P < 0.0001) between the number of stages and the stage transition rate. The senescence tumor suppression factor for 20 non-reproductive cancers is remarkably consistent (0.0099±0.0005); however, five female reproductive cancers have significantly higher tumor suppression. The peak incidence rate for non-reproductive cancers occurs at a younger age for cancers with fewer stages and their carcinogenic stages are of longer duration. Driver gene mutations are shown to contribute on average only about a third of the carcinogenic stages of different tumor types. A tumor’s accumulated incidence, calculated using a two-variable  model, is strongly associated with intrinsic cancer risk. During both early adulthood and senescence, the pace of tumor suppression appears to be synchronized across most cancer types, suggesting the presence of overlapping evolutionary processes.The age-specific trend of cancer incidence rates, but not its magnitude, is well described employing the multistage theory of carcinogenesis by Armitage and Doll in combination with the senescence model of Pompei and Wilson. We derived empirical parameters of the multistage-senescence model from U.S. Surveillance, Epidemiology, and End Results (SEER) incidence data from 2000–2003 and 2010–2013 for The Cancer Genome Atlas (TCGA) cancer types. Under the assumption of a constant tumor-specific transition rate between stages, there is an extremely strong linear relationship ( Cancer incidence rates in adults generally rise exponentially with post-pubertal age, peak around 80 years, and then approach zero after age 100, although estimates are higher in centenarians if incidental tumors discovered at autopsy are included , 3.ASR(t) can be represented by probabilities of occurrence of successive independent stages and by a power function . Dr. DrTP53 t cancer . This isnd KRAS) , althougnd KRAS) .There are many alternatives to the multistage-senescence model of carcinogenesis. A review of carcinogenic models identified five mathematical model types, namely mutations, genomic instability, non-genotoxic mechanisms, Darwinian cell selection, and tissue organization . A well-The decline in cancer incidence rates in old age has been modeled by Cook et al.  for stomFinally, there is a limited potential to identify temporal trends in multistage-senescence model parameter values as seen in the five data sets consisting of the 1979–1983, 1989–1993, and 1999–2003 data from Harding et al. [The prostate cancer incidence rate is influenced by its detection by transurethral resection of the prostate and by prostate-specific antigen (PSA) . The useA generalized limitation of our analyzes of age-specific rate of cancer incidence is their cross-sectional nature. The age-specific incidences rates emanate from various groups of people, who may have different cancer risks for reasons other than age itself, and these reasons are not accounted for in the carcinogenesis models. Hanson et al.  point ouA multitude of carcinogenic models have been proposed in the scientific literature; however, the multistage-senescence model is unique in quantifying several important parameters of the age-specific incidence rate of different cancer types based solely on the number of development stages. The finding, perhaps controversial, that cancer-suppressive mechanisms counter the aging-dependent escalation in cancer complexity and incidence gives support to preventing cancer by augmenting extant mechanisms. The two-variable model gives us a useful tool to further investigate the biological mechanisms that drive specific cancers, such as those that are affected by ethnicity or ionizing radiation.https://github.com/canghel/cancer-incidence-v5.All statistical analysis was performed using software R version 3.6.0 . The fulThe methods to estimate incidence rates from person-years at risk for the older U.S population and to fit the multistage-senescence model were reproduced as closely as possible from Harding et al. . The ageThe incidence cases of 30 cancer types corresponding to TCGA cancer types  were extFor ages up to 85 years the SEER-provided population estimates were used. The U.S. Census 2000 and 2010 populations  for eachThe crude rate per 100,000 persons in each five-year age category were determined and the associated standard error. Although the assumption of constant 85+ population fractions over time may fail for the oldest populations, especially for males with higher mortality rates, the computed standard errors for the incidence rates in these populations are also large and hence these incidence rates to the multistage beta model fit. For the peak age of incidence and the peak rate of incidence, we restricted the values reported to ages less than 105 to avoid outliers.t in We fit the multistage-senescence model  by non-lBy taking the derivative of The total probability of each cancer type, the cumulative ASR, computed analogously for a is a multiplicative term [Parameter ive term , whose vThe number of driver mutations are taken from Table S6, of Iranzo, Martincorena , with thhttps://github.com/canghel/cancer-incidence-v5.Supplementary information is available in the online version of the paper. The full code and additional documentation at the time of publication, under MIT License, is posted in the GitHub repository at Supplementary Figure 1Supplementary TablesSupplementary Notes


Background: Necroptosis is closely related to the tumorigenesis and development of cancer. An increasing number of studies have demonstrated that targeting necroptosis could be a novel treatment strategy for cancer. However, the predictive potential of necroptosis-related long noncoding RNAs (lncRNAs) in lung adenocarcinoma (LUAD) still needs to be clarified. This study aimed to construct a prognostic signature based on necroptosis-related lncRNAs to predict the prognosis of LUAD.Methods: We downloaded RNA sequencing data from The Cancer Genome Atlas database. Co-expression network analysis, univariate Cox regression, and least absolute shrinkage and selection operator were adopted to identify necroptosis-related prognostic lncRNAs. We constructed the predictive signature by multivariate Cox regression. Kaplan–Meier analysis, time-dependent receiver operating characteristics, nomogram, and calibration curves were used to validate and evaluate the signature. Subsequently, we used gene set enrichment analysis (GSEA) and single-sample gene set enrichment analysis (ssGSEA) to explore the relationship between the predictive signature and tumor immune microenvironment of risk groups. Finally, the correlation between the predictive signature and immune checkpoint expression of LUAD patients was also analyzed.Results: We constructed a signature composed of 7 necroptosis-related lncRNAs . The signature could serve as an independent predictor for LUAD patients. Compared with clinicopathological variables, the necroptosis-related lncRNA signature has a higher diagnostic efficiency, with the area under the receiver operating characteristic curve being 0.723. Meanwhile, when patients were stratified according to different clinicopathological variables, the overall survival of patients in the high-risk group was shorter than that of those in the low-risk group. GSEA showed that tumor- and immune-related pathways were mainly enriched in the low-risk group. ssGSEA further confirmed that the predictive signature was significantly related to the immune status of LUAD patients. The immune checkpoint analysis displayed that low-risk patients had a higher immune checkpoint expression, such as CTLA-4, HAVCR2, PD-1, and TIGIT. This suggested that immunological function is more active in the low-risk group LUAD patients who might benefit from checkpoint blockade immunotherapies.Conclusion: The predictive signature can independently predict the prognosis of LUAD, helps elucidate the mechanism of necroptosis-related lncRNAs in LUAD, and provides immunotherapy guidance for patients with LUAD. Lung cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths worldwide . Lung cavia releasing damage-associated molecular patterns (DAMPs), cytokines, and/or chemokines in the tumor microenvironment, resulting in tumor-promoting or anti-tumor effects . Necroptosis is characterized by early loss of plasma membrane integrity, leakage of intracellular contents, and organelle swelling . Recent  effects . On one  effects . RIPK1 e effects . Consist effects . On the pression . What isvia functioning as competitive RNAs to influence the expression of target genes. lncRNA-107053293 was demonstrated to regulate necroptosis by acting as a competing endogenous RNA of miR-148a-3p  are non-coding RNAs with transcripts of more than 200 nucleotides. Growing evidence has ascertained that lncRNAs are involved in the progression and metastasis of NSCLC and were associated with the immune pathway . LINC017-148a-3p . The dep miR-185 . Based o miR-185 . NeverthIn this study, we constructed a novel predictive signature based on NRlncRNAs to forecast the prognosis of LUAD. We also validated its clinical value and confirmed that this signature can be used as a predictor of immunotherapy, which may offer a guiding function for clinicians.https://portal.gdc.cancer.gov/). Meanwhile, we obtained the corresponding clinical parameters of these patients and excluded patients with missing overall survival (OS) or poor OS (less than 60 days) to reduce statistical bias in this analysis.We downloaded the transcriptome RNA sequencing data of LUAD samples from The Cancer Genome Atlas (TCGA)  should conform to the standard of correlation coefficients (|Pearson R|) >0.4 and p <0.001. Then, we obtained 1,061 differentially expressed lncRNAs  after screening the synthetic data matrix by Strawberry Perl V-5.30.0 (https://www.perl.org/) and R software V-4.1.2 (https://www.r-project.org/) with limma R package.A list of 67 necroptosis genes was obtained from previously reported literature . The corp < 0.05). Subsequently, we performed least absolute shrinkage and selection operator (LASSO) Cox analysis  via the glmnet R package to screen out optimal lncRNAs associated with LUAD prognosis. This method aims to prevent over-fitting during modeling. Finally, a prognostic risk signature based on the optimal lncRNAs was established with the multivariate Cox (multi-Cox) regression analysis, and the risk score of every patient with LUAD was calculated based on the following formula:The entire 481 TCGA set of LUAD was divided into a train risk set and a test risk set randomly by the caret R package. The ratio was 1:1. The train set was used to construct a necroptosis-related lncRNA signature, and the test set and entire set were applied to validate the signature. Combined with the clinical information of LUAD in TCGA, we screened and obtained 40 NRlncRNAs linked to OS significantly by univariate Cox (uni-Cox) regression analysis (Coef(i) and Expr(i) represent the regression coefficient of the multi-Cox regression analysis for each lncRNA and each lncRNA expression level, respectively. The patients were stratified into low- and high-risk groups, with the risk score as the cutoff. Kaplan–Meier method and log-rank test were conducted to analyze whether there is a difference in the OS of LUAD patients between the low- and high-risk groups using the survival R package.via chi-square