Translational Precision Medicine approach to drug discovery and development. Key components of Translational Precision Medicine are multi-omics profiling, digital biomarkers, model-based data integration, artificial intelligence, biomarker-guided trial designs and patient-centric companion diagnostics. In this review, we summarize and critically discuss the potential and challenges of Translational Precision Medicine from a cross-industry perspective.In the era of precision medicine, digital technologies and artificial intelligence, drug discovery and development face unprecedented opportunities for product and business model innovation, fundamentally changing the traditional approach of how drugs are discovered, developed and marketed. Critical to this transformation is the adoption of new technologies in the drug development process, catalyzing the transition from serendipity-driven to data-driven medicine. This paradigm shift comes with a need for both translation and precision, leading to a modern  However, in a rapidly evolving healthcare ecosystem, new technologies and innovative concepts of how to leverage them are needed to accelerate clinical trials, lower attrition rates, mitigate research and development (R&D)-related risks and overall improve pharmaceutical R&D productivity , 2. Critht time) , 4. Whilht time) –7.translation [Translational Gap [National Center for Advancing Translational Sciences, a core hub to drive and integrate innovative translational activities across academia, industry and non-profit organizations [European Society for Translational Medicine [Translational Precision Medicine to refer to this emerging discipline.One of the major bottlenecks for drug development is nslation , particuonal Gap –10. To cizations . TranslaMedicine  integratTranslational Precision Medicine concept integrates core components from both translational medicine (mechanism-based early drug development) and precision medicine (patient-centric late drug development) into an end-to-end biomarker-guided drug development cycle. Critical success factors for Translational Precision Medicine are (i) the translation of mechanisms from research to early clinical development (forward translation/bench-to-bedside), (ii) the back-translation from late clinical development insights to drug discovery (reverse translation/back-translation/bedside-to-bench) [Translational Precision Medicine Digital biomarkersPatient engagement.The o-bench) , (iii) do-bench) , (iv) tho-bench)  and (v) o-bench) . Here weine Fig. :Multi-omphenotyping (or phenomics), which is the traditional and most common approach to classify diseases irrespective of the biological origins of disease. Utilizing datasets to define disease subtypes at the molecular level can be referred to as endotyping, as exemplified in respiratory medicine [Clinical data can be classified as phenotypic  or molecular . Capturing comprehensive phenotypic data associated with a certain disease can be referred to as medicine , 17 or omedicine . The Natmedicine . Howevermedicine , 21. Witmedicine  or mutatmedicine , but mosmedicine , particumedicine . Translamedicine , 27 are medicine . Unlike medicine , typicalmedicine , 30–33. multi-omics profiling platforms are emerging, including genomics, epigenomics, transcriptomics, proteomics, lipidomics, metabolomics, microbiomics and others [N-of-1 trials [Omics technologies assess large numbers of genes/proteins, often in a semi-quantitative manner, and are highly sensitive to pre-analytical processes  such as Merging different multi-omics datasets  into a sClinical trial feasibility , 48, espBeyond single omics technologies, d others , 35. Muld others , and netd others . Multi-o1 trials , for und1 trials –41. Despa characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” [5R) framework) demonstrated that the inclusion of biomarkers into early drug development (Ph2 studies) was associated with active or successful projects in contrast to comparable projects without biomarkers [Biomarkers are defined by the Biomarkers Definitions Working Group of the NIH/FDA, as “vention” . In drugvention” , 10, 52,vention”  such as context-of-use (CoU) [BEST  [prognostic biomarkers at baseline are indicative of disease outcome independent from interventions , whereas predictive biomarkers at baseline are indicative of response to a specific treatment (response prediction).The development of a new biomarker is a complex, multistep and iterative process, including biomarker discovery (often based on omics data), pre-analytical validation, assessing different biofluids , analytical validation and finally clinical validation and utility . For eacse (CoU) , 55, 56.r Tools) . Prognosspecific drug development program or (2) the official FDA biomarker qualification program (BQP). The specific drug development program path is the most common strategy pursued in pharmaceutical industry, where the drug developer / sponsor includes selected biomarkers in clinical trials, mainly for internal decision-making and is responsible for all aspects of the biomarker development. The FDA BQP is required to qualify biomarkers as general drug development tools [There are two basic paths how to integrate biomarkers in drug development: (1) within the context of a nt tools  to make nt tools . Of notesurrogate endpoints, which have a major relevance for diseases with outcomes that take a long time to capture using traditional clinical endpoints. Examples here are systolic blood pressure for occurrence of stroke or low-density lipoprotein cholesterol levels for occurrence of heart attacks. For implementation of biomarkers in pharmaceutical industry trials, several drug development aspects have to be further taken into account, including informed consent/data protection considerations, clinical trial logistics/feasibility, impact on clinical decision-making and cost-effectiveness [In general, the level of impact that biomarkers can have depends on three key factors: (i) the validation and qualification status of the biomarker, (ii) the CoU and (iii) the scientific evidence linking the biomarker with the CoU. Biomarkers in clinical trials are mostly used as exploratory endpoints to explore new mechanistic hypothesis and inform internal decision making. If biomarkers are deemed more impactful and clinically relevant, biomarkers are used as secondary or primary clinical endpoints. Typical examples here are physiological biomarkers like blood pressure, clinically-established protein biomarkers such as C-reactive protein, or imaging readouts. If there is convincing evidence from independent epidemiological studies and clinical trials that biomarkers correlate closely with clinical outcome assessments, biomarkers can be considered to substitute for a clinical endpoint as tiveness , 59–61.biomarker-guided trial designs are the method of choice [BiGTeD online resource [stratification [enrichment [biomarker-strategy designs [Master observational trials) [To actively guide clinical trial flows, f choice , 63, whif choice . For a cresource . For biofication  and enririchment . Biomarkrichment  should b designs . All bio trials) .Precision Medicine Initiative [N-of-1 trials [companion diagnostics [numbers-needed-to-treat and/or to spare patients exposure to drugs that have a high probability to fail or even cause harm. The development path for an exploratory biomarker to a full IVD companion diagnostic is complex, requires at-risk investments and should start early in drug development in close alignment with health authorities [Biomarker-guided trial designs ultimately pave the way towards precision medicine, i.e. tailoring drug development to specific patient characteristics , 70. In itiative , 72. Pre1 trials , ideally1 trials . Biomarkgnostics –75, maingnostics . Companihorities , 78. Comhorities . Companihorities  are strihorities  and are horities , 83 as tTo increase the benefit-risk ratio of drug candidates, safety aspects are increasingly becoming an integral part of biomarker-guided precision medicine approaches. For example, the observation that patients receiving checkpoint inhibitor therapy experiencing immune-related adverse events also exhibit an improved treatment response was recently shown to be related to a polygenic risk score . Furtherphysician-centric to a data-driven and patient-centric precision medicine approach [When viewed in combination, biomarker-guided trial designs provide ideal tools to catalyze the transition from an empirical and approach , 86, 87.approach , 88.Given the small size and low number of samples per subject in pre-clinical experiments as well as early clinical trials, analyzing and leveraging biomarker data in translational medicine remains challenging. One way for improving the statistical power for detecting signals is to use longitudinal (i.e. time-dependent) model-based data integration. Mathematical models are used to describe the time course of PK and PD/biomarker results for better understanding of the pharmacology and to predict future experimental outcomes . Alreadyex-vivo). When considering chemotherapies, like methotrexate, questions arose about the tumor-relevant tissue distribution of the compound [Population models most often use simplified model structures to model the observed data  within and between species using power functions with fixed exponents. This works well for PK parameters , yet canFDA is conducting a Model-Informed Drug Development (MIDD) Pilot Program to facilitate the development and application of exposure-based, biological, and statistical models derived from preclinical and clinical data sources, referred to as MIDD approaches. MIDD approaches use a variety of quantitative methods to help balance the risks and benefits of drug products in development. When successfully applied, MIDD approaches can improve clinical trial efficiency, increase the probability of regulatory success, and optimize drug dosing/therapeutic individualization in the absence of dedicated trials.” This MIDD pilot program is based on joint discussions between the pharmaceutical industry and the European health authorities/EMA, which led to a paper in 2016 on good modeling practices [Finally, the FDA has established a new framework for model-informed drug development : “FDA isractices . The latractices  indicateThe amount of data generated and collected in pharmaceutical R&D is increasing at an unprecedented pace. Combined with improvements in information processing and more powerful hardware, machine learning, deep learning and AI in general are positioned to disrupt drug discovery and development towards an algorithm-based R&D . Deep leTranslational Precision Medicine [early mature phase of using AI in R&D. However, an increasing number of healthcare companies have now started their digital journey, building up AI competencies and data literacy across many areas of R&D [Innovative Science and Technology Approaches for New Drugs (ISTAND) that incentivizes the use of AI-based algorithms to evaluate patients, develop novel endpoints, or inform study designs. Moving forward, it will be critical that pharmaceutical organizations continue to constructively engage early on with regulatory authorities on innovative ways to design and assess clinical trials, including a more widespread use of AI technologies in drug development.Given the rigidity of traditional serendipity- and forward translation-based drug development frameworks, shifting to a new mindset embracing the use of AI for the discovery and development of drugs is a critical success factor for Medicine . A comprMedicine , coming s of R&D . For exas