteristics of the white matter connections.


Before the establishment of an adaptive immune response, retroviruses can be targeted by several cellular host factors at different stages of the viral replication cycle. This intrinsic immunity relies on a large diversity of antiviral processes. In the case of HTLV-1 infection, these active innate host defense mechanisms are debated. Among these mechanisms, we focused on an RNA decay pathway called nonsense-mediated mRNA decay (NMD), which can target multiple viral RNAs, including HTLV-1 unspliced RNA, as has been recently demonstrated. NMD is a co-translational process that depends on the RNA helicase UPF1 and regulates the expression of multiple types of host mRNAs. RNA sensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP) composition. HTLV-1 has evolved several means to evade the NMD threat, leading to NMD inhibition. In the early steps of infection, NMD inhibition favours the production of HTLV-1 infectious particles, which may contribute to the survival of the fittest clones despite genome instability; however, its direct long-term impact remains to be investigated. HTLV-1 is a delta-retrovirus infecting approximately 10 million people worldwide ,3,4. HTLThe roles played by innate and adaptive immune responses during HTLV-1 infection are extensively studied. Models of how HTLV-1 successfully escapes or dysregulates these immune responses in order to extend the infection and provoke diseases have been proposed and are regularly updated ,9,10. HoThe nonsense-mediated mRNA decay (NMD) pathway is an interferon (IFN) independent process that has been recently demonstrated by convergent studies to degrade several viral RNA ,39,40,41In this review, we thus propose an analysis of the interplay between HTLV-1 and NMD. In order to give a better insight about this process since NMD does not have a wide audience in the HTLV-1 field to the contrary of other antiviral process mechanisms, we first describe the mechanism, its regulation and the RNA features triggering its initiation. Then, we analyse how HTLV-1 protects itself against NMD and suggest how the consequences of NMD inhibition might converge with known cellular dysfunctions associated with HTLV-1 infection, highlighting a relationship more complex than meets the eye.NMD is a co-translational pathway conserved through evolution from yeast to mammals and leads to the degradation of mRNA. It is initiated when translation is terminated in a specific unfavourable environment. The slowing of the translation terminating steps due to mRNA sequence, organization and/or mRNP composition greatly favours the retention of the NMD central factor UPF1 at the stop codon with the stalled ribosome, leading to NMD activation. UPF1 is a helicase that binds RNA without sequence specificity. It appears to be displaced by the translating ribosome and subsequently accumulates on the mRNA 3′UTR ,45. It iClosely related to translation termination, NMD efficiency is naturally modulated by mRNP composition. Notably, the presence of an exon-junction complex (EJC) is a critical parameter: when located downstream of the stop codon, the EJC promotes the enrichment of the 3′UTR in NMD factors such as UPF3B and UPF2, leading to strong stimulation of NMD . According to the above description, any stop codon, as soon as it is read by the ribosome, might be able to initiate NMD, with the outcome being dependent on the equilibrium between the stimulators and the inhibitors at each step. While the unique function of NMD is to degrade mRNA, two main functions are usually associated with NMD depending on the origin of the stop codon initiating NMD: RNA quality control and gene expression regulation at the post-transcriptional level .Historically, NMD was described as a RNA quality control process, degrading mRNA with a premature termination codon (PTC). It is estimated that such RNA represents 5%–30% of human transcripts and may be the result of genomic mutations (nonsense and frameshift), faulty alternative splicing  or translational errors. The truncated protein can be deleterious for the cell since it is non-functional, or it may even have a dominant negative effect. PTCs are present inside the open reading frame (ORF), creating a longer 3′UTR with a greater distance between the physiologic stop codon and the related translation termination regulatory environment. PTC may also be upstream of exon-exon junctions marked by an EJC. As described above, these parameters greatly favour NMD triggering and lead to the elimination of the PTC-harbouring mRNA.In addition to the quality control function in which NMD prompts the cell to systematically degrade “aberrant” mRNA, NMD can also periodically regulate specific “non-aberrant” mRNA. This intermittent sensitivity to NMD is a programmed cellular protocol based on the local modification of an mRNA sequence, organization and/or mRNP composition. Among the different parameters influencing those local modifications, we can find uORF (upstream open reading frame), which is a translation that may trigger NMD due to the EJC downstream in the main ORF. CREB-2/ATF4 mRNA is a well-documented example ,71,72. AHTLV-1 is a complex retrovirus with multiple proteins coded in a single genomic RNA. The overall sensitivity of HTLV-1 retroviral RNA to NMD has been known for a few years ,43. AlthDirect regulation: As described above, the half-life of unspliced HTLV-1 RNA is increased under NMD inhibition. How can this sensitivity be explained? Since the mRNA is un-spliced, it should not have bound an EJC, especially in the 3′UTR, which is expected to hamper its sensitivity