test. Uni-Cox and multi-Cox regression analyses were performed to explore whether the prognostic signature was a potential independent prognostic indicator for patients with LUAD, and the results were visualized with two forest maps. Several receiver operating characteristic (ROC) curves were generated, and the area under the ROC curve (AUC) was calculated by the survival, survminer, and timeROC R packages to validate the predictive value of the prognostic signature.We evaluated the prognostic value of the established risk signature between the model and the clinical characteristics We combined the risk score with the clinical variables of age, gender, N stage, T stage, M stage, and tumor stage to set up a nomogram for the 1-, 3-, and 5-year OS of LUAD patients by the rms R package. Correction curves based on the Hosmer–Lemeshow test were applied to illustrate the uniformity between the actual outcome and the signature prediction outcome.http://www.gsea-msigdb.org/gsea/index.jsp) to carry out GSEA and to identify significantly enriched pathways between the low- and high-risk groups. Values of p <0.05 and FDR <0.25 were considered the thresholds for statistical significance. The results were visualized by the gridExtra, grid, and ggplot2 R packages.We used gene set enrichment analyses (GSEA) software 4.1.2  was conducted for scoring LUAD-infiltrating immune cells to quantify their relative content via the “GSVA” package. The scores of immune cells and pathways in different groups are shown on multi-boxplots, respectively. Finally, we also made comparisons about the immune checkpoint activation between low- and high-risk groups by the ggpubr R package.To figure out the relationship between this signature and TIME, firstly, we calculated the infiltration values for TCGA-LUAD dataset samples based on 7 algorithms: XCELL , TIMER , QUANTISThe detailed flow diagram of our study is exhibited in via multi-Cox regression analysis. Of those lncRNAs, 6 lncRNAs were regulated positively by necroptosis genes in the Sankey diagram  was an independent predictor of OS in LUAD patients , T cell co-inhibition, T cell co-stimulation, and type II IFN response were higher in the low-risk group  significantly linked to the OS of LUAD patients to construct the necroptosis-related lncRNA signature. Among those lncRNAs, AC099850.3 has been reported to be highly expressed in tumors and closely related to the development and procession of NSCLC ; AC09985sis axis . Other lResearchers have demonstrated that necroptosis is strongly associated with tumorigenesis, tumor immune response, and poor prognosis , especiavia autocrine interleukin-6 (IL-6) production, and that blockade of the IL-6R/JAK1/STAT3 signaling pathway potentiated sensitivity to those EGFR TKIs in NSCLC cells  and immune functions  were mainly active among the low-risk groups, some of which were closely linked to necroptosis. Necroptotic cells can provide both tumor-specific antigens and inflammatory cytokines to DCs for antigen cross-priming which activates cytotoxic CD8 systems . Wang X trophils . These rtrophils . Our fintrophils . In additrophils . Therefovia more external experiments in our following work.However, our research has several limitations and shortcomings. Firstly, it was better to include more clinical databases for external validation. Secondly, the underlying molecular mechanisms of the NRlncRNAs in LUAD should be further validated by experiments. Thus, we will recollect and expand clinical samples and attempt to validate the accuracy of this model In conclusion, the necroptosis-related lncRNA predictive signature can independently predict the prognosis of LUAD patients, helps elucidate the process and mechanism of NRlncRNAs in LUAD, and provides immunotherapy guidance for patients with LUAD, but it still needs further experimental verification in the future.


The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets. The number of publicly available proteomics datasets is growing rapidly, but a standardized approach for describing the associated metadata is lacking. Here, the authors propose a format and a software pipeline to present and validate metadata, and integrate them into ProteomeXchange repositories. ProteomeXchange (PX) is a consortium of proteomics resources, including the PRIDE database2, PASSEL and PeptideAtlas3, MassIVE4, jPOST6, iProX7, and Panorama Public8. As of July 2021, over 27,000 datasets have been submitted to PX data repositories. PX datasets cover the whole spectrum of protein mass spectrometry (MS) analytical methods and experimental designs, which enable biologists and clinicians to study different aspects of the proteome. In parallel to the generalization of data deposition, reuse of public datasets is becoming increasingly popular. However, thus far, data reuse has largely been limited to benchmarking studies and applications related to peptide and protein identification, with resources such as PeptideAtlas3 and GPMDB9 systematically reanalyzing data from PX10. Recently, new efforts like ProteomicsDB11, MassiVE.Quant4, and Expression Atlas12 have started to include reanalyzed quantitative public datasets to present baseline and differential protein expression. However, the scalability and broad reuse of public quantitative experiments have been limited by the lack of sample metadata annotation, which unambiguously associates the samples included in each dataset with the corresponding data files14.The amount of proteomics data in public repositories is growing at an unprecedented rate1. The files included in each dataset are, on one hand, the output of the corresponding instrument , and on the other hand, the processed results, which can be represented, e.g., in standard file formats such as mzIdentML15 or mzTab16. Currently, all PX partners mandate two types of information for each dataset: a general dataset description and the files containing the different required data types. Unfortunately, the experimental design and sample-related information are frequently missing in the datasets or are stored in ad hoc ways and/or formats1. Information about the biological samples such as the analyzed organ, tissue, disease, or cell line, and the links between the samples and the corresponding data files are often lacking.Since 2012, PX resources have been capturing a general dataset description, including the dataset title, description, instrument, protein modifications included in the search, and submitters/principal investigators, among other data17 and MAGE-TAB (MicroArray Gene Expression Tabular)18, which are used in metabolomics and transcriptomics, respectively. As of May 2021, ArrayExpress has stored over 74,000 functional genomics datasets in the MAGE-TAB format19. In both formats, a tab-delimited file is used to annotate the sample metadata and link the metadata to the corresponding data files. While MAGE-TAB was originally designed for microarray experiments, it has been successfully adapted to high-throughput RNA-sequencing and single-cell RNA-Seq experiments20.Sample-related metadata and their relationship with the data files are well captured in two widespread file formats called ISA-TAB21. We have also developed general guidelines about what information needs to be encoded in MAGE-TAB to improve the reproducibility and enable the reanalysis of proteomics datasets. In addition, we have crowdsourced the annotation of over 200 existing public datasets according to these guidelines, covering different analytical methods and experimental designs. Finally, we have developed an ecosystem of tools to validate MAGE-TAB-Proteomics files and integrate the metadata in the PRIDE database, the most popular PX resource. The full specification document describing all aspects of MAGE-TAB-Proteomics version 1.0, the current implementations, as well as application examples, is available at the PSI website (https://psidev.info/magetab).Here we introduce an extension and implementation of the MAGE-TAB format for proteomics (MAGE-TAB-Proteomics). The format has been developed in collaboration with the Proteomics Standards Initiative (PSI), the organization in charge of developing open-standard formats in the fieldMAGE-TAB encodes the sample metadata annotations and the information linking the metadata to the corresponding data files in two different files: the Investigation Description Format (IDF) and the Sample and Data Relationship Format (SDRF). In the following, we describe how we adapted these formats to the specific needs of proteomics.http://proteomecentral.proteomexchange.org/schemas/proteomeXchange-1.4.0.html), an XML file format that captures equivalent information to the ones included in IDF, making both files easily exchangeable , while most of the data-file properties are included in PSI-MS-controlled vocabulary (https://www.ebi.ac.uk/ols/ontologies/ms) and the PRIDE ontology (https://www.ebi.ac.uk/ols/ontologies/pride).All the properties in the SDRF must be encoded as ontology terms, whereas the values of the properties can be encoded as ontology terms, numerical values, or free text. To facilitate the annotation, validation, and processing of SDRF files, a list of ontologies has been defined that can be used for encoding each property. For example, most of the sample properties are included in the Experimental Factor Ontologycharacteristics . Each data file also has a unique identifier (assay name), and every file property has the prefix comment . Finally, the variables under study must be specified with the prefix factor value . The MAGE-TAB-Proteomics specification defines the minimum information that should be provided for every sample and data file (https://github.com/bigbio/proteomics-metadata-standard/raw/master/psi-document/HUPO-PSI-MAGETAB-Proteomics_latest.docx). For all proteomics experiments, the following properties must be provided: organism, organism part, and biological replicate accession. For every data file, the following properties are required: fraction identifier, technical replicate accession, label (in the case of labeling methods), and data-file name. Biological and technical replicates should be explicitly included using the terms characteristics[biological replicate] and comment[technical replicate], respectively , and every sample property is encoded using the prefix ely Fig. . The biocharacteristics[cell line]) is a recommended metadata item for cell-line experiments; for every human dataset, the disease under study should be provided in the field characteristics[disease] and the control samples should be labeled with the value “normal”.A second category of fields includes information that is not mandatory but recommended. Each PX repository can define which of the recommended fields must be provided in their resource, depending on the experiment types. The current PX templates request the submitters to provide the following properties for every data file: instrument model, cleavage agent, fragment-mass tolerance, precursor-mass tolerance, and mass modifications  and artifactual modifications considered in the analysis). Most of the values of these properties can be encoded as a combination of multiple key/value pairs . We believe that this represents the minimum set of information that is necessary for practical terms for understanding and re-using MS-based proteomics