of R&D , 114. Tos of R&D . While ts of R&D , 117 andOverall, the impact of AI in drug discovery and clinical development will largely depend on the underlying data, and its intrinsic limitations. AI-based analysis of both multi-omics as well as EHRs depends critically on the quality and quantity of the provided molecular and clinical datasets, key limitations and challenges that need to be overcome in the future.The near future will show whether and how these emerging AI algorithms will help scientists to (i) identify novel targets or new indications for existing drugs, (ii) uncover latent factors that can inform on disease pathogenesis or drug response, (iii) discover predictive biomarkers enabling patient stratification strategies that can optimize clinical trial designs, and (iv) ultimately impact the drug development value chain. For more detailed overviews of AI in drug discovery and development, we refer the reader to dedicated reviews in this field , 118.The recent evolution of sensor technologies and the widespread use of smartphones and other connected digital products are enabling the comprehensive collection and analysis of health-related data –121. ProSuccessful examples of digital biomarkers are in the field of neurodegenerative diseases, where traditional clinical outcome measures are sparse, highly variable and rater-dependent . SmartphWhile the number of studies involving digital technologies is growing and extending to more technologies, biomarker categories and therapeutic areas , 121, thClinical Trials Transformation Initiative (CTTI) [Digital Medicine Society [Digital Biomarker Discovery Pipeline [Transforming digital device data into validated clinical endpoints is a lengthy process, which involves the collaboration of multiple disciplines, from engineering, machine learning, data science, clinical research and regulatory interactions. An open validation framework based on transparency, metadata standards, external validation and data sharing is necessary to harmonize approaches and evaluate and improve digital biomarkers in clinical settings . Recentle (CTTI) , the Dig Society  and the  Society ; (ii) pr Society , 141;  opPipeline ; (v) andPipeline , IDEA-FAPipeline  and RADAPipeline .Future opportunities for digital biomarkers towards patient-centric precision medicine are (i) algorithms based on longitudinal/real-time composite biomarkers from multiple connected technologies and contextual information in real-world settings , (ii) inPatient-Focused Drug Development initiative led to the guidance for industry on how to best identify what is important to patients. In Europe, the EMA formed its Patients’ and Consumers’ Working Party. Engagement with patients, their caregivers, patient experts and patient advocacy groups have been shown to yield benefit for both patients and the industry [International Alliance of Patient’s Organizations, the Patient-Centered Outcomes Research Institute [Patient-focused Medicines Development [Since the AIDS pandemic in the 1980s, the way industry interacts with patients has changed fundamentally, from passive recipients to active contributors along the whole drug development value chain. This has been particularly evident in the last decade, where most pharmaceutical companies have started patient engagement groups to actively listen to the patient voice , 151. Inindustry , 154. Inindustry . Patientnstitute  and the elopment  among sepatient-led research [Traditionally, patient engagement has been mainly considered once a new drug is already on the market. The majority of decisions about the molecule and its clinical development path, including unmet medical needs, have then already been taken by the company. Studies, however, demonstrated that the early integration of the patient perspective, particularly in preclinical research and early development, has the biggest impact on value creation for patients, business and society . As precresearch . A recenresearch . In interesearch . A key rresearch .Personalised healthcare (PHC), precision medicine and stratified medicine have been used interchangeably to describe the concept of tailoring treatment to patients based on their individual pathology. With the rise of new diagnostic and data-driven approaches that deepen our understanding of the molecular basis of disease, this centuries-old dream has come closer to reality. Nowadays, the awareness of the potential of PHC is also emerging in the patient community and its meaning goes far beyond precision medicine. PHC comprises everything that allows to tailor treatment and medical care by combining conventional clinical datasets, molecular signatures (such as genetics), environment, lifestyle and personal needs. Some of the key innovations in that area include digital healthcare solutions with technologies connecting digital patient information/EHRs with wearable devices, mobile Apps, telehealth and digital assistants using AI , see alsTranslational Precision Medicine comes with a paradigm shift from a one-size-fits-all to a biomarker-guided patient-centric medicine. Key success factors for adoption of this principle in pharmaceutical drug development include the combination of forward and reverse translation, the classification of disease conditions as multi-omics-defined endotypes, the integration of AI- and algorithm-based R&D concepts, the implementation of digital biomarkers as clinical endpoints and the development of companion diagnostics. The rise of data-driven and algorithm-based R&D necessitates the establishment of a new mindset of how data mining and AI tools can be used effectively to discover and develop new drugs [ew drugs . The neaTranslational Precision Medicine evolution comes with distinct challenges: (i) multi-omics data are mainly useful to drug discovery and development if they reveal new drug targets or biomarker signatures that correlate with disease outcome and/or treatment response [digital biomarkers as clinical endpoints provide objective and quantitative measures yet still require broader clinical use and health authority acceptance; (iv) biomarker-guided trial designs and precision medicine approaches are already widely implemented in oncology and rare diseases, while other non-oncology areas have just started to pursue these concepts and (v) precision medicine/companion diagnostics approaches come with substantial development costs and reimbursement hurdles. One important question is how these novel technologies and assessments are perceived by patients, as acceptance and adherence to clinical read-outs is key for patient trial recruitment and long-term engagement. Novel patient-centric interaction approaches are currently implemented to engage patients more pro-actively in R&D, RWE networks and clinical trials. New cloud-based data systems and platforms for interactions with regulatory agencies [Information Exchange and Data Transformation (INFORMED) initiative of the FDA [The response ; (ii) muagencies , for sha the FDA  will furThe COVID-19 era substantially disrupted the traditional pharmaceutical R&D approach at several layers –172: (i)Translational Precision Medicine as for drug development overall remains how to leverage and embrace new molecular and digital technologies in a way that is feasible for larger clinical trials, accepted by regulators and, most importantly, by patients.Emerging therapeutic modalities, including CAR T-cells , gene th


The existence of “sex phenotype” in migraine is a long-standing scientific question. Fluctuations of female sex hormones contribute to migraine attacks, and women also have enhanced brain activity during emotional processing and their functional brain networks seem to be more vulnerable to migraine-induced disruption compared to men. Periaqueductal grey matter (PAG) is a core region of pain processing and modulation networks with possible sex-related implications in migraine. In our study, sex differences of PAG functional resting-state connectivity were investigated in the interictal state in 32 episodic migraines without aura patients (16 women and 16 men). A significant main effect of sex was detected in PAG connectivity with postcentral, precentral, and inferior parietal gyri, and further differences were found between right PAG and visual areas , supplementary motor area, and mid-cingulum connectivity. In all cases, PAG functional connectivity was stronger in female migraineurs compared to males. However, higher average pain intensity of migraine attacks correlated with stronger connectivity of PAG and middle temporal, superior occipital, and parietal gyri in male migraineurs compared to females. Migraine-related disability is also associated with PAG connectivity but without sex differences. Our results indicate that sex differences in PAG connectivity with brain regions involved in sensory and emotional aspects of pain might contribute to the “sex-phenotype” in migraine. The stronger functional connectivity between PAG and pain processing areas may be a sign of increased excitability of pain pathways even in resting-state in females compared to male migraineurs, which could contribute to female vulnerability for migraine. However, pain intensity experienced by male migraineurs correlated with increased connectivity between PAG and regions involved in the subjective experience of pain and pain-related unpleasantness. The demonstrated sex differences of PAG functional connectivity may support the notion that the female and male brain is differently affected by migraine. Despite the extensive research in recent decades and successful drug developments, migraine remains one of the major causes of disability worldwide and the first cause among young women . MigrainIn addition to hormonal factors, recent structural and functional imaging studies revealed that the brains of women and men are differentially affected by migraine . A meta-2) and calcitonin gene-related peptide (CGRP) levels in PAG , a core region of a pain processing and modulating network. First, PAG dysfunction contributes to migraine pathogenesis , whereins in PAG , represeBased on the above findings, we hypothesised that PAG functional connectivity may show differences between female and male migraineurs. In addition, we assumed that sex might have an impact on the relationship between PAG connectivity and migraine attack frequency and migraine-related disability. To test this hypothesis, we conducted a resting-state fMRI study with migraine patients.Thirty-two age-matched episodic migraine patients without aura were included in the study, 16 men (mean age = 29.73 ± 6.12 years) and 16 women (mean age = 29.36 ± 6.20 years). To achieve an adequate sample size, data from two studies were pooled together. Participants were pain- and medication-free 48 h before the examination and did not use any daily medication, except contraceptives. Before entering the study, the mental health of participants was checked by senior researchers using the Mini-International Neuropsychiatric Interview  and episThe study protocol was approved by the Scientific and Research Ethics Committee of the Medical Research Council (Hungary). The entire study was conducted according to the Declaration of Helsinki and with the written informed consent of each participant.