to NMD. However, according to the above findings and our knowledge of HTLV-1 RNA organization, the 3′UTR size stands out as a possible factor. While the median human 3′UTR size is ~750 nt [ ~750 nt , the gag ~750 nt . In cont ~750 nt ,67,80. A ~750 nt . In the  ~750 nt . OkadaicIndirect regulation: In cells transfected with an HTLV-1 molecular clone, the knockdown of UPF1 led to a 3-to-4-fold homogeneous upregulation of all viral RNAs in the steady state [dy state . This hody state .While the sensitivity of HTLV-1 to NMD is well documented, additional work is needed to clearly demonstrate which stop codons are the most likely to induce NMD. Moreover, to maintain its capacity to replicate, HTLV-1 had to evolve solutions to evade the NMD threat. These solutions are discussed in the next section.Tax/Rex mRNA was expressed [The arms race involving HTLV-1 and NMD led to the evolution of two viral countermeasures. The viral proteins Tax and Rex were shown to target the NMD process in what is called “trans-inhibition”, incapacitating the decay of viral as well as cellular NMD targets. This evidence emerged from observations that HTLV-1-infected lymphocytes were able to specifically stabilize globin mRNA with a PTC . Nakano xpressed .Tax was first described as the main viral transactivator . It has The Rex protein was also shown to inhibit NMD. Similarly to Tax, several host mRNAs known to be NMD sensitive had increased half-lives upon Rex expression. Rex is known to bind viral RNA at the RxRE motif. Upon binding to RxRE, Rex controls viral mRNA splicing. It also contacts the CRM1 export system to ensure the nucleo-cytoplasmic shuttling of the unspliced viral mRNA ,89,90. Tgag mRNA and the production of the gag protein necessitate the formation of a favourable environment: NMD inhibition might contribute to this initial condition. Notably, the results from kinetics experiments performed with RNA(+) virus infections suggest that the impact of NMD inhibition on viral RNA is greater in the early steps of the infection [During infection, HTLV-1 is spread in two different ways: viral propagation is initially dependent on cell-to-cell transmission, then it evolves towards polyclonal and monoclonal expansion (reviewed elsewhere ). Cell-tnfection ,39. Finahbz RNA appears to play specific roles in T cell proliferation  and innate immune pathways (with an immunosuppressive effect) . Knoiewed in ).Although it is widely accepted that HTLV-1 infection leads to cell proliferation, it has been frequently observed to cause apoptosis and senescence of lymphoid and non-lymphoid cells in a Tax-dependent manner. It has been proposed that cells responding by senescence or apoptosis have very high levels of expressed Tax and Rex, are associated with robust replication of the virus and are subjected to great oncogenic pressure. This viral hyperactivity might be dependent on the site of integration, stimulating LTR transactivation and thus may be specific to each clone. Senescence induction has been shown to be linked with the stabilization of the p27 protein and cyclin-dependent kinase inhibitor p21CIP/WAF1 mRNA because of NF-κB hyperactivation ,97,98 re. HoweverWhile in the early steps of infection when HTLV-1 directly benefits from NMD inhibition to produce infectious particles, it is conceivable that, in a second step, this NMD trans-inhibition also participates in the selection of a clone with attenuated expression of Tax and Rex that is more suited to avoid the immune response and thus to maintain infection.As introduced above, HTLV-1 is the etiological agent of ATL and HAM/TSP. ATL is a malignant lymphoproliferative syndrome established after decades of latency and characterized by genetic instability combined with checkpoint adaptation. A favorable environment enabling malignant proliferation is dependent on the establishment of an immunosuppressive state. The viral protein Tax plays a major role in these steps. HAM/TSP is an immune-mediated inflammatory disease associated with the accumulation of HTLV-1-specific CD8+ T cells and infected CD4+ T cells in cerebrospinal fluid and neural tissues. It is characterized by a chronic inflammatory state due to elevated cytokine expression and production (reviewed in ). We wonupf1 gene allowing the synthesis of mutated dominant negative p53, functionally correlating NMD inhibition with cancer [In 2015, genomic sequencing of 400 ATL samples showed that the mutation rate for ATL was relatively high compared to other hematologic malignancies, with an average of 2.3 mutations per mega base in coding regions ,112. Noth cancer . Loss-ofh cancer ,117,118.h cancer ,120. Hyph cancer . Moreoveh cancer . In Arabh cancer . The rolHowever, to clarify whether there is an effective link between NMD and HTLV-1-associated pathologies, the status of NMD in the later stages of the infection and in patient cells, ATL and HAM/TSP, has yet to be analysed. The absence of (+) strand transcription in most ATL cells due to epigenetic repression and genomic alterations raises the following questions: Is the burst of (+) strand expression sufficient to inhibit NMD? HBZ, the protein expressed from the HTLV-1 minus strand, is a critical component of cell proliferation and tumorigenesis and maintains constant expression during infection, in contrast to Tax and Rex; could it also be involved in NMD inhibition? NMD is also sensitive to bivalent cation concentrations , and intAlthough HTLV-1 infects its host on the long term and can spread into the organism by inducing clonal expansion of infected cells, a strong immune response is usually directed against some of its