datasets. Furthermore, other properties such as labeling can be provided. Importantly, the set of mandatory properties can be readily expanded in case the PX community decides to extend its metadata requirements in the future. For now, we have defined additional templates  labeling), multiple samples can be related to the same data file Fig. . In thescomment[fraction identifier] = 1).When fractionation is used, the sample information should be repeated for each data file. A property called fraction identifier is used to make clear which fractions correspond to a given data file  set up a collective effort to annotate existing PX datasets using MAGE-TAB-Proteomics14. Volunteers from multiple institutes joined the task of annotating, discussing, and improving the SDRF format, openly and collaboratively. Each annotator created an issue in GitHub about a project of interest, forked the main project repository (https://github.com/bigbio/proteomics-metadata-standard), and annotated the corresponding dataset locally in their computers. Then, a pull request was submitted to include the added annotations. Prior to approval, an independent group of reviewers checked that the proposed SDRF conformed to the guidelines. This collaborative peer-review system allowed the identification of potential issues in the file format, which is now compatible with the main MS experiment types. Additionally, a file validator was developed to automatically perform a semantic and structural validation of created SDRF files (see next section for details).Crowdsourcing has been successfully applied to address key problems in bioinformaticsAs of July 2021, over 200 public datasets have been annotated, covering a broad spectrum of organisms, enrichment/fractionation strategies, quantification approaches, and data-acquisition methods Fig. . For moshttps://metacpan.org/release/Bio-MAGETAB). In principle, Bio-MAGETAB can also be used to validate MAGE-TAB-Proteomics files because they are valid MAGE-TAB files. However, Bio-MAGETAB cannot perform extra validations specific to proteomics data, especially the SDRF-Proteomics rules. To enable the validation of the SDRF-Proteomics of MAGE-TAB-Proteomics files, we implemented two libraries in Java and Python (see below). After validation, parameters such as cleavage agents, post-translational modifications, and mass tolerances (precursor and fragment) are translated into MaxQuant and OpenMS parameters in their corresponding configuration files.To facilitate automatic data analysis and reuse, we developed a set of software libraries that enable the validation and conversion from MAGE-TAB-Proteomics format to parameter files Fig. . For trahttps://github.com/bigbio/sdrf-pipelines), enables the validation of the structure and also of the semantic rules applied to SDRF. The sdrf-pipelines package can be installed from different package managers like BioConda25 or BioContainers26. It validates the files according to the different experiments and data types, as defined in the templates. For example, if the template corresponds to a human dataset, the software validates that the sample metadata comply with the human template . In addition, sdrf-pipelines allows users to convert SDRF files to configuration/input files of other popular proteomics analysis tools such as MaxQuant27, OpenMS28, and MSstats29, to facilitate the automation of dataset reanalyses (https://github.com/bigbio/proteomics-metadata-standard/wiki).The newly developed Python package, called sdrf-pipelines (https://github.com/bigbio/jsdrf) we developed also enables the validation of SDRF files. For example, the library can validate that the SDRF contains each sample and data-file relationship, labeling information, fraction identifiers, and the sample and data accessions. It also includes a generic data model that can be used by Java applications to validate and handle SDRF-Proteomics files.The jSDRF Java library (30 (https://github.com/bigbio/proteomics-metadata-standard/wiki/Annotating-Sample-terms-using-OntoMaton), which allows the annotation of Google spreadsheets using ontology terms coming from the Ontology Lookup Service (OLS)31. Submitters can search ontology terms in OLS and add them to the sample-property columns. The Google spreadsheets provide the functionalities to copy ontology terms across samples, and to add or remove columns and samples. New versions of the tool will include the possibility to add protein modifications, online validation, and loading existing templates among others.While the PX resources have not yet developed any tool to create SDRF-Proteomics files, existing tools for transcriptomics MAGE-TAB and ISA-TAB can be used to facilitate the process. At the moment of writing, we recommend OntoMaton2  and a video that introduces the file format (https://www.youtube.com/watch?v=TMDu_yTzYQM).Resources such as MassIVE.Quant, ExpressionAtlas, and ProteomicsDB have recently started to systematically reanalyze proteomics public quantitative datasets34. As an initial step in this direction, the PRIDE group has started to reanalyze proteomics datasets represented in SDRF and integrates them into the multiomics resource Expression Atlas .MAGE-TAB-Proteomics will facilitate the integration and annotation of proteomics studies, thereby enhancing their discoverability, reproducibility, and reusability. It holds great potential to facilitate the use of standardized workflows for the automated reanalysis of currently available and future annotated public proteomics datasets. This will increase the inherent value of proteomics datasets by making them more amenable for large-scale meta-analysis, systems biology, and multiomics projects that can take advantage of large hardware resources in cloud-based environments. The proposed standard metadata representation will also facilitate the development of common submission systems to streamline the deposition of multiomics datasets to resources for different types of omics data. It may even foster the creation of new multiomics resources, enabling submission, validation, and visualization of multi-omics data in one placehttps://github.com/bigbio/proteomics-metadata-standard/blob/master/sdrf-proteomics/use-cases-under-development.adoc). Researchers interested in use cases that are currently not supported are encouraged to bring them to our attention by creating an issue in GitHub using the above hyperlink. Analogous to the implementation process of other PSI formats, we aim to engage software tools and developers to support the MAGE-TAB-Proteomics format as an input file for data processing and as output file for the dataset-submission process. Notably, the MAGE-TAB-Proteomics format has already triggered interest in the MS metabolomics field. First-metabolomics data have already been annotated using MAGE-TAB-Proteomics (Table 35 have been added to the annotation repository, underscoring the open, collaborative, and community-driven approach of this project. In this spirit, we invite all interested parties to join the MAGE-TAB-Proteomics initiative.Expanding the MAGE-TAB-Proteomics format to additional use cases and more types of MS-based experiments, including metaproteomics and xenograft proteomics experimental designs, is currently under discussion (cs Table  and moreSupplementary Information


Effects of chemical substitution and interstitial dopants are clarified. We also discuss how data-driven approaches are used for studying multinary systems. Chemical composition can be optimized with fewer trials by the Bayesian optimization. We also present a data-assimilation method for predicting finite-temperature magnetization in wide composition space by integrating computational and experimental data.First-principles calculation based on density functional theory is a powerful tool for understanding and designing magnetic materials. It enables us to quantitatively describe magnetic properties and structural stability, although further methodological developments for the treatment of strongly correlated 4f electrons and finite-temperature magnetism are needed. Here, we review recent developments of computational schemes for rare-earth magnet compounds, and summarize our theoretical studies on Nd  T. T101].  formula . Indepen5.Although first-principles calculation is a powerful tool for the development of magnetic materials, there are remaining problems to be fixed. As we discussed, accuracy of the current theoretical methods is still limited. Magnetism is essentially outcome of quantum many-body effects. In particular, treatment of rare-earth 4f electrons is to be improved. Quantitative description of magnetism and stability at finite temperature is also under development.Another direction is materials exploration. First-principles calculation, coupled with machine-learning techniques, has developed rapidly in the last decade. Machine learning is particularly efficient in the optimization of chemical composition. When completely new material is concerned, however, current techniques are not sufficient. Methods for crystal structure prediction and finding new phases is actively investigated, and application to magnetic materials is a hot topic ,103. Dat


Microfluidics is an emerging technology that can be employed as a powerful tool for designing lipid nano-microsized structures for biological applications. Those lipid structures can be used as carrying vehicles for a wide range of drugs and genetic materials. Microfluidic technology also allows the design of sustainable processes with less financial demand, while it can be scaled up using parallelization to increase production. From this perspective, this article reviews the recent advances in the synthesis of lipid-based nanostructures through microfluidics . Besides that, this review describes the recent microfluidic approaches to produce lipid micro-sized structures as giant unilamellar vesicles. New strategies are also described for the controlled release of the lipid payloads using microgels and droplet-based microfluidics. To address the importance of microfluidics for lipid-nanoparticle screening, an overview of how microfluidic systems can be used to mimic the cellular environment is also presented. Future trends and perspectives in designing novel nano and micro scales are also discussed herein. The development of lipid nano-microsystems has initiated in the middle of the last century with Alec Bangham. He developed the thin-film method in a laboratory protocol to produce lipid structures that mimic the lipid bilayer . These sLiposomes are self-aggregated colloidal systems in a bilayer structure composed of phospholipids, forming a spherical vesicle with an aqueous core . Convent®  was the first U.S. Food and Drug Administration (FDA)-approved PEGylated nano-liposomes [Later, at the end of the last century, liposomes were investigated as drug delivery systems due to their amphiphilic nature, allowing the incorporation and conventional encapsulation of different drugs. Doxiliposomes  to treatiposomes . Since iiposomes ,12. In piposomes . Gene thiposomes ,15. The iposomes ,17. Thesiposomes ,19.