*-weighted echo-planar (EPI) pulse-sequence were as follows: repetition time (TR) = 2,500 ms, echo time (TE) = 30 ms, field of view (FOV) = 240 mm2; with a 3 × 3 × 3 mm resolution. The imaging dataset acquisition parameters of MAGNETOM Prisma were as follows: TR = 2,220 ms, TE = 30 ms, FOV = 222 mm2, with a 3 × 3 × 3 mm resolution. High-resolution (1 × 1 × 1 mm) structural data were acquired before the resting-state session using a T1-weighted 3D turbo field echo (TFE) sequence with Philips scanner and 3D MPRAGE sequence with Siemens scanner. The possible effect of the scanners was taken into account during the statistical analysis.Resting-state fMRI was obtained using two different types of 3 Tesla MRI scanners: half of the participants (eight women and eight men) was scanned with an Achieva 3 T Philips Medical System , while the other half of our sample with a 3 T MAGNETOM Prisma Siemens Syngo . The study protocol and the imaging data preprocessing steps were the same in both cases. Participants were required to close their eyes, but remain awake. The imaging dataset acquisition parameters of Philips Achieva T2On a scale of 0–10, on average how painful were these headaches?”) was used to measure the average pain intensity of migraine attacks. Two participants  did not complete the MIDAS questionnaire.The first five items of the MIDAS questionnaire  were sump < 0.05.The characteristics and sex differences of participants in self-reported data were analysed using SPSS  with a significance threshold set at Freesurfer version 5.3 (https://surfer.nmr.mgh.harvard.edu/)  was usedZ-scores using Fisher transformation.Seed-to-voxel analysis was conducted with left and right PAG as seed regions defined after Mainero et al.  Left PA. Seed-bahttp://www.fil.ion.ucl.ac.uk/spm12/) implemented in Matlab 2016a . An explicit grey matter mask was applied in every analysis for noise-reduction, the template was provided by the Brain Imaging Centre . To be able to evaluate the scanner effect the first factor in the full factorial ANOVA was scanner type. The second factor was sex and we investigated the main effect of the scanner, the main effect of sex, and their potential interaction. To follow up the ANOVA results, post-hoc t-tests were performed.These individual connectivity matrices were entered to a second-level full factorial ANOVA using Statistical Parametric Mapping (SPM12) software package  were investigated with two-sample t-tests. The effect of oral contraceptives on PAG-FC was also investigated in a two-sample t-test comparing the group of females using birth control pills to those who are not using them.To investigate the menstrual cycle differences on PAG-FC in female participants, the PAG-FC of females in a luteal phase were compared to PAG-FC of females in the follicular phase using two-sample T-values for our analysis in clusters where family-wise error corrected pFWE < 0.05 significance threshold were reached in at least 20 contiguous voxels , and midcingulate cortex (MCC) refer to .post-hoc t-test revealed a significantly stronger PAG-FC in female migraineurs compared to males with the above-mentioned regions. There was no stronger PAG-FC in males compared to females  was compared between female and male migraine patients to gain new insights into sex-related differences of the “migraine brain.” Some important migraine-related characteristics  were also taken into account to achieve a more specific exploration of the mechanisms involved in altered PAG-FC in migraine in general, and specifically, in association with sex.For the first time, we were able to identify sex-related differences in PAG-FC among migraineurs in the interictal state: female migraineurs compared to males showed a stronger resting-state PAG-FC with somatosensory and motor cortex, visual areas network , SMA and MCC. Among the measured migraine characteristics, only the average pain intensity interacted with sex on PAG-FC: higher pain intensity correlated with stronger PAG-FC with middle temporal gyrus, superior occipital and parietal gyrus in male migraine patients compared to females. Migraine-related disability is also associated with PAG-FC but without sex differences.Sex showed a main effect on PAG –FC with many regions of bottom-up pain processing . The ascFurther analyses revealed that PAG-FC with somatosensory and motor cortices were significantly stronger among female migraineurs compared to males. Our results are in concordance with the Mainero et al. study, where they also detected increased connectivity between PAG and somatosensory and motor cortex in migraine patients who were mainly women . In a prPreviously, in healthy females, PAG showed increased pain-induced connectivity with SMA compared to men . SMA alsIn a previous study, healthy women compared to men also have stronger resting-state PAG-FC with MCC , involveWe also found a stronger PAG-FC with visual cortex including cuneus, calcarine and superior occipital gyrus. In a previous study, these regions had a stronger pain-induced PAG-FC in migraineurs compared to controls , and ourFurthermore, we have to note that PAG also plays a critical role in autonomic processes, behavioural, cardiovascular, and respiratory responses to stressors and the different connectivity of PAG subregions may regulate different physiological functions , 46. TheIn accordance with the general notion that migraine intensity is similar in males and females , our parIn patients with fibromyalgia, increased activity of the angular gyrus and its stronger connectivity with pain- and analgesia-related brain areas correlated with pain intensity analgesia after listening to music . In addiThe superior parietal, middle temporal, and occipital gyri are also involved in pain processing, their activity correlated with subjective pain in a study using a thermal heat task . InteresThe PAG-FC with the above-mentioned regions may be also implicated in pain-related unpleasantness . Men genIn general, women are more likely to report severe headache-related disability compared to men . In our In general, female migraineurs have more frequent migraines than males . In our There are some limitations of our study that we should mention in this study. First, we have a small sample size in each studied group. Second, we did not have healthy control groups. Instead, we used literature results to compare our results to the sex differences in non-migraine subjects. Third, the fMRI data acquisition was assessed in two different types of MR scanners. However, we did not detect any scanner-related effects. The resolution of MR scanners could be also a potential limitation because scanning brainstem structures such as PAG is highly affected by physiological noise and the anatomical complexity of small this small region . In a coFinally, female hormones might affect pain processing and PAG function , 17 thusIn this study, a stronger PAG functional connectivity with regions of ascending pain pathways in female migraineurs indicates increased brain excitability that might be a risk factor for migraine. Therefore, the PAG network may contribute to greater sensitivity and migraine vulnerability distinctively in women. However, the average pain intensity of migraine attacks exhibits stronger PAG connectivity with regions involved in the subjective experience of pain in males which may represent a consequence of increased subjective pain experience. Based on this observation, male-specific mechanisms of migraine should gain more attention. The association between other migraine characteristics and PAG connectivity seems to represent general pain-related features of migraine without sex differences. PAG connectivity shows alterations in association with migraine-related disability suggesting a decreased descending pain control. In conclusion, we were able to identify general and sex-specific differences in PAG functional connectivity among migraineurs, and thereby, show with novel results how the “migraine brain” may be differently affected in females and males even in the interictal state.Preliminary data from this study were presented at the following event: 34th ECNP Congress 2–5 October 2021, Lisbon, Portugal poster presentation.juhasz.gabriella@pharma.semmelweis-univ.hu.The datasets presented in this article are not readily available because they contain information that could compromise the privacy of research participants. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by Scientific and Research Ethics Committee of the Medical Research Council (Hungary). The patients/participants provided their written informed consent to participate in this study.The study was designed and conceived by GJ and GK. ES, NK, and AG were responsible for subject recruitment and data collection. Data analysis was performed by KG with assistance from GJ, GK, CSA, and ME. GB, DD, DB, and GJ contributed to the interpretation to the data. KG, DB, and GJ wrote the first draught of the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Hungarian Academy of Sciences (MTA-SE Neuropsychopharmacology and Neurochemistry Research Group); the Hungarian Brain Research Program ; the National Development Agency (Grant: KTIA_NAP_13-1-2013- 0001); by the Hungarian Academy of Sciences, Hungarian National Development Agency, Semmelweis University and the Hungarian Brain Research Program (Grant: KTIA_NAP_13-2- 2015-0001) (MTA-SE-NAP B Genetic Brain Imaging Migraine Research Group); by the Thematic Excellence Programme  of the Ministry for Innovation and Technology in Hungary, within the framework of the Neurology and Translational Biotechnology thematic programmes of the Semmelweis University; by the National Research, Development and Innovation Office, Hungary (2019-2.1.7-ERA-NET-2020-00005), under the frame of ERA PerMed (ERAPERMED2019-108); and by the ÚNKP-20-3-II-SE-51 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund. The sponsors had no role in the design of the study, in the collection, analysis, interpretation of data, and in the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The association between miRNA-499 rs3746444 and a variety of autoimmune diseases has been reported. However, these results were contradictory and just focused on one or two autoimmune diseases. The present study aims to examine the possible association between rs3746444 polymorphism and the risk of autoimmune diseases.The studies that evaluated the association between miRNA-499 gene polymorphism and autoimmune diseases were retrieved. Five different genetic models were used to evaluate the association. The random-effects model was used to pool the effect sizes. