Alterations of the lipid compositionin cells, tissues, or organelles have been associated with a largenumber of diseases, including inflammation, cancer, and degenerativeor metabolic disorders.19 Among them,high-resolution mass spectrometry (MS) currently represents the mostpopular technique due to the unprecedented sensitivity, mass accuracy,and resolving power offered by modern MS instruments.20 In addition, separation techniques such as liquid chromatography(LC) can be conveniently coupled to the MS analyzers to attain a priorsample separation that reduces ion suppression phenomena and increasessignal-to-noise ratio for low-abundance analytes.22 Moreover, the recent development of MS devices with ion mobilityspectrometry (IMS) can support further characterization of the measuredlipid species and increase the identification confidence in untargetedstudies.22 Despite these technologicaladvances, only a few epilipid classes 23 have been extensively studied over the past decade, and our globalunderstanding of the “epilipidome” (bio)chemistry isstill very limited.24 The main obstacle to a comprehensiveepilipidome analysis and annotation arguably lies in its intrinsicchemical complexity, which causes a series of both analytical- andcomputational-related challenges:Several analytical approaches have been applied to lipidsanalysisand have been extensively reviewed elsewhere.Low abundance. Epilipids generally occur withan inherently low abundance and transient nature within biologicalsystems .25 As a consequence, despite the exceptional sensitivity reached bymodern MS detectors, the signal produced by modified lipids is oftenclose to the instrumental limit of detection.16 This represents a major obstacle to the use of shotgun and/or MS-imagingmethods in epilipidomics studies as they suffer from low overall sensitivitydue to scarce ionization efficiency of low-abundance metabolites.Hyphenated techniques  not only provide an additionalseparation dimension but also can increase the overall dynamic rangeby reducing ion suppression effects. For these reasons, LC-MS currentlyrepresents the technique of choice for the study of epilipids.(1) High structural diversity. The nature andlikelihood of lipid modifications are largely determined by the molecule’schemical structure. In fact, most of the reactions occur at energeticallyfavorable sites of the molecule.26 Forexample, double bonds are deemed the most susceptible sites to electrophilicaddition in polyunsaturated fatty acids (PUFAs), while allylic andbis-allylic positions are prone to radical attack. In addition, chemicalmodifications may also involve the lipid headgroup, vinyl ether moieties,and ester functional groups.29 Considering the lipids’remarkable structural diversity, the large number of potential modificationsites and the range of potential reactions , an enormous number of chemically distinct derivativesis possible.26 In this regard, Ni et al.estimated the epilipidome size by knowledge-based and systematic enumeration,only considering a few main oxidative modifications  ications 1.16 IndeLarge number of isomers/isobars. A directconsequence of the vast chemical space covered by the epilipidomeis the significant number of isobaric  and isomeric(same exact mass) species. As an example, querying the exact mass649.40 with a tolerance of ±0.05 Da in the LIPID MAPS StructureDatabase (LMSD)32 returns three distinctoxidized phosphocholines characterized by different elemental composition Different fragmentation compared to native lipids. Fragmentation mass spectra can be used as “molecular fingerprints” to assign atentative annotation to the unknown detected metabolites.33 Reference libraries of MS/MS spectra can beused to automatize the metabolite annotation using a spectral matchingapproach . Althoughthis currently represents the most effective approach for metaboliteannotation,35 two main obstacles severely limitits application to epilipids. First, comprehensive collections ofcommercially available analytical standards are currently lackingfor these species. This prevents the acquisition of reference MS/MSspectra from the pure compounds and their integration in existinglibraries. Second, the fragmentation patterns of epilipids may significantlydiffer from those of parent molecules. In fact, even “small”modifications in the lipid chemical structure can produce substantialdifferences at the MS/MS fragmentation level. For instance, both 5-hydroxy-PGI1and 6-keto-PGF1α originate from 5Z,8Z,11Z,14Z-eicosatetraenoicacid (arachidonic acid) and even share the same elemental composition; nevertheless, theyproduce significantly different fragmentation spectra.36 As a consequence, MS/MS libraries of nativelipids cannot be effectively exploited for the annotation of modifiedspecies as it would produce unreliable results.(4) Nomenclature. Lipid nomenclature has historicallyrepresented an arduous task due to the lipidome’s structuraldiversity and complexity.40 A systematic shorthand notation for MS-based lipidstructure annotation has evolved in the past two decades to allowtheir unequivocal reporting in a clear and succinct way.42 However, despite the recent efforts of LIPID MAPS,38 a unified nomenclature scheme for modified lipid species throughoutall the lipid categories is still lacking. As a consequence, improperannotation and over-reporting is commonly found among research papers.43 As an example, 1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine (LMGP20010008) has been reportedwith at least six different, but similar names in recent publications.49 Although these names may be very similar and/or differ only in onecharacter, a simple string matching would fail when parsing lipidnames . This reduces the usability of referencedata and complicates the unified computational treatment of lipidnames.50 Common abbreviation systems have been adoptedby the community only for a few oxylipin species -HpETE,and PGE2).53 However, these abbreviations carry almost no structure-relatedinformation and require the researcher to possess prior knowledgefor their correct interpretation.(5) 55). We placed our emphasis on GUI-based software as they provide moreusable solutions for researchers lacking programming skills.56 Finally, emerging computational tools that webelieve can support the analysis of LC-MS epilipidomics data are described.Although the above-mentionedchallenges are currently hamperingthe study of the epilipidome through MS-based methods, they can beaddressed (or at least mitigated) by software-assisted data analysispipelines. Dedicated tools have started to be available; yet, theinformation on the existing solutions is rather unsystematic, hinderingtheir large-scale implementation by the community. In this context,the present review focuses on the computational tools and approachescurrently available for the analysis of LC-MS epilipidomics data.In the following sections, a general epilipidomics LC-MS workflowis described with a focus on the data analysis steps as well as thechallenges that computational tools are expected to overcome lipidomicsstudy including all data processing steps is described 3. We divomics experiments, the term feature typically refersto a two-dimensional signal  that represents a chemicalcompound. The first step in untargeted LC-MS data processing is normallythe “feature detection” (a.k.a. “peak picking”),where boundaries  and intensities are determined for all the features detectedin the analyzed samples.57 The final goalis to turn the complex LC-MS raw data into a list of detected featuresready for further downstream data analysis.58Feature detection normally involves several consecutivesubsteps, including extracted ion chromatograms (EICs) constructionand deconvolution, feature filtering , alignment,etc. We identified two of these substeps as potential pitfalls whendealing with epilipidomics data.In LC-MS-based feature. Intuitively,this threshold has to be chosen with particular care in epilipidomicsapplications, due to the natural low abundance of modified lipid species.In fact, an overly high noise cutoff will lead to the erroneous discardingof relevant signals. On the other hand, an overly low noise thresholdunavoidably produces an exponentially larger number of backgroundsignals to be retained in the final data table, which extends thecomputation time/cost and might complicate downstream processing steps.This problem can be mitigated by fine-tuning the feature detection algorithm parameters and by setting additional constraints  for an LCsignal to be considered an actual feature.62First, during the EIC buildingstep, the user is normally asked to set a noise level threshold whichdetermines the minimum signal intensity  foran LC peak to be retained as a 64 manual tuningand visual inspection of the results is usually needed to ensure resultsfidelity. However, this requires the user to have a good understandingof the  algorithm parameters to be set, since evenlittle changes in these settings have been shown to have a dramaticimpact on the results.66Second, untargeted LC-MS analyses typically rely on genericchromatographiccolumn chemistries and gradients. Under these conditions, positionalisomers often exhibit similar chromatographic behavior, producingcoeluting and/or shoulder peaks. The EICs deconvolution step aimsat correctly splitting neighboring LC peaks that do not present afull baseline separation. Although robust algorithms have been developedfor this purpose,16 MS-based identification of lipids can be carriedout at six levels of structural information as described by Liebischet al.,37 with the highest achievable levelbeing the “fatty acyl/alkyl/sphingoid base structure”.As for the annotation of small molecules in general, assignment ofthe elemental formula to the (unknown) m/z signals of interest is the first committed step towardits structural identification.67 As discussedabove, the epilipidome covers a remarkably vast chemical space overa relatively narrow molecular weight range, which inevitably leadsto the ample occurrence of isobaric species. Therefore, the sole exactmass match is usually not sufficient for an unequivocal elementalcomposition assignment as