®  in 2018 by the FDA, which is small interfering RNA (siRNA) LNPs for amyloidosis treatment, LNPs have been extensively studied as potential gene carriers for vaccines. In this sense, it is possible to highlight the recently approved Pfizer COVID-19 vaccine in the US [After the approval of Onpattron the US ,22. Evenn the US . In thisMicrofluidics is defined as the science and technology that investigate and/or apply the study of behaviors of fluids, controlled fluid manipulations, and the design of such devices or systems that can reliably perform such tasks in microchannels with typical dimensions of micrometers ,25. CommThe most significant advantage of synthesis processes using microfluidics relies on developing sustainable technologies with less financial demand. Microchannels also can be parallelized for amplification purposes and increased production. This fact reduces costs and time for translation technology from bench to bedside . AccordiIn the field of pharmacology and pharmacy, there is a myriad of applications using lipids and microfluidic technology, as lipid nano-micro sized structures for drug and gene delivery and revolutionary platforms for lipid-based nanocarrier assays. This review will describe the recent advances in the production of lipid nanostructures and their application. The main characteristics of lipid nanostructures discussed in this review are illustrated in This paper also introduces the microfluidic approaches to synthesize lipid micro-sized structures, as GUVs, and new strategies for the controlled release of lipid nanostructures from microgels produced by droplet-based microfluidics. This review will also give some insights into recent trends on microfluidic platforms for lipid nanoparticle screening and some brief and relevant information about the main manufacturing techniques and materials regarding lipid nano and microstructures synthesis.There are mainly two methods for producing lipid nanoparticles: top-down and bottom-up processes ,32. Top-In most cases, microfluidics is based on a laminar flow regime, defined by a low Reynolds (Re) number ,35; see Box 1Reynolds (Re) number: A dimensionless number that indicates the type of flow, laminar or turbulent, through the ratio between inertial and viscous forces, defined by the multiplication of the density of the fluid, characteristic length, and velocity, divided by the viscosity of the fluid.Laminar regime: Fluid dynamics is characterized by the smooth flow. On a flat surface, the flow occurs in parallel lamellae. Low Re numbers describe it , and the mixing process is governed by the phenomenon of molecular diffusion and characterized by highly predictable kinetics.Chaotic advection: The mixture phenomenon where the advected particles suffer repeatedly transverse vortices, leading the fluids to wrap into one another, commonly called “stretching and folding”.Regarding the laboratory scale, polydimethylsiloxane (PDMS) and soft lithography are the most common microdevice material and manufacturing techniques because of their low cost and being suitable for rapid prototyping ,40,41. FBox 2Microfluidic devices materials: The basic manufacturing materials need to be the suitable with the most common fabrication techniques. The main materials include PDMS ,44, polyMicrofluidic devices manufacturing techniques: There are several techniques capable of manufacturing microdevices. It is worth mentioning photo ,51 and sIn general, lipid nanostructures can be formed by mixing two inlet streams containing lipids in a water-soluble solvent and another central inlet with an aqueous solution. As the streams flow in parallel, the mixing process is initiated, and polarity changes, favoring the lipid autoaggregation in a reproducible manner ,64. The HFF can be mainly applied through 2D or 3D hydrodynamic flow-focusing (HFF) devices using three inlet streams with a cross-shaped geometry A. In thiCA-M method was created to overcome the productivity limitation of HFF, allowing the production of lipid nanostructures at higher flow rates and improving the mixture efficiency ,73. BarrThis method produces highly monodisperse droplets using immiscible phase fluids (water/oil emulsions and liquid/gas). Droplet-based microfluidics may be used to produce micro-sized particles, such as GUVs and microgels ,81. In tMicrofluidic research has been focused on producing efficient drug and gene carriers. The recent advances in medicine have driven discoveries of potential delivery carriers. The main types of nanocarriers synthesized by microfluidics will be discussed .A liposome’s amphiphilic nature allows the transportation of soluble and low-solubility compounds, which can be entrapped in the aqueous core and bilayer membranes. In general, the encapsulation of drugs into liposomes can be used to (i) control drug release and modify biodistribution; (ii) protect drugs from in vivo degradation; (iii) enhance solubility and bioavailability; and (iv) deliver to a specific tissue using specific ligands ,107. As In this perspective, Kastner et al.  designedIn another study, SHM devices were used to produce a liposomal formulation for curcumin (CUR) entrapment, a hydrophobic drug, which can be used as chemosensitizers in cancer treatments. The authors noted that CUR-loaded liposomes given at a single and low dose exhibited a significant anticancer effect. Additionally, they found that the antitumor efficacy in tumor-bearing EMT6 and B16F10 mice could be increased by combining CUR-loaded liposomes with cisplatin (CIS) and alkylating antineoplastic drugs at a relatively low dose. The drug-loading efficiency found was around 17 wt%, a superior loading value (700-fold) than other liposomal systems reported in the literature. Besides that, the treatment with CUR reduced CIS nephrotoxicity .Another potential application is shown by Gkionis et al.  Figure B, who prControlling the pharmacokinetic drug distribution profile to improve therapeutic efficacy in liposome drug delivery systems is still a challenge. The pharmacokinetics and tissue distribution of the liposomes may affect their therapeutic effect and toxicity . One altFocus on translating liposomes from the bench to the clinic, Forbes et al.  used a mThe proper delivery of genetic material to cells can be difficult due to several hurdles. In general, naked nucleic acids delivery (DNAs and RNAs) are susceptible to rapid elimination from circulation due to several factors, such as biological barriers (nucleases and intracellular compartments), the size, and the negative charge of the genetic material ,110. TheLNPs follow the non-bilayer theory and are not designed to have multilamellar structures, which induces the proper release of genetic material inside cells and increases the biological response. This feature is modulated by changing the lipid composition, which is carried out in a one-step synthesis ,113,114.A typical plasmid DNA (pDNA) needs to be substantially condensed to achieve a suitable size for proper delivery, which relies on about 100 nm or less sizes. To encode the therapeutic protein, DNA has to reach the cell nucleus to allow access to the transcriptional machinery ,110.To deliver pDNA safely, Balbino et al.  produced®  microfluidic device to incorporate pDNA pWRG/c7d11 in LNPs. The results showed that LNPs could successfully be applied as DNA prophylactic Andes and Zika virus DNA vaccines capable of producing elevated neutralizing antibodies in rabbits and nonhuman primates.Kulkarni et al.  studied To generate pDNA (GL3)-LNPs, Quagliarini et al.  used a YApplying a T-junction mixer, Kulkarni et al.  analyzedAlthough DNA-LNPs are gaining attention, the necessity to cross the membrane barrier of the nucleus invigorates the advantages of RNA-LNPs .The main difference between RNA and DNA delivery is related to the intracellular local delivery site. The RNAs mostly used for therapies, such as messenger RNA (mRNA), silencing RNA (siRNA), microRNA (miRNA), and self-amplifying mRNA (SAM), only need to reach the cytoplasm to promote protein expression or inhibition ,110,116.Despite being a simple molecule structure, the non-replicating messenger RNA (mRNA) presents limited in vivo stability and activity due to the limited duration of protein expression inside cells. Possible toxicity also can arise from the protein expression at off-target sites, leading to unwanted protein expression ,118. HensiRNA and miRNA are double-stranded RNA molecules that silence target genes via RNA interference (RNAi) and enable specific silencing of virtually any gene in the human genome . After r®. This effect is due to the ability of ionizable LNPs to adsorb apolipoprotein E in the circulation, giving rise to a natural targeting ligand that binds with high affinity to the low-density lipoprotein receptor that facilitates the release of siRNA into the cytoplasm by endocytosis [The capacity of siRNA to silence hepatic genes in vivo has been well-established since the approval of Onpattroocytosis ,125. So ocytosis ,128,129.ocytosis  synthesiAnother type of therapeutic RNA that has gained attention using microfluidic technology for gene therapy is mRNA ,131. PatAll the advantages in RNA-like LNPs show massive research on technologies to improve RNA-LNPs, which leads to more vaccines and therapeutic candidates in clinical trials ,113,115.Over the years, the focus of nanoparticle design has evolved toward complex single delivery systems that combine multiple functionalities within the same nanoscopic architecture. These hybrid structures, such as core-shell lipid-polymer hybrid nanoparticles, have emerged as a robust and promising delivery platform. These integrated systems, also known as core-shell nanoparticles, have been introduced to mitigate some limitations associated with liposomes and biodegradable polymeric nanoparticles. The lipid shell presence overcomes the main limitations of polymeric nanoparticles, such as the burst release and reticuloendothelial absorption ,133,134.From this perspective, microfluidics has gained substantial attention to prepare hybrid nanoparticles as an alternative to the one-step bulk method. Typically, the convectional one-bulk approach combines single-step nanoprecipitation and self-assembly processes. Thus, this review section focuses on the current research trends on core-shell nanoparticles produced by microfluidics applied for drug and gene delivery.Microfluidic-based core-shell nanoparticles have delivered different nucleic acids such as siRNA, mRNA, and pDNA ,96,97. WEven though microfluidic-based lipid/polymer hybrid nano assemblies demonstrated promising clinical application delivery results, achieving the biomanufacturing requirements is still a challenge. Many current nanoformulation methods, such as bulk mixing and hydrodynamic focusing, still require continuous, scalable, and reproducible technologies to increase their limited yield. In this context, robust self-assembly technologies that decrease heterogeneity and batch-to-batch variation are still needed. In this perspective, flash technologies facilitate nanoparticles’ self-assembly and formulation in a low-cost, high-throughput, and controllable manner. Flash nano complexation (FCN) and flash nanoprecipitation (FCP) involve rapid mixing in confined impingement jets mixers (CIJM) or multiple inlet vortex mixers (MIVM)  Figure .FCN and core-shell nanostructures have been combined for oral gene delivery application. For instance, Nie et al.  developeRecent studies also showed the potential of microfluidics to produce core/shell particles to