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the associations. Stratification analyses were performed by ethnicity and type of autoimmune diseases. False-positive report probability (FPRP) was performed for determining noteworthy associations.Seventeen articles (twenty studies) involving 4,376 cases and 4,991 controls were identified and included in our meta-analysis. The pooled ORs of all eligible case-control studies indicated a significant association between miRNA-499 gene polymorphism and autoimmune diseases: . Stratified analysis indicated a significant association across both Caucasian  and Asian  populations. There was also a significant association in Behcet’s disease, rheumatoid arthritis, systemic lupus erythematosus, and ulcerative colitis populations.Our meta-analysis suggested that the miRNA-499 rs3746444 polymorphism was associated with an elevated risk of autoimmune diseases in the overall analysis as well as Caucasian and Asian populations. MicroRNAs (miRNAs) are small non-coding RNAs of 19 to 25 nucleotides, and their function is to regulate the expression of their target gene , 4.MiRNAs are present in many biological fluids and can regulate a broad range of physiologic and pathologic processes . VariantAn association between miRNA-499 rs3746444 and a variety of autoimmune diseases including rheumatoid arthritis , systemiThe present study aimed to examine the possible association between rs3746444 polymorphism and the risk of autoimmune diseases.Case-control studies included in this study met these criteria: i evaluated the association between miRNA-499 gene polymorphism and autoimmune diseases; ii available and sufficient data including the distribution of genotype frequency in case and control groups. The study exclusion criteria were: i review papers, editorials, comments; ii studies without controls; iii not provided enough information to calculate the odds ratios (ORs) and 95% confidence interval; iv Hardy-Weinberg Equilibrium (HWE) <0.05 in the control group.PubMed, Embase, Scopus, Web of Science, Wanfang, and Chinese National Knowledge Infrastructure databases were searched with the full electronic search strategy as follows: (“microRNA-499” OR “miRNA-499” OR “rs3746444” OR “Pre-miR-499”) AND  AND . The last search was performed on December 15, 2021. We also searched for additional pertinent studies through the references of all identified publications. There was no language restriction in the literature search.Two authors independently extracted the following data from the selected studies: first author; year of publication; country; ethnicity; sample size; genotype frequencies; type of autoimmune diseases; Genotyping methods; Hardy–Weinberg equilibrium(HWE) for controls. Study quality was assessed according to the Newcastle-Ottawa quality-assessment scale. Quality scores ranged from 0 to 9. The work of extraction of data was operated by 2 independent researchers (Kong and Ma). Any disagreement was discussed and resolved with a third author (Xu).The main meta-analysis compared the presence of miRNA-499 gene polymorphism among patients with autoimmune diseases as cases versus healthy subjects as controls. We assessed HWE via Chi-square test in the control populations. The association between miRNA-499 gene polymorphism and autoimmune diseases was estimated by odds ratios (ORs) and 95% confidence intervals (CIs). The strength of the association was determined based on five different genetic models: allelic model (T vs. C), heterozygote model (TC vs. CC), homozygote model (TT vs. CC), dominant model (TT vs. TC+CC), recessive model (TT+TC vs. CC). Because the random-effects model (DerSimonian and Laird method) can incorporate a heterogeneity parameter and enable the modeling of differences between studies, we used it to pool the effect sizes in our analysis , 20. A POur search initially yielded a total of 244 potential articles . After tAmong these studies, eight studies were carried out in the Asian population , 36, 37.A sensitivity analysis was applied to assess the influence of each study on the pooled ORs. When the association between any systemic autoimmune disease and miRNA-499 gene polymorphism was analyzed in model T vs. C, the exclusion of each study showed a significant association. The res9, 3, 3, and 2 of the included studies analyzed miRNA-499 gene polymorphism in patients with RA, SLE, UC, and BD, respectively. Based on the type of autoimmune diseases, subgroup analysis was performed to assess the relationship between miRNA-499 gene polymorphism and RA, SLE, UC, and BD. In RA studies, there was a significant association in some genetic models . In SLE studies, the result show that there has been a marked association in dominant model. In UC studies, two contrast genetic models (T vs. C and TT vs. CC) showed statistically significant association with random effect model . In BD studies, four contrast genetic models  showed statistically significant association with random effect model.To further minimize random errors to confirm the positive association between miRNA-499 rs3746444 and a variety of autoimmune diseases, we performed an FPRP analysis. The results are shown in Tables Autoimmune diseases include a wide range of human diseases, which are characterized by loss of immune tolerance to autoantigens, and the presence of autoreactive immune cells and/or autoantibodies against healthy cells and normal tissues . The etiThe miRNA-499 gene is located on chromosome 20, which regulates the expression of its target genes including IL-2, IL-6, IL-17RB, IL-21, IL-23a, and so on –46. IL-1Numerous case-control studies suggest that the miRNA-499 rs3746444 polymorphism is related to a variety of autoimmune diseases –14, and Ultimately, Seventeen articles (twenty studies) with 4,376 cases and 4,991 controls were included in our analysis. Our results suggested that the C allele of miRNA-499 rs3746444 T/C variant was associated with an elevated risk of autoimmune diseases under the allelic model. In addition, by subgroup analysis, we found that T allele in the Asian population and TT genotype in the Caucasian population were protective factors for predisposition to autoimmune diseases, respectively under allelic model (T vs. C) and dominant model (TT vs. TC+CC). Subgroup analysis by disease types showed the T allele and TT genotype behave as protective factors for predisposition to RA, SLE, and UC populations under some genetic models. Conversely, both case-control studies on BD showed that the T allele of miRNA-499 rs3746444 T/C was linked to an increased risk of BD.There are some previous genome-wide association studies (GWAS) relevant to Behcet’s Disease , rheumatThere are several limitations in our meta-analysis. First, there were only three studies on ulcerative colitis (UC) and two studies on Behcet’s disease (BD). Only one study each was related to autoimmune thyroid diseases, ankylosing spondylitis (AS), and Graves’ disease (GD). Because of the limited number of studies and types of autoimmune diseases, the results we have obtained so far still need to be updated in the future. Second, there were different geographic areas and genetic backgrounds in the included studies. The environmental factors might influence the pooled results. Moreover, we could not analyze the gene-environment interactions to investigate the association between rs3746444 polymorphism and autoimmune disease risk.Despite the limitations, there are some strengths in our meta-analysis. Seventeen articles (twenty studies) were included in our article. The number of studies was significantly greater than that in other meta-analyses published before. This could increase the statistical power in the overall analysis. Seven studies were related to RA in our analysis. There was sufficient data to fully confirm the association of RA and rs3746444 polymorphism.In conclusion, this meta-analysis suggested that the miRNA-499 rs3746444 polymorphism was associated with an elevated risk of autoimmune diseases in the overall analysis as well as Caucasian and Asian populations. Moreover, significant associations were also found in stratified analysis in Behcet’s disease, rheumatoid arthritis, systemic lupus erythematosus, and ulcerative colitis populations.S1 File(XLS)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


Patients with primary aldosteronism (PA) have cardiac remodeling due to hemodynamic and non-hemodynamic causes. However, component analysis of cardiac remodeling and reversal in PA patients is lacking. We investigated components of cardiac remodeling and reversal after adrenalectomy in patients with aldosterone-producing adenoma (APA).This study prospectively enrolled 304 APA patients who received adrenalectomy and 271 with essential hypertension (EH). Clinical, biochemical and echocardiographic data were collected in both groups and 1 year after surgery in the APA patients. The hemodynamic and non-hemodynamic components of left ventricular (LV) remodeling were represented by predicted left ventricular mass index (LVMI) (pLVMI) and inappropriately excessive LVMI .After propensity score matching, 213 APA and 213 EH patients were selected. APA patients had higher hemodynamic (pLVMI) and non-hemodynamic (ieLVMI) components of LV remodeling than EH patients. In multivariate analysis, baseline pLVMI was correlated with systolic blood pressure (SBP) and serum potassium, whereas ieLVMI was correlated with log plasma aldosterone concentration but not blood pressure. Post-operative echocardiography was available in 207 patents and showed significant decreases in both pLVMI and ieLVMI after adrenalectomy. In multivariate analysis, ΔpLVMI was correlated with SBP, ΔSBP, and pre-operative pLVMI, whereas ΔieLVMI was correlated with Δlog aldosterone-to-renin ratio (ARR) and pre-operative ieLVMI.This study concluded that extensive cardiac remodeling in APA patients occurs through hemodynamic and non-hemodynamic causes. Adrenalectomy can improve both hemodynamic and non-hemodynamic components of LV remodeling. Regressions of pLVMI and ieLVMI were correlated with decreases in blood pressure and ARR, respectively. We investigated cardiac remodeling and reversal after adrenalectomy in 304 patients with aldosterone-producing adenoma (APA) with 271 essential hypertension (EH) through hemodynamic and non-hemodynamic components as predicted left ventricular mass index (LVMI) (pLVMI) and inappropriately excessive LVMI (ieLVMI). APA patients had higher pLVMI and ieLVMI correlated with blood pressure and aldosterone. 207 APA patents showed decreased pLVMI and ieLVMI after adrenalectomy with ΔpLVMI correlated with blood pressure and ΔieLVMI correlated with Δlog aldosterone-to-renin ratio. This study demonstrated improved cardiac remodeling in APA patients through hemodynamic and non-hemodynamic causes after adrenalectomy, which correlated with decreases in blood pressure and ARR, respectively.Primary aldosteronism (PA) is a disease featuring excessive endogenous aldosterone , 2. PrevCardiac remodeling is prognostic and linked to heart failure progression . It maniHypertension plays a key role in the hemostasis of hemodynamics, is a well-known risk factor for LVH , and is Inappropriate left ventricular mass is defined as the ratio between measured and predicted left ventricular mass taking sex, body size, and cardiac workload into account. It represents a non-hemodynamic or neurohormonal cause of LVH, and it has been reported to offer additional prognostic value in patients with LVH , 13. A pIn this study, we compared measured, predicted, and inappropriately excessive left ventricular mass between a prospective cohort of patients with aldosterone-producing adenoma (APA) and EH controls, and evaluated their associations with cardiac remodeling and treatment response after unilateral adrenalectomy. We further investigated the pathogenesis of LVH due to hemodynamic and non-hemodynamic causes.We enrolled patients with PA and EH from October 2006 to January 2016 from National Taiwan University Hospital. The PA patients were all registered in the Taiwan Primary Aldosteronism Investigation (TAIPAI) database . Detailein vitro using a commercially available radioimmunoassay kit . All antihypertensive medications were discontinued for at least 21 days before measuring plasma PRA and PAC as suggested in clinical guidelines. Diltiazem and/or doxazosin were administered to lower elevated blood pressure when clinically indicated.Physiological assessments of systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained using a sphygmomanometer according to clinical guidelines. Plasma aldosterone concentration (PAC) was measured using a radioimmunoassay with a commercial kit , and plasma renin activity (PRA) was measured as the generation of angiotensin-I This study was conducted in compliance with the Declaration of Helsinki, and it was approved by the Institutional Review Board of National Taiwan University Hospital . Informed