many candidates would fall within the massrange search. In this respect, the isotope pattern information representsan irreplaceable means to rule out incorrect candidate formulas andraise the annotation confidence.68 Thenext step is the tentative annotation of the chemical structure of(epi)lipid based on the collected MS/MS data. The fragmentation spectraare either compared to reference spectral libraries or annotated usinga rule-based approach.69 However, epilipids are currentlyunderrepresented in reference MS/MS spectral libraries  and subsequentlymatched against the experimental spectra.71Lipid annotation is unanimouslyrecognized as the primary bottleneck in (epi)lipidomics studies.ries see  due to t72 In this regard, additional separation dimensionsprior to the MS acquisition can provide valuable information to raisethe annotation confidence. For instance, a chromatographic separationcan avoid coeluting isomers being cofragmented and prevent the generationof chimeric MS/MS spectra. Annotation constraints based on RT and/orIMS-derived collisional cross section (CCS) information can be includedin the identification strategy to filter out incorrect candidate structuresand reduce the false discovery rate.73Regardless of the annotation strategy, multiple structurecandidates  are normally returned.74 Targeted applications rely on calibration curvesand/or the addition of internal standards for the accurate quantificationof the analytes of interest,75 althoughthis is clearly limited to those molecular species for which analyticalstandards are available. A semiquantitative approach can be pursuedby using a single internal standard per lipid class.76 In the case of modified lipids, using analytical standardsfrom the same subclass species could represent a viable alternative. In practice, untargeted studiesdo not normally require absolute quantification levels and the measurementof the analyte abundance is simply based on the chromatographic peakarea or height.77 In this regard, it mustbe taken into account that a given analyte can generate multiple ionspecies  that will be recognized as distinct featuresdue to the different parent m/z.As a result, the overall signal will be distributed over multipleentries in the final feature list. Therefore, relying on a singleadduct for the feature quantification could introduce biases in thedownstream analysis. The number and type of generated adducts do notdepend only on the chemical structure and characteristics of the individualmolecule, but also on experimental variables difficult to control. Moreover, certain structural modificationscan change the ionization behavior and efficiency with respect tothe native species .78 For instance, the majority of oxidized phosphatidylcholines  are generally detected as formateor acetate adducts, while truncated oxPCs with terminal carboxylicmoieties  tend to form [M–H]− adducts.44 Algorithms forthe automatic grouping of multiple ion species produced by the samechemical entity can address this problem. In the specific case ofepilipids, tools able to “dynamically” pick the correctadduct species for quantification would be highly desirable.Following the identification step,the analyst is often interested in the absolute or relative quantificationof the annotated metabolites across the analyzed samples.One of the ultimate goals ofuntargeted LC-MS data analysis pipelines is to enable an informativevisualization of the annotation and quantification results. Variousdata displaying strategies have been proposed over the years to inspectidentification results, spot misannotations, etc. In this section,we report two data visualization tools developed in different researchfields and later adapted to (epi)lipidomics applications: Kendrickmass defect and Circos plots.80 The KMD plot enables the representation of the whole LC-MS dataset in a single chart, where compounds containing repeated structuralfeatures form easily recognizable homologous series. Briefly, a repeatingunit of interest  is chosen, and the correspondingKendrick mass factor is computed as the ratio between its nominaland exact mass. This factor is then used to create the Kendrick massscale and calculate the Kendrick mass defect. The KMD plot is thengenerated by plotting the KMD of the selected structural feature againstthe m/z or the Kendrick mass. Moredetails about “Kendrick analysis” in general can befound in Fouquet, 2019.81 Although originallydeveloped for petroleum analysis,81 KMD(CH2) and/or KMD(H) can be used to highlight differences in theacyl chain length and saturation of homologous lipid species.85 Korf et al. created a KMD plot by plotting two KMDs against eachother  and used it to identify more than 100lipid species inChlamydomonas reinhardtii.82 In the case of epilipids, the specificmodification can be used to identify modification products of a lipidclass in complex samples. For instance, Helmer et al. plotted theKMD(H) against the KMD(O) to display cardiolipin oxidation productsanalyzed via LC-MS  can be also integrated in the KMD plot in the form of acolor-coded scale to enable further visualization possibilities.83 The resulting three-dimensional KMD plot canbe particularly useful to confirm identification results and/or recognizefalse annotations. In fact, clear class-specific trends are oftenobservable depending on the applied chromatography .83The Kendrick mass defect (KMD)plot is a graphical data representation designed to assist the identificationof compounds that include repeating units in their chemical structures.ia LC-MS 4A.83 The86 Although originally created for genomic data visualization,86 Circos diagrams can be adapted to other datatypes and have recently found applications in many different fields,including epilipidomics. For example, Jha et al. employed the Circosplot to visualize 96 hepatic lipid measured in 385 mice livers andtheir fold change between control and treated groups.87 Multilayered annotations were also added to highlight thesignificant intergroup correlations, including the strength and approximatechromosomal position of the identified quantitative trait loci. Besidesgeneric packages designed to create Circos plot with any type of data,89 Ni et al. developed the Python package LipidCircos for the generationof Circos charts specifically dedicated to epilipidomics data.44 In particular, LipidCircos intends to displayand highlight relationships between identified epilipids and theircorresponding native precursors, as well as overlaying customizedinformation such as quantification data across samples and/or timepoints.44 In the publication, the authorsshowcased the package capabilities by generating a Circos plot todisplay oxidized phospholipids and their corresponding native species,identified in rat primary cardiomyocytes treated with peroxynitritedonor SIN-1 over 16 h  as they represent a more accessiblesolution for researchers lacking coding expertise.Several freely available tools allow the processingand analysisof LC-MS (epi)lipidomics data. Various R96 Lipostar,60 LipidMatch Flow97 (recently introduced as an update to the LipidMatchR package91), LPPtiger 2,44 MS-DIAL 4,98 and MZmine 3.99 In addition, although not a data processingsoftware per se, LipidLynxX40 was alsoincluded in the review as it is one of the lipid nomenclature harmonizationtools, which has already integrated specific optimization for epilipids.Lipostar is proprietary but free for nonprofit institutions, whilethe other reviewed software packages are open source and freely available.The usability of these software for LC-MS-based epilipidomics dataanalysis is summarized in Our literaturesurvey retrieved a total of six GUI-based softwaresuitable to address various stages of the LC-MS epilipidomics dataanalysis workflow: Lipid Data Analyzer (LDA),Although five of the tools listed in 63 for the feature detection.97 A different approachis used by LPPtiger 2, which does not require a prior feature detectionto be carried out. Instead, the software predicts oxidized lipid speciesstarting from a user-provided list of unmodified molecules of interestand searches for the corresponding MS/MS spectra directly in the rawdata.As explainedabove, epilipidsoften produce signals close to the noise level due to their naturallow abundance. Tunable feature detection algorithms can reduce therisk of erroneously discarding relevant signals while ensuring a reasonablecomputation burden. Four of the software listed in 100 This is generally caused by suboptimal parametersettings, such as overly high noise threshold, inconsistent chromatogramresolving, misalignment, etc. In this context, Lipostar, MS-DIAL 4,and MZmine 3 offer the possibility to automatically reinspect thealigned feature table to cope with false missing values that are artifactsof the processing.99 In these tools, the algorithm examines each missing value in thefeature table individually and checks for the presence of omittedchromatographic signals in the original raw data where the peak isexpected . Ifa meaningful LC peak is found, it is integrated and the retrievedpeak area used. This approach (often referred to as “secondaryfeature detection” or “gap-filling”) can significantlyreduce the presence of missing values in the final feature table,providing more accurate quantification data suitable for a robustdownstream statistical analysis.99During the feature detection process, there are a numberof situations where a feature might not be detected in one (or more)samples even though an LC peak is actually present. In these cases,a zero-intensity value is (erroneously) assigned, producing so-called“missing values” in the aligned feature table.The epilipidome structural diversity,the frequent occurrence of isobaric/isomeric species, and the specificfragmentation patterns of certain modified lipid classes are all hurdlesthat directly hamper the annotation of both known and unknown (epi)lipids.For the sake of convenience and to avoid redundancies in the text,these challenges will be discussed together in this section.16 and is offered by all the reviewed software,except LDA, which uses an approach based on customizable mass lists.The reference MS/MS library