combat the SARS-CoV-2 virus. Yang et al.  developeAnother alternative to produce mimicking natural membranes is by combining lipid nanostructures with molecules presented in mammalian membranes or extracellular vesicles. The same processes used to assemble lipids to form liposomes can be used for biomimetic nanovesicles assembly, in which the lipid alcoholic stream is hydrodynamically focused on the lateral aqueous streams containing the transmembrane proteins. In this case, the amphiphilic nature of the transmembrane proteins is the driving force for its insertion into the lipid bilayer.Using this strategy, Molinaro et al.  producedAnother approach to form biomimetic vesicles is based on the combination of exosomes with conventional lipid nanostructures. Exosomes are a subtype of extracellular lipid nanostructures naturally released by cells. Depending on the cell type and cultivation culture, different compositions  can be found in the exosomes. Exosomes can be used as a natural lipid system to be applied as a drug delivery system . Similarco-glycolic acid) (PLGA) nanoparticle (PLGA-NPs) coating with EM or CCM (Different use of the exosome membrane (EM) or cancer cell membrane (CCM) was designed in a core/shell nanostructure. In this case, Liu et al.  developeM or CCM D. As a cLiposomes are self-assembled phospholipid vesicles with great potential in fields ranging from targeted drug delivery to artificial cells. GUVs are vesicles that are >1 μm in diameter . The large size of the GUVs and their low curvature enable us to analyze them individually through optical microscopes. Lipid vesicles can be composed of a single lipid component or mixtures . Vesicles have also been made using many different surfactants besides phospholipids . The proThe most popular methods for GUV production are lipid film hydration , the eleMicrofluidic-based fabrication may be applied to form uni- or multi-compartment vesicles ,157 FigA. Cell mMulticompartment synthetic cells have been created to encapsulate SUVs and LUVs in a high-throughput manner. Weiss et al.  showed tIn another study, Elani et al.  developeAnother potential application is shown by Yandrapalli et al. , who preThe microfluidic production of a W/O/W double emulsion as templates for the formation of GUVs aiming at food and/or pharmaceutical applications was demonstrated by Michelon et al. . Glass-cSimilarly, the microfluidic production of the GUVs based on the W/O/W double emulsion templates can also be achieved by 2D-PDMS microdevices, using a cross-junction with five input channels and one output channel . In thisDroplet microfluidics has also been reported for multicompartment vesicles production ,159,161.The microfluidic approach for generating giant liposomes has many advantages compared to traditional methods (gentle hydration and electroformation). Among these advantages, it is possible to cite the high-throughput production of monodisperse solutions, high encapsulation efficiency, and asymmetric lipid composition, typically found in biological cell membranes . One disBeyond the classical synthesis of nano and micron-sized lipid structures, different microfluidic microdevices are being explored to study their biological performance in in vitro assays.Another challenge in lipid-based nanocarrier design and application is the development of strategies to in vivo sustained release. In general, conventional drug administration requires high dosages or repeated administration to achieve a therapeutic effect. These approaches can result in severe side effects, toxicity, lower overall efficacy, and patient compliance to the treatment ,164. In Different scaffolds and liposomes have been designed to improve the immobilization, release, and uptake of therapeutic genes ,170,171 Yang et al.  developeAnother study used a continuous two-step glass-capillary microfluidic technique to produce a multistage oral delivery system (liposome-in-microgel). The hybrid system is composed of a chitosan-coated insulin-loaded liposome (InsLip-CHT) and an enteric polymer, hydroxypropyl methylcellulose acetate succinate, which forms the microgel. In the first step, unilamellar Ins-loaded PEGylated liposomes were developed using a co-flow microfluidic device, 144 ± 23 nm, PDI of 0.1, ZP of −0.5 mV, and EE equal to 91%. Before the microencapsulation, InsLip was coated with CHT to improve their mucoadhesion, 363 nm, PDI of 0.3, and ZP of +23 mV. Then, the InsLip-CHT encapsulation into microgels was performed by W/O/W double emulsion using a flow-focusing microfluidic device forming a hybrid system of 19 μm in diameter. In vitro release assay showed Ins release starting above the pKa of microgel, pH 6.8, demonstrating efficient protection under gastric acidic conditions . CollectIn gene delivery, non-viral vectors have also been associated with hydrogels to form scaffold-gene non-viral carriers’ platforms to deliver genetic materials locally in vivo. The hydrogel reservoir system can increase transfection efficiency and allow long-term gene expression. Another advantage is that the controlled release of non-viral vectors may decrease their cytotoxicity and stability typically caused by the direct contact of cationic vectors and cell membranes. Even though gene-loaded liposome-in-hydrogel are often studied ,170,171 Microfluidic technology has also emerged as a tool to improve the complexity of the cellular environment. Microfluidic technology is well-known for many advantages, such as a quick and accurate response, portability, the capacity of handling minimal volumes, and low-cost process need. In drug/gene-delivery applications, all the interaction between drugs and therapeutic agents occurs within a microenvironment . In contIn vitro microfluidic models have been developed to mimic better normal and unhealthy tissue than conventional 2D or 3D culture systems. These microfluidic cell culture platforms, known as organ-on-a-chip (OOC), are designed to mimic mechanical, biochemical, and functional properties similar to the in vivo microenvironment. Microdevices with these functions can operate dynamically, variating the fluid flows and mechanical cues that cells experience in organs and tissues, mimicking physiological functions . This reWang et al.  designedThe same research group developed a TOC model that allows different-sized spheroid loading, formation, long-term cultivation, and drug evaluation. The TOC model provides a platform for screening the anticancer efficacy of liposomes (Lip). In this study, they investigated four different liposome formulations, including PTX-loaded PEGylated liposome (PEG-Lip), FA-Lip, cell-penetrating peptide TAT modified liposome (TAT-Lip), and FA and TAT commodified liposome (FA-TAT-Lip). When compared to all nanocarrier formulations, FA-TAT-Lip had the highest cytotoxicity and tumor accumulation. Adopting FA-TAT-Lip as the main nanocarrier, the tumor spheroid growth curve showed better tumor inhibition capability using lower flow rates. In addition, bigger tumor spheroids decreased the liposome binding and uptake efficiency. TOC model results corroborate better with the in vivo mouse assay than the 2D monolayer cell culture and 3D tumor spheroid models .Despite all efforts in designing different types of OOC, few studies are related to the application of OOC in gene therapy studies ,198,199.®  and FuGene® 6  pEGFP-N1. The platform was used to evaluate simultaneously five transfection conditions generated by a chaotic serial dilution generator (lipoplex dilution from 100–0% with 25% steps). This analysis process helps to save large volumes of reagents while guaranteeing more precise control over cell behavior. The authors reported that this platform could be used to quantitatively assess the transfection efficiency and cytotoxicity in a spatio-temporally tunable microenvironment with a real-time investigation.In this perspective, Giupponi et al.  developeLi et al.  investigFocusing on automated transfection process development, Raimes et al.  designedIn general, microfluidic culture devices can explain how microenvironmental factors may influence tumor cell responses to anticancer therapies. Thus, microfluidic tools have enabled strategies toward whole OOC systems, which may transform the process of drug screening in the future. This new approach helps pre-clinical testing drug substances and toxicological studies while also providing more reliable pre-clinical pharmacokinetic and pharmacodynamic data.Microfluidics has shown to be an emerging technology for the synthesis of nano and microstructured lipid-based systems. It is possible to develop bottom-up processes from simple liposomes to engineered and tailored nano or microstructures, such as core/shell, biomimetic vesicles, and giant liposomes. Microfluidic publications over the last five years have demonstrated the advances in microfluidics for lipid-based structure synthesis; however, successful market products in the pharmaceutic industry are still scarce. Although microfluidics production is still a challenge, it is important to highlight the fast development of lipid mRNA vaccines in 2020 against SARS-CoV-2. mRNA vaccines have demonstrated promising results regarding safety and efficacy. Pfizer/BioNTech and Moderna used a microfluidics-based production method to complex the (N1-methyl-pseudouridine) modified mRNA and LNPs ,208. It ®  platforms. However, microfluidic technologies still need to be improved to expand production to make their industrial application feasible. In this sense, microfluidic platforms can be parallelized to replicate the conditions of single to multiple devices. Nevertheless, scalability remains challenging since it is difficult to guarantee the same operating conditions for hundreds of similar devices [Microfluidic platforms that allow a high throughput and flow rate capacity are already available in the market, such as the NanoAssemblr devices ,32. As aBefore thinking about the industrial scale-up processes, the structural morphology of lipid nano and microstructures should be well elucidated. In this sense, current microfluidic designs can be improved using real-time characterization techniques to monitor any change during the lipid structure formation. Computational modeling and in silico experiments can also bring us trenchant insights into nanoformulation designing and particle interactions. In this sense, recently studies have used advanced on-chip characterization techniques such as DLS, confocal Raman microscopy, X-ray absorption spectroscopy , SAXS 2, and SANAlthough many studies aim to develop lipid systems, microfluidic production is still a technological challenge. This review shows that microfluidics’ development of a biomedical solution involves different research fields such as chemical, biology, pharmacy, and engineering. The development of new solutions may often require further steps for pre-clinical tests such as mimicked systems to simulate in vitro delivery and the development of novel microfluidic devices for drug/gene screening. Searching for newly designed lipids is also necessary while combining specific ligands and biomaterials with other materials. It is expected that more effective and scalable self-assembly strategies in this multidisciplinary field will lead to more nano and micro lipid structures emerging in the next decade.