protein. Over the years, models were proposed to explain this apparent contradiction. However, in these models, the intrinsic immunity operating within virus-infected cells, i.e., antiviral factors, are usually put aside. The very persistence of the HTLV-1 infections suggests that these mechanisms fail to restrict the virus or that HTLV-1 evolved countermeasures, if not completely, at least efficiently enough to allow minimum expression. In this review we have presented that HTLV-1 depends on a polycistronic RNA, which tends to have a long 3′UTR which is a critical NMD determinant. To protect its mRNA, HTLV-1 evolved independent mechanisms to trans-inhibit NMD through two viral proteins: Tax and Rex. The strength of these countermeasures shows the size of the threat that NMD constitutes for viral replication and the place of NMD among the cellular antiviral function. Those conclusions are also supported by comparison with several other viruses, especially retroviruses and (+) RNA viruses, which RNAs are also targeted by NMD and that evolved protective means ,126,127.However, while HTLV-1 and the host cell seem to be engaged in an arms race involving NMD, we wonder which partner truly benefits from NMD inhibition during each steps of the infection. The virus, in the early steps, because it is able to produce viral particles again? The host, since NMD inhibition establishes the immune response? Is the apoptosis induced from NMD inhibition contributing to the clonal selection involved in leukaemia emergence? Could the NMD activity be controlled by an occasional burst of Tax expression and be involved in the latency exit as suggested with HIV ? SimilarFinally, multiple “NMD-like” processes have been recently discovered, and they depend on the RNA helicase UPF1 and factors recognizing specific RNA features. In addition to NMD, UPF1 is involved in replication-dependent histone mRNA decay, miRNA decay, glucocorticoid receptor-mediated mRNA decay and regnase1-mediated mRNA decay (RMD) (reviewed in ). Since Considering HTLV-1 has already evolved counteracting measures to take advantage of the host machinery, it is still a promising strategy to gain insights into these molecular biology processes to better understand the virus biology and eventually develop efficient antiretroviral therapies.


The present study examined the relations among poly-bullying victimization (experiencing multiple forms of peer bullying), resilience and subjective well-being. This study specifically examined late adolescents’ resilience as a moderator of the relation between poly-bullying victimization and subjective well-being. In a region of central Spain, 1430 undergraduate students , aged between 18 and 22 years, completed three self-reported measures, including bullying victimization experiences, self-reported subjective well-being and resilience. A substantial proportion of the participants (16.9%) reported being victims of poly-bullying. The results showed that the poly-bullying victimization group reported the poorest subjective well-being and the lowest resilience levels. The regression analyses revealed that resilience was significantly and positively associated with subjective well-being, and resilience moderated the association between poly-bullying victimization and subjective well-being. However, the relation was very weak and accounted for only an additional 1% of variance in the participants’ subjective well-being. Future research should assess resilience trajectories of youth exposed to multiple forms of bullying victimization in order to better understand the potential protective effect of resilience over negative mental health outcomes. There is a general consensus among researchers about the importance of the social and psychological well-being of relationships between peers in adolescence and emerging adulthood . HoweverOlweus  explaineWang, Iannotti, Luk and Nansel  analyzedBullying victimization, that is both “face to face”, and through the ICT, includes a wide range of serious short-term and long-term mental health problems ,19,20,21Research on victimization experiences has grown over the years. However, much of this research has focused on specific victimization types and their impact on victims . More reExposure to multiple types of violence and victimization has immediate and lifelong effects on physical and mental health ,29,30. PHickman et al.  measuredExperiencing multiple forms of peer bullying has also been simultaneously found to negatively affect youths’ psychological and social well-being. For example, Raskauskas  found thThe primary aim of the present study was to examine the relations among poly-bullying victimization (experiencing multiple forms of peer bullying), resilience and subjective well-being. Although bullying occurs in different settings  and may involve different individuals , our analyses will be center on bullying occurring between students in the university context. As it was described above, bullying behaviors between students can take various forms, including physical behaviors , verbal attacks , social/relational aggression , and cyberbullying (posting someone’s private photos online). Our study examines these four types of bullying between students.The World Health Organization (WHO) defines mental health as a positive state, “a state of well-being in which the individual realizes his or her own abilities, can cope with the normal stresses of life, can work productively and fruitfully, and is able to make a contribution to his or her community”  (p. 12).Defining mental health positively implies conceiving mental health as not only the