consent was obtained from all individuals before enrollment.The confirmation and diagnosis of PA and further identification of APA were made according to previously published protocols and algorithms , 17. PatA standardized echocardiographic ultrasound system  was used, and transthoracic echocardiographic images were obtained in fundamental imaging modes by experienced and qualified technicians blind to the clinical diagnosis. M-mode measurements, two-dimensional imaging with standard views, and Doppler ultrasonography were acquired in each patient.via the parasternal long axis view according to the guidelines of the American Society of Echocardiography (3 - (LV end-diastolic diameter)3]-13.6]  (Left ventricular (LV) dimensions, septal and posterior wall thickness, and LVEF (M-mode) were measured iography . Echocar3]-13.6] , then inemale=2) . Left veemale=2) . Inapproemale=2) . LVH wasemale=2) . The cutemale=2) . In thisAll continuous variables were expressed as mean ± SD if normally distributed and as median with interquartile range if non-normally distributed. The PA group and EH controls were matched using propensity score matching adjusted for age, sex, SBP, DBP, number of antihypertensive medication types, hypertension history using the Python-based extensions, FUZZY and PSM, in SPSS. All continuous variables were compared across groups using the Student’s t test  or the Wilcoxon rank-sum test , while the paired sample t-test was used if two groups were dependent. Categorical variables were presented as counts and percentages and were compared using the McNemar test. Equality of two proportions was assessed using the Pearson chi-square test. Data of PAC, PRA, and ARR were log-transformed due to non-normality as assessed using the Kolmogorov–Smirnov test. Pearson’s correlation tests were performed to determine correlations between LVMI/pLVMI/ieLVMI and ΔLVMI/ΔpLVMI/ΔieLVMI and clinical parameters. Significant determinants found in the Pearson’s correlation test (p ≦ 0.05) were then examined using a multivariate linear regression test with backward subset selection to identify independent factors predicting LVMI/pLVMI/ieLVMI and ΔLVMI/ΔpLVMI/ΔieLVMI. All statistical analyses were performed using SPSS for Windows version 25.0 . All tests were two-tailed, and a p value of ≤ 0.05 was considered to indicate statistical significance.In this study, we enrolled 304 patients with PA, all of whom had APA, and 271 EH patients. The basic clinical characteristics of the patients are listed in Echocardiographic parameters were then compared between the APA and EH groups before and after propensity score matching Table 2.Regression analysis of factors predicting LVMI/pLVMI/ieLVMI in the study cohort are listed in Among the 304 APA patients, echocardiography data 12 months after adrenalectomy were available in 207, and their clinical and echocardiographic data are listed in Comparison of the changes before and after operation of APA according to clinical cure or clinical non-cure was demonstrated in Regression analysis of factors predicting changes in LVMI/pLVMI/ieLVMI after adrenalectomy among the APA group are listed in There are five findings in this study. First, after propensity score matching for age, sex, SBP, DBP, hypertension duration, and use of antihypertensive, the APA patients had greater LVMI, pLVMI, and ieLVMI, with more LVH compared to the EH patients. Second, LVMI was independently associated with SBP, number of antihypertensive medication types, and log-transformed PAC, while pLVMI (hemodynamic component of LV remodeling) was associated with SBP, and ieLVMI was associated with log-transformed PAC (non-hemodynamic component of LV remodeling). Third, significant regressions of LVMI, pLVMI and ieLVMI and reversal of LVH were observed after adrenalectomy. Fourth, ΔLVMI was associated with SBP, ΔSBP, and Δlog-transformed ARR, while ΔpLVMI was associated with SBP, and ΔSBP and ΔieLVMI were associated with Δlog-transformed ARR. These findings clearly showed the factors associated with hemodynamic and non-hemodynamic component of LV remodeling in PA patients, and the factors associated with the regression of both components after adrenalectomy. Last, the study demonstrates that inappropriately excessive left ventricular mass (ieLVM) could be a novel and promising parameter evaluating aldosterone-induced non-hemodynamic left ventricular remodeling.A previous meta-analysis reported that LVH identified by ECG or echocardiography was highly prognostic and an independent risk factor for cardiovascular outcomes . In addiAn inappropriate increase in LVM, which is referred to as an abnormal (non-compensatory) increase in LVM as opposed to a normal (compensatory) LVM increase, has been reported to be more strongly associated with more metabolic risk factors . PreviouSeveral previous studies have reported differences in LVMI and LVH between PA and EH groups and improvements after surgery or medications. Rossi et al. reported increased LV wall thickness and LVMI with higher percentages of LVH and concentric remodeling in PA patients compared to matched EH controls, and that adrenalectomy markedly reduced LV wall thickness and LVMI in APA patients . Catena To the best of our knowledge, this prospective cohort study includes the largest sample size to date, with 304 PA patients  and 271 EH controls, and 213 matched pairs. The cases were matched using propensity score matching for sex, age, SBP, DBP, number of antihypertensive medication types, and hypertension history, with the aim of eliminating as many confounding factors as possible. The use of ieLVMI is different from previous studies and demonstrated the effect of excessive aldosterone on LVH in addition to adaptation to compensate for hemodynamic load. Our findings may help to improve the prediction of cardiovascular outcomes after eliminating the influence of hypertension. The utilization of LVMI, pLVMI and ieLVMI provided further insight into the pathogenesis of increased LVMI, a process involving mixed etiologies, in which pLVMI represented the hemodynamic component and ieLVMI the non-hemodynamic component. Correlation studies of LVMI/pLVMI/ieLVMI between the patients with APA and EH identified the factors associated with increased LVMI, while correlation studies of ΔLVMI/ΔpLVMI/ΔieLVMI after adrenalectomy identified the factors associated with changes in LV morphology and treatment effect of adrenalectomy. Of note, ΔLVMI was independently correlated with ΔSBP and ΔlogARR. Furthermore, ΔpLVMI was independently correlated with ΔSBP, while ΔieLVMI was correlated with ΔlogARR. These findings emphasize the treatment benefits of adrenalectomy, which both reduces blood pressure and aldosterone by removing the aldosterone secreting source in the adrenal gland, thereby resulting in LVH regression in both hemodynamic and non-hemodynamic pathways. The use of ieLVMI has the advantage of excluding the influence of blood pressure on the left structure. In addition, the exclusive association between ΔieLVMI and ΔlogARR consolidates the pathogenesis of aldosterone-induced neurohormonal LVH, which is independent of the influence of hypertension. Moreover, this shows that the treatment effect of adrenalectomy was not just from treating hypertension.There are several limitations to this study. First, this clinical study showed the association between preoperative and postoperative endocrinological and cardiac structural changes, however the study design cannot elucidate the actual causative role of aldosterone in LV remodeling. Second, our follow-up period was 1 year, and longer follow-up periods are warranted to examine whether there is any further late-onset effect of adrenalectomy. Third, we did not perform the saline load test with plasma aldosterone measurement and fibrinogen level on all the APA patients before and after the operation. Fourth, this study only investigated excess aldosterone and showed that elevated ARR increased LVMI, but the effect on cardiovascular mortality or morbidity was not investigated. Further long-term follow-up studies are needed to investigate the clinical impact of aldosterone excess on cardiovascular outcomes.Extensive cardiac remodeling through hemodynamic and non-hemodynamic causes occurs in APA patients. Adrenalectomy improved both hemodynamic and non-hemodynamic components of LV remodeling. The regression of pLVMI and ieLVMI were correlated with decreases in blood pressure and ARR, respectively. These findings provide further evidence of aldosterone-induced hemodynamic and non-hemodynamic LV remodeling in patients with PA, and the effect of adrenalectomy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by National Taiwan University Hospital (NTUH) Research Ethics Committee (REC). The patients/participants provided their written informed consent to participate in this study.C-TP: experimental design and manuscript writing. X-MW, C-HT, Y-YC and ZW-C: patient enrollment and experimental design. C-CC and B-CL: diagnosis helping, AVS, and report. C-WL and Y-LC: patient case management and experimental design. L-CL and Y-RC: diagnosis helping, echocardiography, experimental design. C-SH and Y-HL: patient enrollment, experimental design, funding raising, manuscript editing. All authors contributed to the article and approved the submitted version.This study was partially supported by grants from the Ministry of Science and Technology (MOST 106-2314-B-002 -048 –MY3) and National Taiwan University Hospital (NTUH 109-A141). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years, the new third-generation ultrathin bioresorbable-polymer sirolimus-eluting stent (BP-SES), characterized by some of the thinnest struts among commercially available devices (60–80 μm) and an amorphous silicon carbide coating, has been introduced for the treatment of coronary artery disease (CAD). The present study aimed to assess different clinical outcomes and safety of this drug-eluting stent in male and female patients in a real-world setting.The present study is a retrospective analysis including all patients treated with BP-SES between January 2017 and December 2019 at a single high-volume center. Follow-up data, including stress test results and clinical setting, were collected during outpatient visits or by telephone contact. Patients symptomatic for angina or with a positive stress test were addressed to CT scan/coronary angiogram. The main study outcome was target lesion failure (TLF), defined as a composite of cardiovascular death, target vessel myocardial infarction, or target lesion revascularization.P = 0.003) and with a lower body mass index (BMI)  compared to men, with no other relevant differences in baseline characteristics. Indication for percutaneous coronary intervention (PCI) was acute coronary syndrome in 86 (20.7%) of the cases, with no significant differences between male and female patients. A total of 558 lesions were treated with BP-SES stents, 90 in women and 468 in men ; cumulative stent length , and mean stent diameter  did not differ in women compared to men. Technical and clinical successes were achieved in all patients. Stent thrombosis (ST) occurred in 2 (0.5%) patients, both men. TLF occurred in 10 (2.9%) men and 2 (3.0%) women after a median follow-up of 402 days, without significant differences at log-rank analysis .Overall, 66 (15.9%) female and 349 (84.1%) male patients were included; women were older (median age 70 vs. 66, Ultrathin struts BP-SES showed to be a safe and effective option for the treatment of CAD in both women and men, with a very low ST rate and favorable long-term outcomes. Women