can be already integrated in the softwarepackage, in house-curated by the user, or generated in silico using a rule-based approach. Concerning the latter, class-specificfragmentation rules formulated from experimental data are consideredin the generation of the in silico MS/MS spectra.All tools come with a set of class-specific fragmentation rules thatcan be variably customized by the user . Intensity-free spectra can also be computed by MZmine 3and LPPtiger 2, while intensity relationships between fragments canbe defined in LDA and MS-DIAL 4 and may help in assigning the sn-position of acyl chains. Lipostar also generates intensity-freespectra but allows the user to associate weights to the fragment ionsand take them into account for the spectral match scoring.As discussed in 22 MZmine 3, MS-DIAL 4, Lipostar, and LDA enable the assignment ofspecific RT windows to the different lipid classes, which can be usedto avoid incorrect annotations. Essentially, features annotated asbelonging to a certain lipid class, but eluting outside the suspectedwindow, are discarded. CCS-based annotation constraints can be definedin MS-DIAL 4 and Lipostar. RT- and CCS-based filtering options arenot available in LPPtiger 2 and LipidMatch Flow. Notably, MZmine 3also allows discarding noisy features and/or false annotation basedon the chromatographic peak shape  and additionalfilters .85 The differences in the annotation workflow customizability amongthe reviewed software are summarized in RT-and CCS-based constraints can be included in the identificationworkflow to raise the annotation confidence.in silico MS/MS spectra starting froma user-provided database of lipid chemical structures.Modified lipids are currently underrepresentedin reference spectrallibraries (oxPLs and oxTGs represent the only epilipid classes coveredin public reference MS/MS libraries at the time of writing). Therefore,some packages  also offer the possibilityof generating m/z are matched against the calculated oxidized lipid theoretical masses.When a match is found, the MS/MS spectrum is checked, and fragmentions exhibiting the same shift as the native-oxidized forms are takeninto account in the spectral match. Moreover, a direct connectionbetween LPPtiger 2 and Lipostar has been recently established withthe aim of exploiting the benefits of both software  and adduct type(s). Subsequently,the unknown features’ ware see 5. Specifmass shift level”, indicating the mass shift fromthe parent molecule ; (ii) the “elemental composition level”, pointing out the changein the elemental composition with respect to the parent molecule ; (iii) the “typelevel”, which relates to the type of chemical modification; (iv) the “sitelevel”, referring to the position of the modificationsite on the parent molecule; and (v) the “stereochemistrylevel”, where information about stereochemistry isprovided . As discussed above, althoughguidelines for the shorthand reporting of modified lipid species havebeen recently introduced,38 different stylesand information levels are used by the reviewed software to annotatethe same species and/or modification. For instance, LDA can annotateoxidized species at the “type level”or the sn-position and the software reports themwith an “ox” prefix. However, no specific nomenclatureis used to discriminate between oxo, keto, epoxy, and furan modification.Lipostar and MZmine 3 provide annotation at the “elementalcomposition level” . If the specificacyl chain that underwent the oxidation can be determined from theMS/MS data, Lipostar reports the information in the identificationsummary. LipidMatch Flow uses an “elemental compositionlevel” annotation and can specify the FA residue(s)object of the modification; three different annotation styles areavailable: OxPC(16:0_18:2(2O)), PC 16:0_18:2;2O, and OxTG(14:0_14:0_18:2(OHOH)).Finally, LPPtiger 2 reports the type of modification .Such inconsistency in reporting epilipid species limits the establishmentof automated pipelines for parsing and querying lipid names acrossdifferent resources , reduces the usabilityof reference data, and leads to over-reporting in research papers.In this context, the recently developed LipidLynxX software aims atproviding a unified nomenclature system compatible with differentidentification levels and that is easy-to-understand by both researchersand computer scripts. The epilipids nomenclature scheme proposed byLipidLynxX relies on a multitiered identification matrix reportedin Five levelsof informationcan be used in reporting modified lipid species: (i) the “Databases normally play a central role in any computational methodand/or workflow. In this section, we review those databases that currentlycontain relevant information regarding epilipids.101 MassBank,102 and the MassBankof North America (MoNA)103 constitute avaluable resource to assist (epi)lipid annotation as they containboth experimental and in silico-generated MS/MS datafor a number of oxidized lipids. Other generic databases such as ChEBI104 and PubChem105 containsome epilipids but were not optimized to filter or search for epilipidsspecifically, however, cross-linking from other lipid databases  is available for data integration. A list of databaseswhere structures of epilipids are collected and easily accessibleis provided in Generic metabolitedatabases such as METLIN,108 Established in 2003, it includesdatabases of known lipid chemical structures (LMSD),32in silico-generated structures , computationally generatedoxidized phospholipid species (including the corresponding precursorion’s m/z), experimentalCCS values , as well as arepository of experimental lipidomics data sets.108 Notably, the LMSD also contains nomenclature, references,cross-links to other databases, and experimental MS/MS spectra forseveral lipid species. At the time of writing, the LMSD includes over250 oxPLs, 1000 octadecanoids, 1200 eicosanoids, and 1100 docosanoids(accessed September 2022).LIPID MAPS (which stands for LIPID Metabolites andPathways Strategy)is arguably the most complete and widely used gateway for lipidomics.107 All the informationis curated from peer-reviewed literature. The set of known lipid structuresis also complemented by a library of theoretical structures obtainedby the combination of known building blocks from the curated set.Both known and theoretical lipids are organized into a single commonhierarchy that follows the notation for mass spectrometry-based lipidomicdata proposed by Liebisch et al.38 and is consistentwith the classification developed by LIPID MAPS. Concerning modifiedlipids, a collection of octadecanoids (13), eicosanoids (136), anddocosanoids (13) is included, and a few complex epilipids are includedin the database at the time of writing (accessed September 2022).SwissLipids is a curated collectionof known lipid structures andrelated information about metabolism, interactions, as well as subcellularand tissular localization.109 It includes a large numberof both confirmed and predicted lipid structures. At the time of writing,the HMDB includes 480 eicosanoids species and over 1000 predictedoxPLs.The Human Metabolome Database (HMDB) is a freely accessible databasecontaining information about small molecule metabolites found in thehuman body.31The Riken IMS oxidized phospholipid database containsthe MS/MSspectra acquired in negative ion mode of 386 total molecular speciesof oxPLs obtained by biogenic conversion from oxidized FAs incorporatedinto cellular phospholipids. More details can be found in Aoyagi etal.60Notably, LIPID MAPS LMSD, SwissLipids,RIKEN IMS oxidized phospholipids,and HMDB are downloadable and can be conveniently connected to differentsoftware packages to assist the (epi)lipid annotation pipeline .110 LipidBank111 orCEU mass mediator112) are available onlyonline. Notably, CEU mass mediator 3.0 (CMM 3.0) integrates compoundsfrom different sources and offers a module to annotate oxidized lipids.112 LipidBank is the official database of the Japanese Conferenceon the Biochemistry of Lipids and contains molecular structures, spectraldata , and literature information for more than 7000unique lipid species. Among them, 329 eicosanoids and 5 lipid peroxidesare present (accessed September 2022). LipidPedia is a database ofbiomedical information for over 4400 lipid species retrieved fromthe literature using text-mining strategies with more than 1,500,000associated literature references (accessed September 2022). At thetime of writing, searching the term “oxidised” in thedatabase returns only 6 six entries, although comprehensive informationis provided.Other lipid structure databases  but are finding a wideruse in epilipidomics studies.113 for example, found specific lipoxygenases to be importantfor coagulation and the thrombotic disorder antiphospholipid syndrome(APS) using an epilipid correlation-network approach.Metabolicnetwork analysisis a powerful visualization tool widely used in computational biology.It can be used to display qualitative and quantitative changes inthe lipids profile at a system level. A metric particularly interestingfor epilipidomics applications is the change of  correlationsbetween pairs of species under different experimental conditions.The intuition here is that a reaction connecting two (epi)lipid speciesis more likely to be active when the respective epilipids are highlycorrelated. Consequently, a loss of high correlation between experimentalconditions is a hint toward changes in reaction activity. Similarideas have already been shown to generate novel insights into theregulation of epilipid biosynthesis. Lauder et al.,115 abbreviated as LINEX. It is based on networks that combine fattyacid and lipid class metabolic reactions to compute species-levelbiochemical connections. Due to this modular structure, it is well-suitedto cover epilipids by incorporating position-specific hydroxylationpatterns and reactions.One lipidomics-specificmethod for extensive network analysis is the Lipid Network Explorer,p-values (between sample groups) as well as correlation metrics . By projecting differences in samplegroups onto the network, it allows the exploration of global changesin the lipidome. Since the network