absence of mental illness but also as the presence of different psychosocial resources that contribute to the realization of one’s full individual potential .That is Previous research has suggested that bullying and cyberbullying negatively impact victims’ subjective well-being . With a The same relations have also been found in poly-victimization research. Víllora, Yubero and Navarro  observedAlthough research has shown that victimization experiences have short-term and long-term negative effects on mental health, not all victims experience the same outcomes. Differences in outcomes may result from different resilience levels. Indeed, the term resilience is used to “refer to the finding that some individuals have a relatively good psychological outcome despite suffering risk experiences that would be expected to bring about serious sequelae”  (p. 1). Resilience is understood as a personal trait that is inherent to an individual, but also as a process or phenomenon influenced by the cultural and social context . The facDifferent studies have found that resilience is negatively related to bullying and cyberbullying, and it moderates the relation between bullying victimization and youths’ negative mental health outcomes. For example, Hinduja and Patchin  found thThe present study was designed to analyze the associations among poly-bullying victimization and reports of well-being, and to explore the moderation effect of resilience on the relation between these two variables. Consequently, the purposes of this study were to: (a) explore the prevalence of poly-bullying victims among late adolescents; (b) examine the association between well-being and resilience among late adolescents exposed to multiple forms of bullying; (c) analyze the moderating effects of resilience on poly-bullying victimization and well-being. Therefore, the following hypotheses are proposed:Hypothesis 1.Subjective well-being will be poorer among poly-bullying victims than among non-victims and other groups of victims.Hypothesis 2.Poly-bullying victimization will be related to poorer subjective well-being, whereas resilience will be related with better subjective well-being.Hypothesis 3.Resilience will moderate the relation between poly-bullying victimization and subjective well-being.z value of 1.96 (95% confidence level) and ± 3.4% error margin with an expected proportion (P) of 0.5. The analyses determined that 1019 students were required for the study. We intentionally oversampled.Data used in this study are from a non-probabilistic sample formed by 1430 undergraduate students from a Spanish university located in central Spain with approximately 23.000 students. Specifically, 64% of the participants were females and 36% were males. Their ages ranged from 18 to 22 years . The data in the present study were collected in 2018 and involved convenience sampling from 17 faculties of the University of Castilla-La Mancha with students attending all knowledge areas in years 1 to 3. The university student population in the 2017/2018 academic year was 22.483 undergraduate students. Sample size was calculated considering a The participants provided information about demographic variables, such as gender, age and grade. The following instruments were used to analyze the study variables.Self-reported bullying victimization. Bullying victimization at university was assessed using “The Bullyharm” developed by Hall , which cSubjective well-being. Well-being was measured using “The Mental Health Continuum-Short Form (MHC-SF) developed by Keyes, Wissing, Potgieter, Temane, Kruger and van Rooy , and valResilience. The Scale of Protective Factors-24 (SPF-24)  was usedData were collected by self-reported group class-administered pencil-and-paper anonymous questionnaires. One researcher administered questionnaires to the participants and answered any questions asked by the participants. The procedure took approximately 20 min in each group class. The Clinical Research Ethics Committee of the Virgen de la Luz Hospital in Cuenca approved the study protocol (PI0519). All the subjects signed informed consent forms prior to participating in the study.Based on the identification and study of poly-bullying victimization prevalence, we first analyzed the prevalence of the participants reporting bullying victimization across the four examined forms of bullying  and the groups of victims by considering the number of forms endured. Second, we compared reports of well-being and resilience across the different victimization groups by applying an analysis of variance (ANOVA). Third, a linear regression analysis was used to estimate the relations of poly-bullying victimization and resilience on subjective well-being (Step 1), and to then detect whether resilience moderated the association of poly-bullying victimization on late adolescents’ subjective well-being by introducing the interaction between resilience and poly-bullying. Statistical analyses were done using the software package SPSS 22.0.Students were considered victims of each form of bullying if they reported having suffered one of the behaviors indicated in the questionnaire at least once a week in the last 3 months. The remaining students were considered to not be involved in bullying. Students were also classified according to the number of experienced forms of bullying see , and 16.X2 = 9.175 (3), p = 0.057). A one-way ANOVA was calculated for subjective well-being and resilience by considering the different groups of victims. The analysis was significant for subjective well-being  and resilience . A follow-up analysis using Bonferroni correction showed that