have been historically underrepresented in randomized clinical trials, particularly in the cardiovascular field. Newly developed medical devices and drugs are too often tested in unbalanced populations in terms of gender in controlled studies, which are hardly representative of the real-world populations in which such devices are used . This liResearch and development of coronary DES have led to marked improvements in several stent technology features, from the type of eluted-drugs to polymer and struts design as well. Stent struts thickness has been progressively reduced, with favorable effects in terms of timing for endothelialization, local inflammation, vessel injury, thrombogenicity, and neointimal proliferation , 8. IntrWith the increasing use of ultrathin BP-SES in clinical practice, it is of utmost importance to test the safety and effectiveness of such devices different among gender in a real-world clinical setting, especially because patients treated in routine clinical practice may often present different features and offer different challenges compared to those included in RCT. The present study aims therefore to evaluate the short and mid-term outcomes in women and men treated with the implantation of ultrathin BP-SES during routine clinical practice.The present study is a single-center, retrospective analysis including all patients treated with the implantation of at least one ultra-thin BP-SES between January 2017 and December 2019. All coronary interventions were performed at Centro Cardiologico Monzino, IRCCS, Milan, Italy, a third-level high-volume cardiologic referral center. Choice of the type of implanted stents was left at the operator's discretion. No formal exclusion criteria were defined.For all patients, baseline clinical features, past medical history, indications for PCI, and main laboratory values were collected. Coronary anatomical characteristics, lesion features, and procedural data were registered as well. Chronic coronary syndrome, non-stent thrombosis (ST) elevation acute coronary syndrome , and ST-elevation myocardial infarction (STEMI) were defined according to the current European Society of Cardiology guidelines –13. TechIn hospital and post-discharge, medical treatment was prescribed according to current guidelines, in particular, all patients were on at least one antiplatelet drug before stent implantation. Clopidogrel with a loading dose of 600 mg and a maintenance 75 mg daily dose for 6 months was administered on top of aspirin in patients with chronic coronary syndromes, whereas patients with the acute coronary syndrome (ACS) were given a loading dose of Prasugrel or Ticagrelor followed by a maintenance dose of the respective drug for 12 months.All patients were prescribed a stress test 8–10 months after PCI and then yearly. Follow-up data were collected during the outpatient visit or, whereas not available, by telephone contact. Patients symptomatic for angina or with a positive stress test were addressed to CT scan/coronary angiogram. The main outcome assessed was target lesion failure (TLF), defined as a composite of cardiovascular death, target vessel myocardial infarction (MI), or target lesion revascularization . Secondary outcomes evaluated comprised individual components of the composite outcome, all-cause death, and stent thrombosis, defined according to the Academic Research Consortium criteria.Continuous variables were reported as median with interquartile ranges (IQR) or mean ± SD and were compared by Kruskal-Wallis non-parametric test; categorical variables are reported as numbers with percentages and were compared by Pearsons's Chi-square test or Fisher's exact test, as appropriate.Event rates are reported as per 100 patient-years; survival analyses were evaluated by Kaplan Meier curves and were compared by log-rank test.p-value of 0.05 was set as statistically significant. All analyses were performed using Jamovi version 1.6.21.0.A two-sided P = 0.003) and presented lower body mass index (BMI)  compared to men, whereas other baseline clinical features were similar between genders , whereas 51 lesions were treated with a different kind of DES. Mean stent diameter was similar across genders , whereas mean cumulative stent length was borderline longer in men . No differences in the rate of small vessel PCI were observed . Technical and clinical successes were achieved in all patients; no in-hospital deaths were recorded.As reported in P = 0.80 at log-rank; P = 0.075 compared to the general population), with no significant differences between men and women . In patients with ACS, 4 TLF occurred out of 86 patients , with no significant differences between women and men .After a median follow-up of 402 days (IQR 256-618), TLF occurred in 12 (2.9%) patients  approval of cardiovascular drugs included a significantly lower ratio of enrolled women compared to the diseased population, in particular for ACS and coronary artery syndromes. They calculated the participation to prevalence ratio, i.e., the ratio between the percentage of women in trial participants and the percentage of women in the disease population, and found it to be as low as 0.6 . Gender The third-generation ultrathin BP-SES included in our analysis has shown excellent results in RCTs in terms of effectiveness and safety. It outperformed durable polymer EES consistently across different populations  and diffThe main limitation of the present study is the retrospective design, which does not allow to control for unaccounted confounding. Moreover, the choice to implant the investigated BP-SES was left at the operator's discretion and not guided by the study protocol. The low number of adverse events recorded did not allow to perform a multivariate adjustment.Third-generation, ultrathin BP-SES does not appear to be associated with an increased risk of adverse outcomes in a real-world clinical setting, irrespective of the gender of the treated patients. Continuous post-marketing monitoring and larger-scale studies are needed to corroborate these positive results.https://zenodo.org/record/5236411#.YXbcXBrMKUk.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The studies involving human participants were reviewed and approved by Centro Cardiologico Monzino, IRCCS Ethic Committee. The patients/participants provided their written informed consent to participate in this study.SGi and DT: conceptualization and writing. PM, FF, AB, and PR: revision. GT, GS, and SGa: data collection. SGi: statistical analysis. DT: data analysis and revision. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chiral sulfoxides are valuable organosulfur compounds that have been widely used in medicinal and organic synthesis. Biocatalytic approaches for preparing chiral sulfoxides were developed in the past few years, mainly through asymmetric oxidation of prochiral sulfides. Recently, the application of sulfoxide reductase to prepare chiral sulfoxides through kinetic resolution has emerged as a new method, exhibiting extraordinary catalytic properties. This article reviews the chemical and biological functions of these sulfoxide reductases and highlights their applications in chiral sulfoxide preparation. Sulfoxide is a chemical compound containing a sulfinyl (SO) functional group attached to two carbon atoms. Chiral sulfoxides are noteworthy reagents in a wide range of industrial fields . Opticalrac) sulfoxides. A great number of oxidases, including Baeyer–Villiger monooxygenases  (L-methionine (L-Met) is a proteinogenic amino acid with a sulfide moiety. In living cells, methionine is relatively easily oxidized to sulfoxide (L-Met-O) under mild conditions . For manse (Msr) . In addise (Msr) . It is bse (Msr) .E. coli. Three Msrs are active toward Met-S-O: MsrA that reduces Met-S-O in both free and protein-bound forms, MsrA1 that is only active on peptide-bound Met-S-O, and fSMsr that only reduces the free Met-S-O. Two forms reduce Met-R-O called MsrB and fRMsr, which are mainly active on the Met-R-O in protein-bound and free forms, respectively. The sixth enzyme called mem-R, S-Msr, a membrane-bound protein, catalyzes the reduction of both Met-O enantiomers, either free or inserted into peptides   .-S-O, MsrA could be used as a biocatalyst to perform the kinetic resolution for the preparation of (R)-sulfoxides. Tremendous progress in the application of MsrA enzymes for preparing chiral sulfoxides through kinetic resolution has been achieved in the last 5 years. In 2016, our group reported the first application of MsrA for the preparation of (R)-sulfoxides  was noticed -sulfoxides through the reductive resolution of rac-sulfoxides. After optimizing the conditions, the phenyl methyl sulfoxide and several halogen substitutes on the aromatic ring were obtained with approximately 50% yield and more than 90% enantiomeric excess (ee) in the R configuration  was used as a reductant to regenerate the enzyme after catalysis. The (R)-sulfoxides were obtained with high enantioselectivity  under the substrate concentration up to 200 mM in 4 h. The substrate tolerance of this system was much better than most reported biocatalytic asymmetric oxidation strategies. Moreover, this system was also capable of preparing (R)-sulfoxides with substituents at the aromatic ring efficiently  exhibiting remarkably excellent activity and enantioselectivity toward a series of aryl methyl/ethyl sulfoxides was obtained  at initial substrate concentrations up to 320 mM (approximately 45 g/L)  . The iniE. coli overexpressing MsrA to establish this biphasic system   . Two antequence) . The actectively . In some protein .R enantiomer of sulfoxides, the application of this kind of enzymes for preparation of (S)-sulfoxides through kinetic resolution was possible. Nosek and colleagues have investigated several MsrB homologs, but no candidate was obtained (S)-sulfoxides preparation. Finally, the first MsrB with good activity and enantioselectivity was discovered (S)-sulfoxides in 93–98% ee were obtained through the catalysis of recombinant MsrB from Acidovorax species (named akMsrB), at initial substrate concentration up to 50 mM (S)-sulfoxides. Compared to MsrA, the MsrBs are less active and have higher substrate specificity (S)-sulfoxides.Based on the fact that MsrB is specifically active on the obtained . Considescovered . The (S)to 50 mM . The estcificity , limitin2 to MQ, the transfer of two electrons from the membrane domain to the molybdenum active site, and the reduction of DMSO to DMS , an electron transfer subunit that contains groups of cysteine residues for assembly of [4Fe-4S] clusters (DmsB), and a membrane anchor and quinol binding subunit (DmsC)  . The Dmst (DmsC)   . The sect (DmsC) .trans dithiolene ligand to form the more localized dithiolate type, dithiolene, lastly drives the reformation of Mo (VI) and the release of product DMS -isomer of methionine sulfoxide, instead of MsrB  (Mo-bisMGD) molecule at the active center. During the reduction of DMSO, the Mo atom cycles between the Mo (IV), Mo (V), and Mo (VI) states. Firstly, the Mo (VI) ion accepts one electron from the electron transfer subunit and one proton to form a hydroxideligated Mo (V) intermediate, followed by another one-electron/one-proton addition to yield the Mo (IV) form. The putative weakly bound water molecule is then displaced by DMSO, which is bound exclusively through the oxygen, with concomitantly weakening the S=O bond of DMSO. The electron-withdrawing ability of the duct DMS . Most DMduct DMS . Recentl of MsrB . This reRhodobacter sphaeroides for the kinetic resolution of methyl phenyl sulfoxide and obtained (R)-sulfoxides in 