topology entirely depends on lipidmetabolic reactions, this enables the identification of groups ofreactions with distinct patterns between sample groups as well asgenerating hypotheses on alterations in enzymatic activity.To enable a quantitative analysis oflipidomics results, LINEXutilizes statistical metrics such as fold-changes and 116 and Reactome117), and it is now possible to generate reasonableepilipidome metabolic networks. Nevertheless, additional efforts shouldbe made to integrate the available information, enable the applicationof active module identification algorithms, and set the basis formetabolic modeling of the epilipidome. Furthermore, such networkswill enable the integration of epilipidomics data with data from proteomicsand other omics disciplines. We can therefore expect epilipidomicsdata interpretation to deliver novel insights into disease mechanismsand general biological processes.Epilipid metabolism pathways are partially searchable in databases appear as connected nodes and cluster into “molecular families”.This tremendously assists the visualization of the detected chemicalspace and greatly facilitates the annotation of unknown, structurallyrelated metabolites within a molecular network. Moreover, in combinationwith common feature-finding tools, semiquantitative information (peakarea) can be mapped over each node in the network to infer semiquantitativedifferences among samples or sample groups.119 Although originally developed for metabolomics data analysis toassist the identification of novel metabolites,120 molecular networking has recently been applied in several(epi)lipidomics studies.126 Notably, Watrous et al. applied untargeted LC-MS/MS in conjunctionwith MN networking to expand the repertoire of oxylipins in humanplasma samples by annotating 362 putatively known oxylipins and 46unknown related compounds.121 As demonstratedby this study, the epilipidomics field can greatly benefit from MNand MN-based strategies for the rapid prioritization and annotationof both known and unknown modified lipids through the network connections.Although other solutions exist,127 themost widely used platform to carry out MN analysis is currently theGlobal Natural Products Social Molecular Networking (GNPS) ecosystem,119 which offers a web-based GUI and extensive documentation resourcesfor new users.Molecular networking (MN) is arelatively new computational strategy for the analysis and visualizationof untargeted LC-MS data.131 Another interesting application of ML-based methods is the predictionof RT132 and ion mobility-derived CCS133 values for metabolite identification purposes.The goal of these prediction models is the calculation of these valueswithout the need of their experimental measurement (which requirereference samples and/or standards). Notably, Zhou et al. developeda dedicated model for the prediction of CCS for lipids.134 A further application of ML for metaboliteannotation is represented by tools for the automated interpretationof MS/MS data, such as the SIRIUS software suite135 and MSNovelist.136 In particular,these methods offer the possibility of database-free predictions ofelemental formulas and chemical structures that do not restrict theannotation to searching against structural databases  and/or matching against experimental MS/MS spectral libraries.This can be of particular help in the identification of truly new,unreported metabolites. Notably, the recently released SIRIUS 5 includesa dedicated module for lipid structure predictions.137Machinelearning (ML)approaches to support the untargeted LC-MS data analysis workfloware becoming more and more popular. For example, ML-assisted algorithmshave been developed for the automated peak picking and filtering ofnoise features based on chromatographic peak shape and intensity.Mass SpectrometrySearch Tool (MASST)138 and the Mass Spectrometry Query Language (MassQL),139 have been recently developed by the Dorrestein lab withthe aim of making the ever-growing untargeted MS data repositories an easily accessible resource to assist the annotationof unknown molecules and structural analogues.Deposition of untargeted MS data in the publicdomain is experiencingrapid growth, largely thanks to the increasing adoption of universal,nonvendor-specific MS data formats . The two MSdata mining tools described in this section, the 140 It enables querying of MS/MS spectra across public spectral libraries and repositories of metabolomics MS data. A specific MS/MS spectrum of interest can be searched bycopy–pasting the spectrum peak list and defining a set of searchtolerances within a user-friendly web interface.140 A query result includes all matches to identical or analogousMS/MS spectra in public spectral libraries and repositories alongwith their associated metadata and/or sample information. In particular,metadata can be linked at the data set level , file level  or single annotated spectrum level .The MASST toolis a web-based search engine for MS data.139 Inspired by the SQL programming language, MassQLimplements a consensus vocabulary to search for MS patterns usinghuman-readable query strings. Searchable MS terms include both MS and MS/MS fragmentation patterns , with support for both data-dependent (DDA) anddata-independent acquisition (DIA). Additionally, terms for separationmethods , user-definedtolerances , and boolean conjunctions can be used to define inclusion/exclusion criteriaand create more complex pattern queries. As an example, choline-containingphospholipids normally exhibit diagnostic fragments and neutral lossesin the MS/MS spectra (positive ionization) arising from the glycerophosphocholinegroup: m/z 184.0739 and correspondingneutral loss of m/z 183.0733 (phosphocholine), m/z 125.0004 .143 The following MassQL query string can be formulatedto quickly retrieve all the MS/MS spectra that contain such diagnosticfragments/losses (within a 10 ppm mass tolerance) and likely belongto glycerophosphocholine lipids:QUERY scaninfo(MS2DATA) WHEREMS2PROD= 184.0739:TOLERANCEPPM = 10 ANDMS2PROD= 125.0004:TOLERANCEPPM = 10 ANDMS2NL= 183.0733:TOLERANCEPPM = 10PPMMassQL is a novelquery language for the mining of MS data.The datasearch can be carried out on a single file, data set,or scaled up to entire MS data repositories. As for MASST, MassQLquery results include all matches to identical/analogous MS/MS spectrain spectral libraries and data repositories together with the associatedmetadata.ad hoc strategies for data analysis in recentyears, although currently standard data analysis pipelines are missing.In this review, we first aimed at describing the available softwarefor lipidomics endowed with a graphical user interface (to facilitatetheir use) and freely available for academic use. Though some workflows,which were originally developed for generic unmodified lipidomicsor metabolomics, can be tweaked to analyze certain epilipids, epilipids-specificcriteria still demand extra focus. From the availability of solidstructure- and spectral libraries to the confidence of identificationalgorithms, data processing of epilipids has more challenges thanwell-developed metabolomics workflows. For example, due to the similaritiesof multiple isomeric species  at differentmodification sites, the algorithms for the identification of exactepilipid structures and for accurate quantification of each isomerare required to implement multiple filters and rules that relate tospecific epilipid classes and differ from common metabolomics algorithms,with the risk of becoming time-consuming. These unique challengeslead to adapting or developing computational tools for epilipidomics,which are suitable to deal with the intrinsic complexity of the epilipidome at different levels, as wellas with the analytical challenges in the field. Ouranalysis brings us to believe that, although several steps forwardhave been made to develop software and computational approaches forepilipidomics, the currently available in silico toolsstill suffer some limitations due to experimental-derived and purecomputational issues.The growinginterest in epilipidomics has boosted the developmentof In silico predicted CCS values generated by epilipid-specific algorithms mightbe a potential starting point, but experimental CCS values for epilipidsare needed to validate predictions. Finally, the analysis of the epilipidomeneeds accurate analytical procedures to reduce the risk of analyzingartifacts. Indeed, potential degradation products like oxidized lipidscould form during sample processing if not correctly handled, andthis will hamper any software’s ability to deliver reliableresults.76From the experimental perspective, thelack of a large collectionof standards for epilipidomics has a negative impact both on quantificationand on the accurate definition of fragmentation rules or spectralmatching strategies to be used in identification. Similarly, whileCCS libraries are available for native lipids, the collection of CCSvalues for epilipids is still in its infancy. lipid quantification and results investigation will require only minorimprovements in the existing software, taking advantage from the experiencein the field of native lipids. For example, the main bottleneck in lipid quantification is again the lack of proper standards.In addition, one needs to consider the specific changes of adductdistribution for epilipids, and the use of adduct clustering algorithmsalready available for native lipids have not yet been extensivelyvalidated for epilipids. Therefore, we still recommend reviewing theautomatic assignment of lipid identification together with peak integrationto make quantification reliable. Concerning the feature detection step, we described several tools that facilitate peak picking closeto the noise level , whichwill be very beneficial for epilipidomics. It is noteworthy that,while LC-MS/MS data acquired in DDA mode are widely supported, onlyLipostar, LipidMatch Flow, and MS-DIAL 4 support DIA mode such asAll Ion Fragmentation (AIF), MSE, or SWATH. About the lipid identification step, we believe that large improvementsare still possible to deal with the high number of isomers and isobars.So far, only LPPtiger 