non-victims reported better subjective well-being and higher resilience levels than victims. The poly-bullying victimization group reported the poorest subjective well-being and the lowest resilience levels.As shown in It was hypothesized that resilience would moderate the relation between poly-bullying victimization and subjective well-being. To examine this hypothesis, a hierarchical regression analysis was conducted with subjective well-being taken as the criterion variable. Although gender was not statistically significant in the previous analyses, it was inputted together with poly-bullying victimization and resilience in Model 1. In Model 2, the product of poly-bullying victimization and resilience was inputted to test the interaction effect. The results of Models 1,2 of the hierarchical regression analyses are reported in 2 change: 0.010, p < 0.01).The results for Model 1 showed that gender had no significant effect on subjective well-being but revealed a significant main effect of poly-bullying victimization. That is, the participants who reported having experienced three or four forms of bullying were more likely to also report lower subjective well-being levels. Resilience was significantly and positively associated with subjective well-being. The participants who reported higher resilience levels also reported higher subjective well-being levels. The results in Model 2 showed that resilience moderated the association between poly-bullying victimization and subjective well-being, but the relation was very weak and accounted for only an additional 1% of variance in the participants’ subjective well-being  forms of bullying victimization were classified as “poly-bullying victims”. A substantial proportion of the participants 16.9%) reported being victims of poly-bullying. The prevalence rates in this study were higher than those previously found, when the incidence of poly-victimization did not exceed 11% % reporte,62. One Regarding the association between well-being and resilience among students exposed to several forms of bullying, the results fall in line with the expectations posed in Hypothesis 1. The results showed that the poly-bullying victimization group reported the poorest subjective well-being. The participants who reported having experienced three or four forms of bullying were more likely to also report lower subjective well-being levels. This finding coincides with previous research, which has shown that exposure to multiple forms of victimization has an impact on young people’s mental health, specifically their subjective well-being ,45,49. TThe regression analysis results indicated that resilience was significantly and positively associated with subjective well-being. The participants identified as non-victims reported better subjective well-being and higher resilience levels than victims. On the contrary, the poly-bullying victimization group reported the poorest subjective well-being and the lowest resilience level. These results confirmed Hypothesis 2 and fall in line with previous studies showing how young people who suffer victimization and poly-victimization had poorer mental health and lower resilience than those not victimized ,59.Finally, in line with previous studies ,58,59,63Nevertheless, conceptual discrepancies in the scientific community about the resilience concept make it difficult to evaluate and compare the research and operationalization results of the construct for measurement purposes . The preWhen interpreting these findings, certain limitations in our study need to be considered. First, the study sample was confined to university students from a region of central Spain. Thus, it is difficult to establish whether the study findings are generalizable to Spanish population in other regions. It would be appropriate to replicate these findings in other settings. Second, given the survey’s cross-sectional nature, it did not allow us to establish causal relations between the analyzed variables. Longitudinal studies need to be conducted to begin examining possible causal relations. Third, this study included only an evaluation of positive mental health. It is important for future research to analyze other forms of internalization and externalization problems as consequences of poly-bullying, e.g., suicidal or aggressive behavior, anxiety or depression. It would also be necessary to replicate and validate these results by exploring a wider range of individuals and contextual psychosocial variables, e.g., self-esteem, self-efficacy, cohesion and family communication or positive school experiences. Fourth, it is also important to point out the inherent limitations of asking participants to self-report their experiences of victimization. Thus, collection data from multiple informants rather than relying exclusively on self-reports would be beneficial in future studies.This study examined poly-bullying victimization in a sample of late adolescents. Those who were victims of poly-victimization reported more negative outcomes (worse subjective well-being and lower resilience levels) than the youths who experienced single forms of victimization or were not involved. Previous research and the present study reveal that negative outcomes from bullying victimization experiences differ according to the form of peer victimization and the number of experiences suffered. Although prevention and intervention efforts must deal with all victimization episodes, special attention should be paid to the youths suffering multiple forms of bullying victimization, and interventions need to be tailored to each form of victimization . Therefo