35–42% yield and 50–99% ee  -sulfoxide, whereas DMSO reductases from E. coli and Proteus species reduce the (S)-sulfoxide. Moreover, the same DMSO reductase could also show opposite stereospecificity toward different substrates  . Unlike cificity  -sulfoxides with a variety of functional groups are obtained with high ee (>97%) through this system. In addition, the dynamic kinetic resolution using DMSO reductase combined with chemical oxidant has also been developed to improve the yield and ee of chiral sulfoxide products -sulfoxide to the sulfide, followed by using a heterogeneous Ta2O5-SiO2 catalyst for the oxidation of sulfide to the rac-sulfoxide. The sequential deracemization process applied for rac-sulfoxide is cyclically performed, which leads to 97.5% ee of (S)-sulfoxide and a 56% yield after three deracemization cycles.Based on the fact that DMSO reductase can accept electrons directly from artificial electron donors, studies on electrochemical DMSO reductase systems using an artificial electrode as an electron donor have been reported . For insproducts . EnantioOverall, the DMSO reductases have been proven to be a valid alternative method for synthesizing chiral sulfoxides. Compared to Msrs, the substrate scope of DMSO reductases is much wider. However, the substrate tolerance is much lower and the enantioselectivity is not good enough. Moreover, as a membrane-bounding protein with three subunits, which is kind of too complicated, DMSO reductases have mostly been used as a natural whole-cell form. The commonly used genetic engineering techniques are difficult to apply for improving the yield and activity of the enzyme. In addition, the anaerobic environment is usually needed to ensure the growth of the bacteria and upregulate the synthesis of the reductase. These shortages severely limit the development of DMSO reductases in the application of chiral sulfoxides preparation. Thus, more efficient ways to improve the enzymatic system will be needed in the future.rac-sulfoxides catalyzed by these reductases provides a valid alternative way to prepare enantioenriched sulfoxides besides the conventional asymmetric oxidation strategy. Among these sulfoxide reductases, the MsrA exhibited extremely high substrate tolerance and catalytic rate, wide substrate scope, and low-cost and good operability in the reaction system, which implies very good prospects in industrial applications. However, the limitation in substrate structure  blocked its application. Future efforts in protein engineering like directed evolution for the improvement of substrate adaptability are still needed. Compared to MsrA, the MsrB exhibited relatively poor catalytic properties in the aspects of catalytic efficiency, substrate tolerance, and substrate scope. Therefore, screening homologs with better catalytic properties are the primary issues to be resolved. Compared to Msrs, the DMSO reductase displayed better substrate adaptability but worse substrate tolerance and catalytic rate. Moreover, the complexity in composition and structure and the difficulties in protein expression limited the improvement of DMSO reductase through biological techniques. Efforts in developing novel catalytic systems would probably offer new ways to synthesize enantioenriched sulfoxides using this enzyme. At last, although these enzymes provide excellent alternatives for the preparation of chiral sulfoxides, the natural defect of 50% maximum theoretical yield for kinetic resolution cannot be ignored. Developing dynamic reduction-oxidation enzymatic cascade reaction might be a solution to overcoming this drawback in the future.This mini-review mainly focuses on developing sulfoxide reductases and identifying their chemical and biological functions and applications in chiral sulfoxide preparation. The properties and prospects of these enzymes, especially MsrA, have been highlighted and discussed. The asymmetric reductive resolution of


Escherichia coli.An elaborate network of sRNA-mRNA interactions controls the pathogenicity of enteropathogenic  Escherichia coli (EPEC). The emerging network comprises hundreds of sRNA-mRNA pairs, including mRNAs of virulence-associated genes interacting with known sRNAs encoded within the core genome, as well as with newly found sRNAs encoded within pathogenicity islands. Some of the sRNAs affect multiple virulence genes, suggesting they function as hubs of virulence control. We further analyzed one such sRNA hub, MgrR, and one of its targets identified here, the major virulence-associated chaperon, cesT. We show that MgrR adjusts the level of EPEC cytotoxicity via regulation of CesT expression. Our results reveal an elaborate sRNA-mRNA interactome controlling the pathogenicity of EPEC and reinforce a role for sRNAs in the control of pathogen-host interaction.Small RNAs (sRNAs) exert their regulation posttranscriptionally by base pairing with their target mRNAs, often in association with the RNA chaperone protein Hfq. Here, integrating RNA-seq–based technologies and bioinformatics, we deciphered the Hfq-mediated sRNA-target interactome of enteropathogenic  Escherichia coli (EPEC) is a human bacterial pathogen causing conditions ranging from asymptomatic colonization to persistent life-threatening infantile diarrhea   , 0.3 to 0.6], or overnight growth of static culture on LB medium at 37°C. The former condition strongly induces the expression of the virulence genes, and the latter represses their expression. These conditions are termed hereinafter “activating” and “nonactivating,” respectively. Our data showed that the levels of Tir and BfpA were elevated in the Δhfq mutant (fig. S1). However, the overexpression of BFP protein due to the lack of Hfq appears to impose stress (perA (encoding the BFP master regulator) nonfunctional, through a frameshift mutation (fig. S1). These results suggest that Hfq, presumably in concert with sRNAs, acts to prevent overexpression of the T3SS and BFP genes, where overexpression of the latter might lead to instability.To evaluate the roles of Hfq and sRNAs in virulence, we aimed at comparing the transcriptome of wild-type EPEC with that of an isogenic Δhfq mutant, using the mutant version where pMAR2 was stabilized by the acquisition of the perA mutation (fig. S1). RNA was extracted from cultures grown under either the activating or nonactivating condition and compared between wild-type and Δhfq strains by DESeq2 . We further noted that Hfq has a clear repressive effect on the expression of the LEE genes , encoding a LuxR-type transcription factor, involved in advancing the transition of E. coli from planktonic to adhesive lifestyle   to wild-type and Δet al. (http://RILseqDB.cs.huji.ac.il), a database of Hfq-mediated RNA interactions determined by RIL-seq.To explore the involvement of sRNAs in EPEC virulence at a global scale, we used RIL-seq, our recently developed methodology for in vivo detection of the Hfq-dependent RNA-RNA interactome (fig. S3) . We searched by blastn . The genomic context of glnZ binding site differs among species, i.e., there are large insertions/deletions and the distances of the predicted binding site from the stop codon and the Rho-independent terminator of glnA are not fixed (fig. S4). This highlights the conservation variability of 3′UTR-derived sRNAs, where some are evolutionarily conserved throughout their length [e.g., the recently discovered CpxQ . A total of 479 of these interactions were previously revealed using RIL-seq in et al. . We selected for experimental verification five of the accessory genome–encoded sRNA candidates, termed here PasA, PasB, PasC, PasD1, and PasD2 . These sRNA candidates ranked high by their number of interactions, including interactions with virulence genes (tables S3 and S5), and three of them showed complementarity to a common motif identified in their target RNA sequences (fig. S5). We performed Northern blot analysis that corroborated the sizes of these sRNAs as predicted by the RNA-seq read coverage  and their Hfq-dependent stability . PasA, Pfp genes . TogetheExamination of the targets of core-encoded and accessory genome–encoded sRNAs revealed that under both conditions, core-encoded sRNAs and accessory genome–encoded sRNAs were found to interact with both core-encoded and accessory genome–encoded targets. Several sRNAs interact mostly with core-encoded targets in one condition and with accessory genome–encoded targets in the other condition . As manyper-bfp genes, and 45 (33%) involved LEE genes. Under the nonactivating condition, the virulence-associated network is less dense, containing only 97 nodes and 90 edges (per-bfp genes and seven (7.8%) involved LEE genes. Notably, several sRNAs, such as MgrR and Spf, are hubs of interactions involving virulence-associated RNAs in the network of activating condition.To obtain a global view of the impact of sRNAs on EPEC virulence, we focused on sRNAs interacting with at least one target that is encoded in the accessory genome of EPEC. To increase the reliability of the results, we considered only RNA-RNA interactions that were identified in at least two individual libraries in addition to their identification in the unified library , 34 . Consistent with the RNA-seq results (table S2), the blots showed a slight increase in Tir, CesT, and intimin levels in EPEC Δhfq compared to the wild-type strain . To characterize the mechanism by which MgrR down-regulates the production of CesT, we fused gfp to the chromosomal cesT, generating two strains that contain either a transcriptional or a translational fusion. Overexpression of a plasmid-born MgrR in these strains caused a substantial decrease in the fluorescence intensity of the translational fusion, while the transcriptional fusion showed no decrease in green fluorescent protein (GFP) intensity .We then applied Western blot analysis to test the impact of MgrR expression on the levels of the three proteins encoded by the LEE5 operon: Tir, CesT, and intimin. We included an e strain . Overexpe strain . When contensity , suggestcesT, we have mutated two adjacent nucleotides in the identified binding site . Overexpression of MgrR in EPEC cesT* mutant strain could no longer repress CesT production  and found that the cesT deletion mutant is highly toxic to the host cell . The cohort of the newly described accessory genome–encoded sRNAs expands the list of unique sRNAs found in EPEC and the closely related EHEC strains. These include sRNA genes located within prophages identified by prediction followed by Northern blot analysis in EPEC , a major regulator of biofilm formation (vpsT) and aphA, which is a major regulator of both biofilm formation and virulence , we find that cesT is ranked at the top with 1791 chimeric fragments , followed by eptB, bfpC, ygdQ, tir, and grlR . Among the 13 targets with >100 chimeric fragments, nine are unique to EPEC, including one encoded on the small plasmid p5217  in a final concentration of 0.9 mg/ml, and immediately frozen in liquid nitrogen. The frozen pellet was subjected to two cycles of thaw and freeze at 37°C and in liquid nitrogen, respectively, and RNA was extracted using Tri-Reagent (Sigma-Aldrich). RNA-seq libraries were constructed by the RNAtag-seq method  (et al. (RIL-seq computational analysis procedure was conducted as detailed in Melamed et al. (et al. (As we realized that the annotation of sRNA-encoding genes in EPEC genome was only partial, we extended this annotation, identifying by blastn (et al. (Following Melamed Additional information related to the data annotation, network construction, and small-scale experiments is provided in the Supplementary Methods.