2 was specifically designed to identify epilipidsfrom a number of classes  with the aim of discriminatingisomers and isobars with the drawback of being a time-consuming analysis,while the other software here discussed propose faster but less accuratealternative solutions. A first attempt of making a step forward inepilipid identification is the recent link between LPPtiger 2 andLipostar 2 that allows having the accurate annotation of epilipidsof the first but in a short time thanks to a preanalysis made by Lipostar2. Similarly, we suggest that other software could connect to LPPtiger2 in the near future. However, at this stage we still recommend manualrechecking of automatically identified epilipids to reduce misannotationsand over-reported species (due to adduct clustering failure) to overallimprove the quality of epilipidomics data analysis. Indeed, takinginto account the well-known issue of the lack of standards, the predictionof epilipids’ properties such as fragmentation pattern, retentiontime, or CCS value could partially consolidate lipid identification.Machine learning tools offer the potential to predict CCS and RT valuesfor small molecules with high accuracy,145 but they have not been suitably explored for lipids or epilipids,where a high degree of rotatable bonds leads to a higher complexityand lower performance. Nevertheless, concerning RT prediction, elutionorder prediction could be preferred in a first attempt, as it providesmore stable results.146From the computationalperspective, referring to the four stepsof data analysis discussed in this review, 147 it is still challenging to develop algorithms that can correctlydistinguish epilipid isomers with high confidence.The scatteredsolutions, each with strong focus in certain fields,call for joining the efforts through collaborative workflow connectingmultiple tools. In addition, there is a trend to adapt new algorithmsfrom metabolomics including molecular networking and machine learningto epilipidomics with additional adjustments. However, unlike severalalgorithms that can be applied to a large number of different metaboliteclasses across highly diverse structures,in primis, a common nomenclature, hampers theeffort of collecting the available information. The use of a standardnomenclature is essential for sharing and comparing lipid annotationsand to browse into databases. LipidLynxX has the merit of being atool able to convert different annotations to unified identifiersbased on a community-accepted shorthand notation system.38 The computational community should work in connecting software tothis tool to have a common nomenclature system . Finally, an increase of the number of epilipidspecies in databases will offer new perspectives for computationalapproaches, although the limited number of standards for epilipidomicshampers the construction of large databases of experimental data.Finally,currently available databases containing information aboutepilipids were also described. Databases containing structures ofepilipids and/or related MS/MS libraries are still very limited andfragmented. Among the various sources, searching for the number ofepilipids considered in a given database is still a challenging task,probably due to the limited attention that has been devoted in thepast to epilipids. The lack of well-defined keywords, labeling systems,and, in silico tools dealingwith new and emerging modified lipids in complex matrices by applyinguntargeted approaches.To conclude, we must emphasize that much of what is shown and discussedin this review is related to oxidized lipids only, which representonly a part of the epilipidome. The new frontier, therefore, willbe to broadly investigate the epilipidome space in its entirety andcomplexity and to develop new


Despite decreasing the prevalence of chronic hepatitis B (CHB), it is still a major health care challenge. Current antiviral regimens aim to suppress hepatitis B virus (HBV) deoxyribonucleic acid (DNA) activity to prevent the risk of hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma (HCC). Currently, pegylated interferon (Peg-IFN) and nucleos(t)ide analogs (NA) are the first-line choices of drugs. Peg-IFN is now discontinued due to its mode of application and side effects. NA is used once daily to suppress HBV DNA activity but has little effect on covalently closed circular DNA (cccDNA), so continuous long-term therapy is required to suppress HBV DNA. Due to this effect, disease remission, relapse, and even clinical flare are common phenomena after the end of treatment (EOT). This review aimed to analyze the current regimens for treating chronic hepatitis B. Their mode of action, duration of treatment, and events after stopping therapy. The review was performed using the preferred reporting items for systematic reviews and meta-analysis (PRISMA) 2020 guidelines. A search was undertaken in PubMed, PubMed Central, Google Scholar, and ScienceDirect. Screening of articles was carried out to find relevant and appropriate articles. Articles were then quality-checked before inclusion.Our analysis showed that long-term finite therapy with nucleoside analogs could improve clinical outcomes and suppress viral DNA activity. However, a functional cure, loss of hepatitis B surface antigen (HBsAg), is rarely achieved. The decision to end treatment depends on quantitative HBsAg level (qHBsAg), alanine aminotransferase (ALT), HBV DNA (deoxyribonucleic acid), hepatitis B e antigen (HBeAg), and fibrosis assessment. It is concluded that patients with HBeAg negative without cirrhosis can be easily withdrawn from treatment if they have long-term viral remission and a high HBsAg loss rate. However, patients with positive HBeAg should continue treatment because there is a high chance of disease relapse and even acute flare. To predict whether patients will benefit from EOT, some immunomodulatory markers are studied, including interleukin , fas ligand (FASGL), and IFN gamma. Although these factors are reliable, none pose an independent effect on disease remission. Combination therapy  is promising but has clinical shortcomings. Hepatitis B virus (HBV) is one of the most serious and prevalent diseases, even with the introduction of a vaccine in 1982. There are still 290 million people worldwide, with the majority residing in the Asia-Pacific region [Chronic hepatitis B (CHB) is marked by persistent HBsAg (surface antigen of the hepatitis B virus) for six months or more. It's a global concern as it can lead to complications of liver cirrhosis, hepatocellular carcinoma, and hepatic decompensation if left untreated . Loss ofCurrently, two regimens are approved for CHB. The first line is pegylated interferon (Peg- IFN-γ), a subcutaneous injection; the other is oral nucleos(t)ide analogs (NA) entecavir (ETV) and tenofovir disoproxil fumarate (TDF). Peg-IFN acts as an antiproliferative and immunomodulatory agent. Whereas NA inhibits viral replication, thus preventing liver necroinflammation and disease progression. However, both the agents do not affect hepatitis B virus DNA (HBV DNA), especially cccDNA, i.e., covalently closed circular DNA in the nucleus. So, treatment cessation almost always leads to viral remission. Thus, an indefinite continuation of NA is necessary for sustained virological response. Treatment duration relies on the desired treatment goals, but there is conflicting data in considering NA cessation .IFN was the only treatment available before the introduction of lamivudine (LAM), but its functional cure after 48 weeks of treatment was less than 5% . It's inLong-term NA therapy may lead to HBsAg loss, but significant seroconversion is seen after stopping NA . A contrHowever, there is a propensity of data to suggest the baseline of stopping NA therapy. The Asian Pacific Association for the Study of Liver Disease (APASL) suggests that among HBeAg-negative patients whose HBV DNA remains undetectable on three separate occasions, each more than six months apart and their ALT is monitored every month in the first three months and then every three months, and have acquired NA treatment for at least two years might stop it . AmericaIn light of the evidence suggested by previous studies regarding the use of peg-IFN and nucleotide analogs in treating CHB, physicians are still interested in investigating the endpoint to stop therapy. Monotherapy or combination therapy still has chances of remission, relapse, or flare-up. With relapse, there are chances of deterioration to hepatic decompensation, so close monitoring is crucial. To clarify, we conducted a systematic review to improvise a standard of care and the potential benefit of these therapies in preventing clinical relapse of CHB.To stop the progression of chronic hepatitis B to anticipated liver damage, peg -IFN therapy and long-term administration of third-generation nucleotide analogs have been suggested . It's clMethodMethodology and Search StrategyOur method and results for systematic review are reported according to the preferred reporting items for systematic reviews and meta-analysis (PRISMA) 2020 guidelines following our screening selection .We used PubMed, ScienceDirect, and Google Scholar to look for articles using Medical Subject Headings (MeSH) and keywords to highlight the most relevant reviews and studies for analysis. The keywords included: "Chronic Hepatitis B", "peg-IFN alpha", "oral nucleotide analogs", "remission" and "relapse". We used Booleans to put together the keywords for an algorithm to use in PubMed. The articles were screened to highlight those most relevant to the search question and selected according to the inclusion/exclusion criteria.Inclusion and Exclusion CriteriaThe selection choice was from randomized control trials (RCTs) published from 2018 to 2022. All selected articles were peer-reviewed and published in the English language. Grey literature was excluded. Our selection for eligibility followed the population, intervention, comparison, and outcomes (PICO) model. The inclusion and exclusion criteria are shown in Table Data ExtractionThe data retrieval and review were completed by two separate researchers independently. In the case of