Results of the early-stage rTMS intervention revealed that right nucleus accumbens played the role as a central hub, and VC was a potentially novel rTMS target region during adolescent schizophrenia. Moreover, the EC of right nucleus accumbens shell and orbitofrontal cortex was demonstrated to be a potential biomarker. To our knowledge, this was the first resting-state fMRI study using GCA to assess the deficits of a visual-reward neural pathway and the effectiveness of rTMS treatment in MAM rats. More randomized controlled trials in both animal models and schizophrenia patients are needed to further elucidate the disease characteristics.The aim of the current resting-state functional magnetic resonance imaging (fMRI) study was to investigate the potential mechanism of schizophrenia through the posterior–anterior cerebrum imbalance in methylazoxymethanol acetate (MAM) rats and to evaluate the effectiveness of repetitive transcranial magnetic stimulation (rTMS) as an early-stage intervention. The rats were divided into four groups: the MAM-sham group, vehicle-sham group, MAM-rTMS group, and vehicle-rTMS group. The rTMS treatment was targeted in the visual cortex (VC) in adolescent rats. Granger Causality Analysis (GCA) was used to evaluate the effective connectivity between regions of interest. Results demonstrated a critical right VC–nucleus accumbens (Acb)–orbitofrontal cortex (OFC) pathway in MAM rats; significant differences of effective connectivity (EC) were found between MAM-sham and vehicle-sham groups (from Acb shell to OFC:  Although the mechanisms of schizophrenia remain unclear, altered brain maturational processes can be found in schizophrenia patients, suggesting schizophrenia as a potential neurodevelopmental disease . AdolescResting-state functional magnetic resonance imaging (fMRI) was widely used to investigate the mechanisms of schizophrenia; indicators such as amplitude of low-frequency fluctuation (ALFF) and functional connectivity (FC) were employed to describe neural activities . AbnormaRepetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique used for treating various mental disorders. It has been clinically approved by the U.S. Food and Drug Administration for treatment-resistant depression , and oneIn order to further clarify the causal connection in the VC–Acb–OFC pathway in MAM rats and to evaluate the effectiveness of rTMS treatment, we used Granger Causality Analysis (GCA) to analyze the effective connectivity (EC) between the regions of interest (ROIs). Granger Causality defined the causality of time series as follows: if preceding time series x could help predict the present time series y better than what preceding time series y could predict about the present time series y itself, then x “Granger caused” y . GCA hasIn summary, the aim of our resting-state fMRI study was to investigate the potential mechanism of schizophrenia and to find an effective early-stage intervention. MAM rats were used to mimic schizophrenia patients. VC was chosen as a novel target region of intervention, and rTMS treatment was delivered during adolescent stage in MAM rats. GCA was applied to demonstrate the causal link between VC, nucleus accumbens core (AcbC), nucleus accumbens shell (AcbSh), and OFC separately in both hemispheres. We aimed (1) to evaluate the abnormal EC from posterior to anterior cerebrum and to find a potential biomarker in schizophrenia and (2) to assess the effectiveness of rTMS intervention on early-stage schizophrenia. We hypothesized significant alteration of EC in the VC–Acb–OFC pathway both in MAM rats and after rTMS treatment.The rats were provided by VITAL RIVER Laboratories, Inc. . On embryonic day 17, SPF level Sprague–Dawley rats were randomly, blindly divided into MAM groups (MAM-sham group and MAM-rTMS group) and vehicle groups (vehicle-sham group and vehicle-rTMS group). The MAM groups were injected with 22 mg/kg MAM, and the vehicle groups were injected with 0.9% saline solution. Pups were ablactated on postnatal day (PD) 21 and regrouped according to gender; male rats were chosen as the experiment material, with 2–4 per cage under controlled environment  to mimic normal circadian rhythm, with full access to food and water. All experiments strictly followed the guidelines of the Animal Care and Use Committee at the Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences.n = 19) and Vehicle group (n = 19) were randomly divided into the rTMS group and sham group. Hence, the rats were separated into the MAM-rTMS group (n = 9), MAM-sham group (n = 10), vehicle-rTMS group (n = 9), and vehicle-sham group (n = 10). For adolescence animals (PD 40–60), stimulation for the rTMS group was carried out through a magnetic stimulator and a ring-shaped animal coil produced by Wuhan Irid Corporation. During the stimulation, the heads of rats were fixed, the coils were tightly adhered to the scalps, and the center of the coils was placed over the anterior interaural line of the skull, targeting VC  was then used with the same localization of anatomical scan to acquire fMRI scan with the following parameters: repetition time (TR) = 1,000 ms, echo time (TE) = 14 ms, matrix size = 64 × 64, field of view (FOV) = 2.4 × 2.4 cm2, slice number = 20, slice thickness = 0.8 mm.During adulthood (PD 64–70), a 7.0-T MRI scanner  was used to scan the rat with a rat brain quadrature surface coil for receiving ; the rat was anesthetized with 2% isoflurane. A Rapid Imaging with Refocused Echoes (RARE) sequence was used to position fMRI slices, and an anatomical scan was acquired with the following parameters: repetition time (TR) = 5,000 ms, echo time (TE) = 12 ms, matrix size = 256 × 256, field of view (FOV) = 2.4 × 2.4 cmrfmri.org/DPABI)  . The anag/DPABI) , and theThe regions of interest (ROIs) were anatomically masked and defined based on the standard anatomical rat template . VC, Acbt-tests between MAM-sham and vehicle-sham groups, MAM-rTMS and MAM-sham groups, MAM-rTMS and vehicle-rTMS groups, and vehicle-rTMS and vehicle-sham groups. Before each comparison, Levene’s tests were conducted to detect homogeneity of variance. When p 0.05, the results were suggested as significant.For each ROI, we calculated the mean and standard deviation values of the time series in four separate groups; then, we made comparisons of the standard deviation values between every two groups to preliminarily confirm the effectiveness of disease and rTMS treatment. We performed two-sample Chen’s extended linear autoregressive model was used in our study for GCA . For twoThe joint-regressive model was defined as below:p was the regression order, A_iand B_i were the GC coefficients, and ℰ and μ were the residuals. If A = 0 and ℰ=μ, then x was not the GC of y, and vice versa. Through calculating the GC coefficient between two ROIs, we could judge their GC. Results of right and left hemispheres were calculated separately. The GC of each connectivity was regarded as an independent outcome. We obtained 32 groups of results.Among them, t tests to evaluate the difference in EC between MAM-sham and vehicle-sham groups, MAM-rTMS and MAM-sham groups, MAM-rTMS and vehicle-rTMS groups, and vehicle-rTMS and vehicle-sham groups; comparisons of the left and right hemispheres were separately made. Before each comparison, Levene’s tests were conducted to detect homogeneity of variance. When p 0.05, the results were suggested as significant , while no significant difference was detected in the left hemisphere , from OFC to AcbC , and from OFC to AcbSh . Increased EC was found from AcbC to VC , from AcbC to OFC , from AcbSh to VC , and from AcbSh to OFC . In the left hemisphere, only increased EC from AcbSh to AcbSh  was found after rTMS treatment , from VC to AcbSh , from OFC to AcbC , and from OFC to AcbSh . Increased EC was found from AcbC to VC , from AcbC to OFC , from AcbSh to VC , and from AcbSh to OFC . In the left hemisphere, only increased EC from AcbSh to VC  was found in the MAM-rTMS group compared to the vehicle-rTMS group  as well as AcbC  was detected in the left hemisphere, while no significant difference was detected in the right hemisphere  targeting Acb to treat obsessive–compulsive disorder (OCD), and one resting-state fMRI study suggested that it could normalize Acb-related connectivity in reward circuits . We postThe right AcbSh-OFC EC showed significant difference between both MAM-sham and vehicle-sham groups and MAM-rTMS and MAM-sham groups; according to our results, we suggest that schizophrenia decreased the causal link from AcbSh to OFC, and early-stage rTMS treatment increased it compensately. As a subregion of the Acb, AcbSh is mostly made up of GABAergic medium spiny neurons (MSNs)  and partThere were several limitations in our resting-state fMRI study. Firstly, no significant difference of EC from VC to Acb was detected between the MAM-sham group and the vehicle-sham group. We hypothesized the reason for this was that in contrast to senior cortex OFC, VC was a primary sensory cortex and Acb was a subcortical region, both of which undertook fundamental functions in brain. During neurodevelopment, deficits from VC to Acb occurred in the early stage of schizophrenia, while the Acb-OFC EC progressed in adulthood. As a consequence, we failed to detect significantly different VC-Acb EC in our results. Future studies targeting early diagnosis could take our hypothesis into consideration and explore the potential alteration of this connectivity. Secondly, GCA was used to reveal Granger causal links in the pathway, and the results suggested synchronization of time series between ROIs; however, the precise mechanism remains unclear. More related studies are needed in the future to verify our