COVID-19 is a disease with a spectrum of clinical responses ranging from moderate to critical. To study and control its effects, a large number of researchers are focused on two substantial aims. On the one hand, the discovery of diverse biomarkers to classify and potentially anticipate the disease severity of patients. These biomarkers could serve as a medical criterion to prioritize attention to those patients with higher prone to severe responses. On the other hand, understanding how the immune system orchestrates its responses in this spectrum of disease severities is a fundamental issue required to design new and optimized therapeutic strategies. In this work, using single-cell RNAseq of bronchoalveolar lavage fluid of nine patients with COVID-19 and three healthy controls, we contribute to both aspects. First, we presented computational supervised machine-learning models with high accuracy in classifying the disease severity (moderate and severe) in patients with COVID-19 starting from single-cell data from bronchoalveolar lavage fluid. Second, we identified regulatory mechanisms from the heterogeneous cell populations in the lungs microenvironment that correlated with different clinical responses. Given the results, patients with moderate COVID-19 symptoms showed an activation/inactivation profile for their analyzed cells leading to a sequential and innocuous immune response. In comparison, severe patients might be promoting cytotoxic and pro-inflammatory responses in a systemic fashion involving epithelial and immune cells without the possibility to develop viral clearance and immune memory. Consequently, we present an in-depth landscape analysis of how transcriptional factors and pathways from these heterogeneous populations can regulate their expression to promote or restrain an effective immune response directly linked to the patients prognosis. COVID-19 derives from SARS-CoV-2 infection, having diverse clinical symptoms according to the infection severity. There are still many unresolved questions regarding the disease pathogenesis and the reasons underlying the high variations on clinical courses, ranging from asymptomatic forms to severe manifestations. So far, illness severity is cataloged according to the clinical manifestations. However, physiological symptoms do not always reflect the capability of patients to overcome the disease. A molecular characterization based on signatures and alterations at the cellular level according to illness severity can streamline the clinical management and treatment. Interestingly, the responses of immune and non-immune cells recruited during the infection determine the clinical outcome of the disease. Recent single-cell studies show the differences of these cells, characterizing the immune landscape during infection . ThroughRecent single-cell data from bronchoalveolar lavage fluid (BALF) of 3 healthy controls and 9 COVID-19 patients described the cellular composition of immune cells and the expression of some cytokine/chemokine and chemokine receptors in each clinical response (Raw scRNA-seq data of Covid-19 patients were obtained from GEO (GSE145926). To sum up, bronchoalveolar lavage fluid (BALF) cells were collected from 3 healthy controls and 9 patients grouped according to their symptoms as moderate, and severe. Samples were sequenced using 10x Genomics technology  for the XGBoost algorithm. Shap values utilizes a game-theoretical approach to proceed with the best interpretation and explanation of the output of our machine-learning model. To visually identify the most relevant genes that contribute to the classification, we plotted the aggregate explanation plot, which comprises the SHAP values for each gene and their average expression levels  algorithm. This is a supervised algorithm to accomplish tree classification widely used in machine-learning , 18. AllTo identify the cell type in each cluster, differentially expressed genes were gathered using the FindMarkers function and compared to marker genes for each cell type. We got the marker genes from two sources: 1) The LM22 compilation containing 22 functionally defined human immune subsets profiled by microarrays ; 2) baseAccordingly with the T & NK separation and using the same parameters, we analyzed the heterogeneity within epithelial cells. We used 14 marker genes to identify five epithelial subtypes: alveolar type I (AT1), alveolar type II (AT2), secretory, squamous, and ciliated , 22  using the top 20 principal components of the PCA and a resolution of 0.5. Data subsets representing each cell type were used to study their regulatory network inferences.via Viper which obtains a p-value and a normal enrichment score when it compares the regulon enrichments score with a null model which is randomly generated by permuting the samples. We took the top TFs value for each population: 50 for macrophages, monocytes and epithelial cells, 30 for T/NK cells and 100 for dendritic and B cells. A differentially expressed analysis and clustering were performed based on the TF activity, the used parameters were: the top 50 principal components of the PCA, a resolution equal to 0.8, uwot method, and the cosine metric. We used the framework provided by Seurat.Functional pathway analysis was performed with PROGENy by the computation of top 500 pathways activity scores , 24 implPathway enrichment analysis was done using the Gene Set Enrichment Analysis (GSEA) . Gene seRecently single-cell RNA studies on patients with COVID 19 suggest that specific immune and non-immune cells determine the disease severity , 9, 28. Methods) and calculated the SHAP value for each gene, a numeric value that ranges from positive to negative values and quantify the level of contribution of the gene into the classification , assessing the model performance, and identifying variables that contribute to the classification. As a result, our machine-learning model correctly classified cells from moderate and severe patients with an accuracy of 0.98 for testing data  . Besidesvia ERK/MAPK pathway  cells take part in the defensive frontline against viruses. They induce self-destruction of virus-infected cells by apoptosis. Additionally, NK cells set the maturation of dendritic and T cells. Studies have reported that positive SARS-CoV-2 patients have decreased circulating NK cells . Moreovevia the non-canonical pathway associated with long-lasting pro-inflammatory mediators production  are a class of bone marrow derived cells originating from lympho-myeloid hematopoiesis that link the innate sensing of pathogens and the activation of adaptive immunity. DCs recognizes and responds to pathogen-associated and danger-associated signals, molding the inflammatory response . To unveHaving identified the single-cells classified as DC, we applied DoROthEA and obtained the TFs activated or inactivated within each disease severity. At a glance, our analysis showed that there was a clear signature dividing a set of active and inactive TFs in moderate patients . This fiIn detail, the host innate immune system is the first line of defense after viral infection. Germline-encoded pattern-recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs) related to viral infection and are responsible for initiating the biochemical signaling cascades that orchestrate the innate immune response. PRRs are a large group of proteins that include either membrane-bound receptors or cytosolic receptors. Some examples of membrane-bound receptors include C-type lectin receptors (CLRs), and the cytosolic receptors compromise the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) , 140. BoFinally, the results obtained using PROGENy . TolerogT cells exert primary functions in viral containment and clearance. Their role on SARS-Cov-2 infection is negatively affected by lymphopenia, which is related to disease severity and majorly impacts these cells , 153–155Functional pathways analysis with PROGENy showed pathway activation among patients and controls . As far in silico study associates JUN as a therapeutic target to reduce fibroblast proliferation and inflammation , as described in infections led by the influenza virus and SARS-CoV-1 . Even thvia matrix metalloproteinase-9 (MMP-9) due to herpes simplex infection  in distinguishing moderate and severe patients samples , 224. FuThe SARS-CoV-2 has a primary entrance to the human body through respiratory epithelial cells. Moreover, the virus also infects and impairs monocytes and macrophages , 230. Thin-vitro study showed that IFN signaling was time-dependent, it takes time to ISG, and IFN signaling mediated by TGFβ developed an antiviral state  promoting IL-6 and innate cell recruitment, which exert a massive pro-inflammatory condition affecting the adaptive response blocking antigen presentation. Carrying on this downfall process, immature DCs impairment induces massive cytokine release, leading to more innate cell recruitment, cytokine release, and fibrosis. T, B, DCs, and neutrophils exacerbate the condition releasing IL-6 deploying an infective adaptive response aggravated by the loss of germinal centers, consequently impairing immune memory. Thus, despite severe patients showing activation IFN-I, their response might not be adequate. NFKβ pathway has been identified impaired in the broncho-alveolar space cells; mostly all the analyzed cells show impairments. Overall, severe patients activated the described cells pushing the system into a hyper-inflammation state that might cause patients to die.Several studies have used the same dataset to evaluate the immunological effect in COVID patients. Among the differences: Park and Lee identified the effect on neutrophils glucocorticoid receptors on severe patients and described in general terms cytokines released by myeloid cells . OverholIt is essential to consider that patients were under pharmacological treatment at the moment samples were taken. Four of six severe patients took interferon, methylprednisolone, and ribavirin. Methylprednisolone is an immunosuppressive drug; it stops or delays pneumonia. Moreover, severe patients did not show an immunosuppressive effect. Cells for this group have activation of pro-inflammatory pathways and cytokine release. Therefore, the drug effectiveness might be reconsidered. All patients except one moderate were under interferon treatment. Despite the implication that the interferon might be beneficial, we considered that these effects are conserved across data, excluding a possible bias.As a final remark, the present study set the integration of cells belonging to the immune landscape for COVID-19 patients. Having each cell’s global and particular contribution made the therapeutic options clearer with their scopes and limitations. Along with this work, we have drawn some future perspectives. First, as soon as more available single-cell RNAseq data from BALF and PBMC, it will allow us to explore the scope of our conclusions and its limitations regarding variables such as ancestry, treatments, and the prevalence of different comorbidities. Second, given the complex nature of the disease, we should move toward integrative studies capable of integrating clinical data and other high-throughput technologies that complement our conclusions and set tactics to control illness severity. Machine-learning and systems biology are indispensable tools to reach this goal , 246. Wehttps://github.com/resendislab/Covid-19_scRNAseq.The scRNA-seq data used in this study is publicly available in GEO GSE145926) (45926 (1)AV-J, UA-PDL, and OR-A conceived and designed the re-analysis of single cell data. AV-J did the computational analysis of single-cell data. UA-PDL and MM-G analyzed single-cell data using progeny and dorothea. AV-J and EM-O performed the enrichment analysis, and the progeny and dorothea analyses. Finally, OR-A applied machine-learning on single cell data. All authors analyzed and interpreted the data. OR-A supervised the research. All authors wrote, discussed, and reviewed 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.