disagreements, the researchers would discuss the data for its relevance and design to eligibility criteria to reach an accord. A third researcher was counseled for objectivity if a decision could not be made.Critical Appraisal of StudiesWe critically appraised our screened articles using the Cochrane risk of bias tool . The biaResultsLiterature Search and Study SelectionThe MeSH strategy generated five hundred eighty articles from keywords, eligibility criteria, and databases. This systematic review was conducted following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. Fifty articles were excluded due to duplication. Upon reading the titles and abstracts, 320 articles were excluded. After filtering to include articles from the last five years only, as the guidelines vary consistently, we were left with 20 articles. Of the remaining articles, five of them were journal guidelines, and one didn't meet the quality check criteria. Eight final articles met the criteria and were included. Our PRISMA flow diagram is shown below in Figure Baseline characteristics: These characteristics and definitions are generalized to all reviewed articles -22.Treatment IndicationsIf HBV DNA>2000 IU/ml and ALT > two times the Upper Limit of Normal (ULN) or HBV DNA >2000 IU/ml and ALT >ULN with moderate necroinflammatory activity and fibrosis.Patients were excluded if they had detectable HBV-DNA or positive HBeAg at treatment cessation, presence of liver cirrhosis, co-infection with hepatitis C or human immunodeficiency virus, chronic kidney disease, hepatic decompensation, hepatic, or non-hepatic malignancy, received an organ transplant, or used immunosuppressive drugs in the last few months.Virological remission: Virological remission is defined as undetectable HBV DNA and negative HBeAg.Clinical relapse: Clinical relapse was defined as serum ALT greater than two folds the upper limits of normal (40 U/L) plus serum HBV DNA rising above 2000 IU/mLVirological relapse: Elevation of HBV DNA >2000 IU/mlExpected outcome: Loss of HBsAg in serum, which was defined as a measurement below the lower limit of detection (0.05 IU/mL)A systematic review of the management paradigm is shown in Figure A brief overview of the studies included is given in Table DiscussionTreatment Goals After Stopping NA TherapySince available regimens can only suppress the viral replication but cannot target the cccDNA in the nucleus, relapse is most likely seen after stopping NA in HBeAg-negative patients. The relapse rate varies greatly among studies, and whether all patients need retreatment is also unclear. In one of the studies , particiAlthough the loss of HBsAg is considered a functional cure and a predictor of therapy cessation, another study in Taiwan explored the role of HBcrAg along with HBsAg in clinical relapse . The stuDuration of TreatmentDuration of treatment, stopping treatment, and observing remission is crucial. Among Asian patients in a study, more disease remission was observed in 1.3 years 72 weeks) after stopping NA . In the Immunomodulatory Markers to Predict Disease RelapseIt is proposed that immunomodulatory markers can be used to assess and predict the disease course in CHB patients in the clinical setting . IL-21 iCombination Therapy The use of IFN-ALPHA has been largely suppressed upon the introduction of NA due to its side effects and unfavorable mode of administration. It's used by only <5% of patients who either do not tolerate long-term NA or have immune-active CHB . The mosFinally, there is a need to develop new drugs that target different phases of the HBV life cycle and cccDNA reservoir and focus on innate host immunity. Using finite NA has become a high HBsAg loss rate standard, but future drugs should aim at an even higher rate.Limitations of this systematic review are that all included articles were published within the last five years as guidelines change rapidly. Studies on CHB patients with other comorbidities, acute flare, seeking treatment for other comorbidities, and baseline advanced liver disease were not analyzed. Studies that were not in the English language and were not in the database search, articles that were unpublished, not peer-reviewed, or grey literature.Treatment for chronic hepatitis B is evolving rapidly with new antiviral drugs and regimens but is still unsatisfactory. Our goal was to study the current treatment regimen and assess its role in the possibility of disease remission and relapse. The paradigm is shifting from infinite long-life NA therapy to finite NUC therapy. Although this therapy has succeeded in suppressing HBV DNA, there are few chances of HBsAg loss. Moreover, reliable factors are studied to predict outcomes in patients who stop NA therapy. These include IL-21, CCL-4, IFNg, IL-8, FASLG. However, as studies continue to predict off-treatment events and predictors, the best possible is very low HbsAg and undetectable HBV DNA. Close monitoring of HBV DNA and ALT post-treatment can predict retreatment options. Retreatment options then depend on the patient's strict predefined criteria baseline characteristics to avoid lifelong retreatment decisions.


Regulatory T cells work to suppress the response of immune cells. In ovarian cancer, tumors may exploit immune suppression to escape destruction by the immune system. We propose that targeting regulatory T cells with medications could improve ovarian cancer survival by preventing immune suppression.Regulatory T cells (Tregs) have been shown to play a role in the development of solid tumors. A better understanding of the biology of Tregs, immune suppression by Tregs, and how cancer developed with the activity of Tregs has facilitated the development of strategies used to improve immune-based therapy. In ovarian cancer, Tregs have been shown to promote cancer development and resistance at different cancer stages. Understanding the various Treg-mediated immune escape mechanisms provides opportunities to establish specific, efficient, long-lasting anti-tumor immunity. Here, we review the evidence of Treg involvement in various stages of ovarian cancer. We further provide an overview of the current and prospective therapeutic approaches that arise from the modulation of Treg-related tumor immunity at those specific stages. Finally, we propose combination strategies of Treg-related therapies with other anti-tumor therapies to improve clinical efficacy and overcome tumor resistance in ovarian cancer. Ovarian cancer is the deadliest gynecological cancer, with patients in advanced stages surviving for five years less than 30% of the time. This poor survival stems from late diagnosis, frequent metastasis into the abdomen, and a high recurrence of treatment-resistant disease; furthermore, in 70% of cases, malignancy is not identified until an advanced stage ,2. One o+FOXP3+ and other Treg populations, such as CD8+ subsets, have been identified but much of their functions remain uncertain. Having an increased amount of effector CD8+ T cells infiltrating a tumor may indicate improved patient survival . Tu. Tu26]. Tregs may directly target and destroy other immune cells via the secretion of granzymes, which induce apoptosis in the target cells . In cancTreg cells associated with ovarian tumors have been found to highly express PD-1 while circulating Tregs in those patients with expressed PD-1 at lower levels . PD-1 pr+ Tregs, leading to a more stable suppressive phenotype ..71].Immunotherapies can be a versatile way of modulating Treg function during cancer treatment. They may function alone or in combination with other therapies.+ effector cells in the tumor microenvironment and, in fact, IL-2 injections have been found to increase survival in human trials .,91.84,91Combining immunotherapies with Treg-targeting drugs may work to improve the poor response rate of some treatments. Checkpoint inhibitors are one such immunotherapy that have several undesirable side effects during long-term treatment and that have been less effective in ovarian cancer than in other forms of cancer. Despite this, combination therapies utilizing immune checkpoint inhibitors have been found to reduce the size of ovarian tumors in some patients . Checkpo+ T cells to destroy cancer cells, an action which is hindered by Tregs in the areas surrounding tumors and, as such, would benefit from reduced Treg activity [Reducing the immune suppression by Tregs may improve the outcomes of chimeric antigen receptor (CAR)-T cell therapy. This treatment involves modifying the patient’s own T cells to better attack cancerous cells for solid tumors and blood cancers. For some patients, CAR-T does not yield positive results due to suppressed anti-tumor immunity and failure of the modified T cells to infiltrate tumors . Modifyiactivity ,94.Inhibiting DNA repair mechanisms via poly (ADP-ribose) polymerase (PARP) inhibitors as a treatment for ovarian cancer is being investigated by several clinical trials as both a monotherapy and a combination treatment . These d+ T cell to Treg ratio noted and increased immunosuppressive cytokines found in the tumor microenvironment [In addition to simply targeting cancerous tissue or the patient’s immune system, potential treatment could include treatment of other factors that may modulate cancer growth or any immune response, such as potential viral infection . For insironment . TreatmeThe role of Tregs in ovarian cancer is complex and not fully explored. Targeting Tregs when treating ovarian cancer may lead to improved outcomes and less toxicity than directly treating malignant tissue. This can be achieved either by targeting secreted factors, such as cytokines and chemokines; by targeting surface molecules, such as receptors; or by epigenetic modification of Treg function and development. Care must be taken when utilizing these treatments as they may lead to unwanted systemic side effects or inappropriate drug responses. Locating potential new targets for these types of drugs may go a long way to improving treatment outcomes in ovarian cancer patients. These treatments can then be combined with more traditional methods of treatment to improve the overall efficacy of a course of treatment.