results. Finally, significant lateralization was found after rTMS treatment. Although our intervention chose the whole VC as the target region, rTMS treatment mainly came into effect in the right hemisphere. This might be attributed to the inhibitory effect of rTMS in the right side, and partly explained why decreased VC-Acb EC was found after intervention . Future We found a critical right VC–Acb–OFC pathway in the MAM rat model using GCA, indicating that abnormal posterior–anterior EC might be the substantial mechanism of schizophrenia; rTMS treatment was suggested as an effective early-stage intervention, among which right Acb played the role of a central hub, and VC showed its potential of becoming a novel rTMS target region during adolescent schizophrenia. Moreover, right AcbSh-OFC EC was shown to be a substantial biomarker. To our knowledge, this was the first resting-state fMRI study using GCA to assess the deficits of a visual-reward neural pathway and the effectiveness of rTMS treatment in MAM rats. More high-quality, multiple-center randomized controlled trials in both animal models and schizophrenia patients are needed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Care and Use Committee at the Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences.FW: conceptualization. HG, DS, JY, HW, and YZ: methodology. CP: software. HG, DS, and JY: validation. YX, CP, CL, and PZ: formal analysis. HG, DS, and JY: investigation. JW: resources. CL: data curation. YX: writing—original draft preparation. YX, YZ, JFW, and FW: writing—review and editing. YX and JFW: visualization. JW, XZ, and FW: supervision and project administration. DS and FW: funding acquisition. 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.


The diagnosis of high-grade relapse of lymphomatous disease was made. In this context, the homogenous and symmetric lesion of the cavernous sinuses, without any other encephalic or meningeal lesions, raised the hypothesis of a paraneoplastic origin. A plausible paraneoplastic link between the neuro-ophthalmological lesion and the malignant disorder is IgG4-related disease, a condition that may be associated with lymphoma. As in our case, this diagnosis is often presumptive because histopathological confirmation is difficult to obtain.A 73-year-old man with a history of marginal zone lymphoma was admitted to the emergency room for diplopia and ipsilateral headache. The Fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography ( Painful ophthalmoplegia refers to periorbital or hemicranial pain associated with ipsilateral oculomotor nerve palsy. It may be caused by any process exerting a mass effect simultaneously on the internal carotid artery and the cavernous sinus and/or the superior orbital fissure. It comprises several etiologies, including neoplastic, vascular, inflammatory, or infectious disorders , firstly described in 1954, is a diagnosis of exclusion. It is characterized by a unilateral headache associated with a paralysis of the ipsilateral third, fourth, and/or sixth cranial nerves caused by nonspecific granulomatous inflammation of the cavernous sinus, the superior orbital fissure, or the orbit . This inflammatory process is of unknown etiology and rapidly resolving after corticoid therapy  treated by splenectomy in 2012.At the admission, the patient had completely normal parameters with a blood pressure of 120/60 mmHg, heart rate of 65 bpm, temperature of 36.3°C, and oxygen saturation in ambient air of 95%. The clinical examination revealed a systolic heart murmur irradiating in both carotids. Neurological examination showed oculomotor paralysis of the left eye with ptosis, unreactive mydriasis, and divergent strabismus. Blood tests only showed severe hyperleukocytosis (with 95% of lymphocytes) and signs of cytolysis. The brain CT was unremarkable.In the context of a discontinued anticoagulation, an ischemic stroke of cardio-embolic origin located in the left mesencephalic region was suspected and the patient was admitted to the stroke unit.Among the additional biological dosages made during the hospitalization , lymphocyte typing revealed a pathological population of B lymphocytes.Brain magnetic resonance imaging (MRI) demonstrated no recent ischemic lesion and no brain mass.18F-FDG PET/CT) demonstrated intense and symmetrical hypermetabolism of the cavernous sinuses .The cerebrospinal fluid examination was unremarkable .Transformation of a MZL to a DLBCL was then concluded.Concerning the neuro-ophthalmological symptoms, a local homogenous and symmetrical lymphomatous infiltration of the cavernous sinuses without any other sign of encephalic or meningeal lesions seemed unlikely. Therefore, we raised the hypothesis of an IgG4-RD. Indeed, IgG4-RD may cause an orbital inflammatory disease and an etiological link is suspected between IgG4-RD and lymphoma  and 4 cycles of high-dose methotrexate (HD-MTX).The patient was treated by corticoid therapy with rapid and complete resolution of the neuro-ophthalmologic symptoms. The cerebral Fig.  and wholSix months later, the patient presented a DLBCL recurrence in the supra- and sub-diaphragmatic lymph nodes with no neurological symptom. He was treated by Rituximab- with Gemcitabine/-, Dexamethasone, and Cisplatine (R-GDP), shifted to Rituximab with Gemcitabine, Dexamethasone, and Carboplatin (R-GDCarbo) for nephrotoxicity, with complete response after 3 cycles.Unfortunately, before the end of this therapy, the patient died from a septic shock accompanying a SARS-CoV-2 infection (COVID-19).THS is a painful ophthalmoplegia related to the so-called orbital pseudotumor , a nonspecific granulomatous inflammation of the orbit, the superior orbital fissure, or the cavernous sinuses (Gladstone 18F-FDG PET/CT images since this examination provided a clear demonstration of a homogenous and symmetric involvement of the cavernous sinuses that had not been seen on the CT and MRI.The case here reported illustrates the value of cerebral Without any other encephalic or meningeal lesions, this cavernous sinus involvement could not be reasonably attributed to an isolated local lymphomatous invasion. This was support by the rapid clinical response to corticoid therapy, as expected in THS. Therefore this neuro-ophthalmological manifestation had most likely a paraneoplastic origin. Since paraneoplastic syndromes are generally related to autoimmune processes, we considered the hypothesis that THS in this case was a manifestation of a lymphoma-induced IgG4-RD. Indeed, on the one hand, THS has been described in association with IgG4-RD (Lindfield et al. A diagnosis of IgG4-RD is often difficult to establish. Wallace et al. have demonstrated that only 51% of patients with active disease have elevated serum IgG4 concentrations (Wallace et al. THS is a diagnosis of exclusion in the presence of painful ophthalmoplegia. It is characterized by a local nonspecific granulomatous inflammation.Painful ophthalmoplegia requires investigation by MRI to exclude a local mass, but this method may not demonstrate and localize all inflammatory lesions in this clinical setting.18F-FDG PET/CT may be valuable in the work-up of painful ophthalmoplegia. Its high sensitivity helps to localize the causing lesions, including when the origin is an inflammatory process.As shown in our patient, 18F-FDG PET/CT has a dual advantage in this context. First, it helps detect and characterize a local pathological process producing the syndrome. Second, it may potentially reveal a malignant disorder at the origin of the painful ophthalmoplegia.Considering the reported association of THS with malignancies, 18F-FDG PET/CT performed.Since the link between malignancy and painful ophthalmoplegia may be an IgG4-RD, other manifestations of this disorder may also become apparent on the


Indicator condition (IC) guided testing for HIV is an effective way to identify undiagnosed people living with HIV, but studies suggest its implementation is lacking. This systematic review provides an overview of the adoption of IC-guided testing in Western countries.Seven ICs were selected: tuberculosis (TB), malignant lymphoma, hepatitis B, hepatitis C, cervical/vulvar carcinoma/intraepithelial neoplasia grade 2+ , and peripheral neuropathy (PN). Embase and Ovid MEDLINE were searched up to November 20th, 2020. Publications of all types, using data from ≥2009, reporting on HIV test ratios in patients ≥18 years in all settings in Western countries were eligible. HIV test ratios and positivity were reported per IC. A random effects-model for proportions was used to calculate estimated proportions (ES) with 95% CIs. This study was registered at PROSPERO, registration number CRD42020160243.Fifty-seven references, including 23 full-text articles and 34 other publications were included. Most (28/57) reported on HIV testing in TB. No reports on HIV testing in VC/VIN2+ or PN patients were eligible for inclusion. Large variation in HIV test ratios was observed between and within ICs, resulting from different testing approaches. Highest HIV test ratios  and positivity  were observed among TB patients, and lowest among CC/CIN2+ patients .IC-guided HIV testing is insufficiently implemented in Western countries. The large variation in test ratios provides insight into priority areas for implementing routine IC-guided HIV testing in the future.HIV Transmission Elimination in Amsterdam (H-TEAM) consortium and Aidsfonds . Identifying and treating people living with HIV is key to control the HIV epidemic. Opportunities to identify undiagnosed HIV through indicator condition guided testing are being missed in various healthcare settings. We have found no systematic review on the extent to which indicator condition guided testing has been