There is a clear clinical need for efficient cartilage healing strategies for treating cartilage defects which burdens millions of patients physically and financially. Different strategies including microfracture technique, osteochondral transfer, and scaffold-based treatments have been suggested for curing cartilage injuries. Although some improvements have been achieved in several facets, current treatments are still less than satisfactory. Recently, different hydrogel-based biomaterials have been suggested as a therapeutic candidate for cartilage tissue regeneration due to their biocompatibility, high water content, and tunability. Specifically, magnetic hydrogels are becoming more attractive due to their smart response to magnetic fields remotely. We seek to outline the context-specific regenerative potential of magnetic hydrogels for cartilage tissue repair. In this review, first, we explained conventional techniques for cartilage repair and then compared them with new scaffold-based approaches. We illustrated various hydrogels used for cartilage regeneration by highlighting the magnetic hydrogels. Also, we gathered in vitro and in vivo studies of how magnetic hydrogels promote chondrogenesis as well as studied the biological mechanism which is responsible for cartilage repair due to the application of magnetic hydrogel. Cartilage injuries occur due to degenerative disease and surgical and traumatic injuries , 2. The Traditional techniques for curing articular cartilage defects include microfracture, osteochondral autographs, and allographs . ShortcoThe application of the magnetic hydrogel for cartilage tissue regeneration is still at the beginning of the road and much has not been done in this area. There is a need for a detailed study of magnetically induced chondrogenesis and magnetic hydrogels. In this review, a study was done on the fabrication of magnetic hydrogels that induce chondrogenesis. Furthermore, the influence of magnetically labeled stem cells in enhancing the cartilage matrix synthesis was investigated. In addition, the results of in vitro and in vivo studies of magnetic hydrogel for articular cartilage defects were discussed.A variety of natural and synthetic polymers have been studied for the fabrication of hydrogels for cartilage regeneration. Commonly used polymers for cartilage tissue regeneration are summarized in Hyaline cartilage is a nonvascularized connective tissue and the most abundant cartilage type of the body which is found in the costal cartilage, trachea, and articular cartilage , 37. TheSeveral pathological conditions such as infectious disease, osteoarthritis, cancer, or traumatic injuries can lead to cartilage defects. Articular cartilage has a low intrinsic capacity for repair so treatment of defects of articular cartilage whether from trauma or degenerative disease is a significant challenge for orthopedic surgeons , 47. AltMicrofracture is a safe, effective, minimally invasive, and marrow-stimulating technique that aims at triggering vascular response to injury . This teWhen the articular defect size is bigger than 15 mm, the transplantation of autogenic and allogenic tissue is preferred over marrow-stimulating techniques . This teACI is the first cell-based biological approach to the treatment of grade III and grade IV cartilage lesions , 59 thatThree-dimensional scaffolds are becoming popular because they are cost-effective, time-efficient, and require a single-stage procedure. Furthermore, they provide a high capacity for cell attachment as well as adjustability for having appropriate mechanical properties , 67. FurSince the beforementioned treatments  have limitations, the hopes are in scaffold tissue engineering. However, because the chemical and mechanical properties of the cartilage tissue are not consistent through the entire tissue, it is complicated to create a scaffold that can fully mimic the natural cartilage . TherefoDifferent materials have been suggested for cartilage regeneration purposes  and they can be used in various physical forms of fibers, meshes, and hydrogels. Hyaluronan and collagen-based scaffolds are polymers widely used for the fabrication of cartilage scaffolds due to their similarity to the natural cartilage tissue , 64. ScaRecently, multidisciplinary effort has been put into the fabrication of scaffolds that can specifically and molecularly interact with the cartilage microenvironment. Biological signals such as growth factors and signals are often incorporated into the hydrogels to enhance chondrogenesis in large cartilage defects . For exaThe mechanical properties of hydrogels are considered their main limitation for load-bearing tissues . DiffereHydrogels are good candidates for the construction of three-dimensional structures due to their tunable biomechanical properties and biocompatibility. Hydrogel scaffolds can regulate different materials to promote cartilage repair . The hydHydrogels can be used as filling agents, a delivery vehicle for bioactive molecules, and a three-dimensional structure for organizing cells , 88. DifIn order to generate MSC-based functional hyaline cartilage, MSCs should be directed to chondrogenic lineage and initiate the formation of cartilage matrix including collagen type II and glycosaminoglycan (GAGs) . AlthougThere have been several types of research to expand and improve the hydrogel that induces chondrogenesis; however, researchers are looking for physical cues that can promote cartilage regeneration after hydrogel is added . The eleThe electromagnetic field (EMF) and pulsed electromagnetic field (PEMF) are FDA-approved techniques that are previously shown to promote osteogenic differentiation of MSC . Human mTherapeutic applications of the magnetic field keep expanding because magnetic nanoparticles can apply remote magnetic-induced physical stimulation which enables targeting of the specific site as well . MesenchIt is shown that an early essential step for initiating the chondrogenic differentiation of the stem cell is its condensation . For exa3O4) induced the expression of late chondrogenic differentiation markers including COL2 and aggrecan [Magnetic nanoparticles can induce chondrogenic differentiation under the magnetic field when they bind to the cell surface. For example, Pulse electromagnetic fields (PEMFs) was applied to bone marrow mesenchymal stem cell (BMSC) cultured on the electromagnetic hydrogel. The hydrogel composed of gelatin, beta-cyclodextrin (beta-CD), and magnetic iron oxide (Feaggrecan . In addiSuperparamagnetic iron oxide nanoparticles (SPIONs) can guide cells and serve as physical stimulation. In one study, SPIONs were encapsulated in a hydrophobic shell of hyaluronic acid-graft-amphiphilic gelatin (HA-AGMCs) microsphere. Hyaluronic acid is one of the major components of the chondrocyte extracellular matrix (ECM) which provides a backbone for aggrecan aggregation . FurtherIn another study, magnetic nanoparticle-vesicle (MNVP) was assembled by cross-linking of phospholipid vesicles and magnetite nanoparticles. Chondrocytes and nanoparticle-vesicle assemblies were coimmobilized within a calcium alginate hydrogel. This smart biomaterial responds to the alternating magnetic field by translating noninvasive magnetic signals into cellular responses . Their rThe other advantage provided by the incorporation of magnetic nanoparticles into the hydrogel is they can serve as a contrast agent for imaging . Magnetiβ-cyclodextrin and inserted them into the rabbit cartilage defect. MSCs seeded in hydrogel exposed to the PEMF promoted the differentiation of the stem cells by promoting COL2 and aggrecan [The cartilage microenvironment can be triggered by magnetic stimuli through being exposed to the external magnetic field or magnetic scaffolding . Approacaggrecan . Similaraggrecan  first coaggrecan .The additive manufacturing technique is promising for the fabrication of magnetic hydrogels due to their potential to be customized and be reproduced . De Santα2 (ITG α2), integrin α6 (ITG α6), integrin β3-binding protein (ITG β3BP), intercellular adhesion molecule–2 (ICAM-2), and platelet/endothelial cell adhesion molecule–1 (PECAM-1) have been upregulated in magnetically labeled MSCs. Increased adhesion rate of MSCs can make them engraft to the defect area biologically [Magnetic hydrogels can be used to influence cellular mechanotransduction and translational and paracrine responses of the cells to encourage cartilage repair through different cellular and molecular mechanisms . The mecogically .β) and it results from the activation of the Wnt1/LRP6/β-catenin signaling pathway [Also, the electromagnetic field can induce cartilage tissue regeneration by stimulating chondrocyte maturation –115. For pathway . Moreove pathway . Parate  pathway  have dem pathway .  Table Magnetic hydrogels have been shown to be promising for the treatment of cartilage lesions due to their influence on the stem cell fate as well as chondrocyte behavior . Magneti