adopted in Western countriesWe searched Embase and Ovid MEDLINE for evidence published up to November 20th, 2020 for evidence on the extent to which indicator condition guided testing for HIV is implemented in all healthcare settings in the Western world. All publication types, including full-text peer reviewed articles, as well as abstracts, short communications, and correspondence reporting on HIV testing in a selection of seven indicator conditions in or after 2009 in all healthcare settings in Western Countries were eligible for inclusion. No language restrictions were applied. The search included terms for HIV testing, the selected indicator conditions, and the term ‘indicator condition’.This systematic review revealed that indicator condition guided testing for HIV is unevenly and insufficiently adopted across indicator conditions and healthcare settings in Western countries. We found that even in AIDS defining conditions, such as tuberculosis or cervical cancer, HIV testing strategies need to be further improved. Additionally, for some indicator conditions such as peripheral neuropathy, no evidence on HIV testing was identified, suggesting that in some conditions, this testing strategy might be lacking even more. Overall, this review revealed that adopting these strategies is an effective way to identify undiagnosed HIV, as in most conditions positivity percentages exceeded the established cost-effectivity threshold of 0·1%.Indicator condition guided testing for HIV remains insufficiently practiced, and results from settings where this strategy is best implemented reveal opportunities for improvement. Lessons on effective implementation can be learned from these settings, such as the high HIV test ratio observed when opt-out testing strategies are used. Adopting these strategies could lead to improved indicator condition guided HIV testing strategies across healthcare settings in Western countries.Alt-text: Unlabelled box13) is reported in almost half of all new cases. In our global efforts to complete the ‘last mile’ towards ending the HIV epidemic, timely diagnosis remains an important challenge. In the European Union/European Economic Area (EU/EEA), an estimated 14% of people living with HIV (PLHIV) is unaware of their diagnosis and late diagnosis  are defined as conditions that are either (1) AIDS-defining, (2) associated with an undiagnosed HIV prevalence of >0·1%, the cut-off for cost-effective screening for HIV, ,5 or (3)In recent years numerous studies have shown that IC-guided HIV testing is an effective approach to identify undiagnosed PLHIV. [25]The main objective of this systematic review was to assess the proportion of patients presenting with indicator conditions that are tested for HIV (i.e. the HIV test ratio). The secondary objective was to assess the outcomes of this testing strategy (i.e. the percentage positive).22.1supplementary appendix 1), and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (supplementary appendix 2).The protocol for this review was published at PROSPERO , cervical cancer (CC) or cervical intraepithelial neoplasia (CIN) grade 2+, vulvar cancer (VC) or vulvar intraepithelial neoplasia (VIN) grade 2+, malignant lymphoma, hepatitis B (HBV), hepatitis C (HCV), and peripheral neuropathy (PN). These ICs were selected as they are diagnosed and managed by various medical specialties , ensuring a wide scope of the extent to which IC-guided testing is adopted, and they can all be objectively diagnosed using diagnostic tests.2.3supplementary appendix 3). No language or date restrictions were applied. Additionally, all articles referring to the HIDES studies, HIV positivity was highest among TB patients, followed by HBV, HCV, and malignant lymphoma, respectively, but again large variation was observed. Among CC/CIN2+ patients, one study reported a positivity of 0·2%, This review confirms previous reporting on missed opportunities for earlier diagnosis through IC-guided testing. A barrier to optimal IC-guided testing might be the large number of ICs, the large variety in types of conditions and the many medical specialties involved. This variety requires tailored strategies to assure routine IC-guided testing is implemented across ICs. Moreover, an evaluation of IC-guided HIV testing recommendations in specialty guidelines in the UK and Europe revealed that the majority of IC guidelines do not recommend HIV testing, and physicians are not always aware of current HIV testing recommendations. ,37 This A major strength of this review is the variety of settings and countries included. Second, by including not only published full-text articles, but also other publication types, we gained a more comprehensive picture of actual IC-guided HIV testing practices.Although the retrospective design of included studies posed a potential risk of bias, most full-text studies were assessed as low risk of bias. We further addressed this possible limitation in a sensitivity analysis including only full-text articles with low risk of bias and found lower estimated proportions, suggesting that the IC-guided HIV test ratio outside study settings might be even lower. Very large heterogeneity was observed in the meta-analyses by IC, probably reflecting true heterogeneity across settings and Western countries. Thus, exact inferences on HIV test ratios by ICs could not be made, but conclusions can be drawn from the heterogeneity itself; testing practices are both inconsistently reported and inconsistently adopted. These findings should be considered when evaluating efforts to improve HIV testing strategies. Finally, a selection of only seven ICs was included in this review. Although not all ICs were included, it is unlikely that the HIV test ratios in other ICs will be much higher, as well-established and guideline-supported ICs such as TB and HCV were included in this study, and it is evident that even in those improvement is still needed.This systematic review shows that a decade after its introduction, IC-guided testing for HIV is still insufficiently implemented in Western countries. Lessons on effective strategies from ICs with the highest test ratios, such as universal testing strategies, should be used to design effective implementation strategies for optimal IC-guided testing, to reduce underdiagnosis and late presentation of HIV.Aidsfonds (Grant No. 2013169), Stichting Amsterdam Dinner Foundation, Bristol-Myers Squibb International Corp. (study number: AI424–541), Gilead Sciences Europe Ltd (Grant No. PA-HIV-PREP-16-0024), Gilead Sciences , Janssen Pharmaceutica (reference number: PHNL/JAN/0714/0005b/1912fde), M.A.C AIDS Fund, ViiV Healthcare  and ZonMw (Grant No. 522002003). This review is further funded by Aidsfonds (Grant No. P- 42702). The funders of the study had no role in the study design or execution.The H-TEAM initiative, of which this review is a project, is being supported by The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.Geerlings acquired financial support for the project leading to this publication. Geerlings, Schim van der Loeff, van Bergen and Bogers were involved in the conceptualisation and design of methodology of the study. Bogers performed the literature search. Bogers and Hulstein performed data curation including screening of search results, data extraction, and performing quality assessments. Bogers analysed the data and designed the figures. Schim van der Loeff performed quality controls and validation on all data analyses. De Bree and Reiss provided commentary and revisions on the original draft. All authors were involved in the interpretation of the data and the preparation of the final manuscript.www.hteam.nl). There are no personal fees or payment involved. H-TEAM is not involved in the design or conduct of this work. S.H.H. Hulstein has nothing to disclose. Dr. Schim van der Loeff reports other from Merck, non-financial support from Stichting Pathologie Onderzoek en Ontwikkeling (SPOO), outside the submitted work. Dr. de Bree reports grants from AIDS Fonds, grants from Stichting AmsterdamDiner Foundation, grants from Gilead Sciences, grants from Janssen Pharmaceutica, grants from ViiV Healthcare, grants from ZonMW, grants from M.A.C AIDS Fund, during the conduct of the study; grants and other from Gilead Sciences, outside the submitted work. Dr. Reiss reports grants from AIDS Fonds, grants from Stichting AmsterdamDiner Foundation, grants from Gilead Sciences, grants from Janssen Pharmaceutica, grants from ViiV Healthcare, grants from ZonMW, grants from M.A.C AIDS Fund, during the conduct of the study; grants and other from Gilead Sciences, grants and other from ViiV Healthcare, grants and other from Merck, outside the submitted work. Dr. van Bergen reports grants from RIVM: national institute for public health and the environment, during the conduct of the study; and reports being a member of the board of the H-TEAM, a consortium of all actors involved in hiv-care and prevention in Amsterdam, with the ultimate goal to pursue the end of new hiv-infections in Amsterdam. The H- team is sponsored by a mix of organisations, including the Municipality, AIDS-funds, and several farmaceutical companies (www.hteam.nl). There are no personal fees or payment involved. Dr. Geerlings reports grants from Aidsfonds, during the conduct of the study.Dr. Bogers reports grants from Aidsfonds, grants from H-TEAM, during the conduct of the study; and this work is partially funded by H-TEAM, a consortium of all actors involved in hiv-care and prevention in Amsterdam, with the ultimate goal to pursue the end of new hiv-infections in Amsterdam. The H- team is sponsored by a mix of organisations, including the Municipality, AIDS-funds, and several farmaceutical companies (