Keyword filters were used to identify texts discussing flavored ENDS products . These were further narrowed down to texts discussing flavor policy workaround strategies (n = 7429) and N-gram analysis was performed. Finally, findings from the N-gram analysis were triangulated through qualitative review of a separate sample of texts (n = 488) from the flavor policy-related posts and comments. Results: Overall activity on the subreddit r/JUUL peaked around the time of the EVALI outbreak (September 2019) and when FDA issued guidance restricting flavored ENDS product sales (January 2020). The N-gram analysis revealed an active discussion of banned products one can “still get” or “JUUL compatible” alternatives, including specific brands, brick and mortar locations, and specific flavors. Ten dominant themes emerged from the qualitative review, with some posts containing more than one theme. Conclusion: Many users turned to Reddit for information related to the shifting regulatory landscape concerning flavored ENDS. Discussions focused on both legal alternatives to banned products as well as illegal means of acquiring JUUL pods, including residual retail supply, online, and mail vendors.Objective: To examine conversations among JUUL users on Reddit related to restrictions on flavored ENDS and the shifting policy landscape. Methods: Posts and comments ( Ongoing efforts to restrict the availability of flavored tobacco products necessitates careful examination of the public response to a rapidly evolving electronic nicotine delivery systems (ENDS) marketplace. In the wake of the e-cigarette or vaping use-related lung injury (EVALI) outbreak in September 2019, electronic cigarette manufacturer JUUL voluntarily removed flavored pods aside from tobacco and menthol from the U.S. market ,4. This Flavored Tobacco Product (FTP) policies play a critical role in reducing the access and availability of these products , particuSince the initial ban on flavored pods, many users simply switched to alternative flavored products . Bans thThe abrupt change in the product landscape caused by the flavored pod ban likely lead users to rely on social media platforms for up-to-date information. Changes at the societal level, such as a shift in policy banning a popular product, can lead people to mediated channels , particuNot surprisingly, a robust community of JUUL users on Reddit, a popular moderated, forum-based social media platform, provided lots of discussion about the new FTP restrictions, as users sought information regarding how the newly imposed ban affects them, as well as instrumental support regarding the residual availability of newly banned products, replacement alternatives, and other means of adjusting to or even evading the consequences of the new flavor policy. Examining these discussions can help to evaluate the effectiveness and limitations of this policy. Though by no means immune to the limitations of data from other social media platforms, posts from Reddit are often detailed and candid and focus on shared experiences rather than punchy sound bites , and thuThis study aims to leverage Reddit discussions among JUUL users to better examine how they responded to real-time changes in FTP policies. Although the rise in popularity of disposable flavored products is likely the direct result of the most recent wave of FTP policies , understTo examine how users of flavored ENDS responded to shifting FTP policies, we collected, parsed, and analyzed posts and comments from r/JUUL using the third-party archive of Reddit content, pushshift.io API, to scrape all posts and comments between 1 May 2019 and 31 May 2020. Although we were primarily interested in the time period from August 2019 to January 2020, which included EVALI, JUUL’s voluntary removal of flavored pods, and the FDA’s guidance to ban flavored pods, we gathered the whole year of data to place these events within the context of the whole year. Two keyword filters were validated and used to narrow the total sample of texts down to those related to flavors and then further to those associated with FTP policies. Analyses of post and comment volume over time, N-gram analysis of relevant content identified by our keyword filters, and qualitative analysis of content identified by human coders were used to systematically examine the most common responses to the recently imposed regulations. All validated search parameters are available in the n = 166,169 texts (posts and comments) was retrieved from r/JUUL for further classification. Keyword filters were first used to identify texts discussing flavored ENDS products, and then further filtered to identify FTP policy-related content. For the flavor filter, n = 2500 posts were dual coded for correct identification of flavored ENDS product relevance . The filter performed well in accurately identifying flavor-related content . This initial filter yielded a flavor-related corpus of n = 33,884 texts. We then used a keyword filter to identify policy-related texts from within the flavor-related corpus. To evaluate the accuracy of this keyword filter, n = 200 posts were dual coded for the correct identification of relevant content. The coders were reliable , and the filter was sufficiently accurate . N-gram analysis was then performed on n = 7429 texts that our filters categorized as about FTP policies to provide insight into the content of these discussions through identifying the most common one, two, and three-word phrases.An initial corpus of n = 2000 texts from the 33,884 flavor-related texts for human coding. Two independent coders reliably  identified n = 488 texts related to FTP policy workarounds which were set aside for qualitative examination. All discrepancies were adjudicated by the study’s lead analyst. This extra round of human coding was performed to assure that qualitative examination would be conducted only on relevant content. In doing so, we both corroborate and add context and nuance to findings from our machine-driven approach.To triangulate findings from our N-gram analysis, we randomly sampled n = 7429 texts identified by our relevance filters to better understand the content of FTP policy-related discussions. Finally, to triangulate inferences based on N-gram analysis, we qualitatively examined a subsample of n = 488 texts identified by human coders as about FTP work around strategies.Three sets of analyses were conducted to further examine our data. First, using our filters, we graphed text volume by month for flavor and FTP policy-related content as a proportion of total content on the r/JUUL subreddit to visualize the relationship between the inciting event, EVALI, the ensuing ban on flavored pod devices, and the resulting discussion on Reddit. Next, we identified the most frequent words, bigrams, and trigrams (two and three word sets) among the The qualitative review revealed ten dominant themes across workaround-related content, with some posts containing more than one theme. More than one in three posts (39%) were seeking advice on disposable or refillable substitute brands that offered newly unavailable flavors, including 5.9% which named specific refillable pod device brands including Caliburn (9), Smok (7), Voopoo (1), Novo (7), and Vuse (5). One post mentioned OVNS, a refillable pod brand that is compatible with JUUL devices. Advice on product availability in localities where the ban had recently gone into effect  was sought in 23% of posts. Furthermore, 20% of posts inquired about how to acquire products online, while 16% concerned the ability to import products from abroad . Other topics were present in fewer than 5% of reviewed posts. For example, 4% discussed stockpiling or bulk buying and 2.5% sought advice on counterfeit, third-party, and off-brand pods. Fewer than 1% of posts concerned petitioning authorities regarding the newly imposed regulations, substituting other tobacco products including cigarettes and smokeless products, obtaining products from friends or family, and modifying unflavored pods using accessories or “slip-tips”. Thematic analysis reaffirmed findings from the N-gram analysis showing an overwhelming majority of discussions related to the newly imposed flavor restrictions were focused on the availability of alternatives and how to obtain them. Discussions of a “black market” identified in N-gram analysis were revealed to focus on the role of illicit market THC pods in the EVALI outbreak, decrying that a ban on flavored pods would inevitably lead to an illicit market, as well as seeking potential illicit market sources. The prominence of “mango” and “mint” in our N-gram analysis was indicative of the two most popular JUUL pod flavors  also beiThis study describes the content of discussions by flavored JUUL pod users to the evolving flavored product restrictions in real time. Significant spikes in usage of the r/JUUL subreddit corresponding to EVALI, JUUL’s voluntary removal of flavored pods, and again following issuance of FDA guidance suggest that the r/JUUL subreddit is a popular online platform for users to discuss product and policy shifts related to ENDS. The period between the EVALI outbreak and the FDA guidance removing flavored pods from the market saw a substantial increase in both overall activity on the subreddit as well as discussions related to the implications of the flavor ban. Qualitative review of these posts revealed that 95% of them concerned either alternative means of acquiring newly banned flavors or soliciting similar alternative products. Although there was some discussion of illicit markets, most discussions blamed the illicit market of THC cartridges for the new restrictions on JUUL pods. These findings highlight both the utility of Reddit for evaluating the public’s response and the potential limitations of newly enacted FTP policy.The findings also highlight the need for comprehensive flavor restrictions given the almost instantaneous response of JUUL pod users to identify alternative flavored products. Although JUUL was credited with the popularity of ENDS with youth, disposable flavored ENDS brands quickly filled the void left by JUUL pods . DiscussAlthough discourse surrounding tobacco regulation spans a variety of social media platforms, Reddit may be of particular use in examining more in-depth discussions. Reddit currently has over 440 million active users , but mosAlthough this study has many strengths, it is not without limitations. First, the selection biases inherent in social media sampling limits the generalizability of these findings. However, triangulating the findings in this study with data from other sources serves to support these findings, which highlight the quick market shift to disposable flavored brands such as Puff Bar . SecondlFlavors are consistently identified as the leading reason for tobacco product trial and use, particularly among young people ,30. As a


Individuals presenting with complex behavioural and mental health needs may not receive the provision of care needed. Those presenting with a more complex clinical presentation may have a history of self-harm and suicide attempts. A common risk factor for preceding suicide is previous self-harm, suicide attempts or discharge from inpatient units. Understanding the descriptive symptom domains for inpatients and those treated in the community and the relationship between them could inform suicide prevention. The aim of this study was to explore the extent of self-harm and suicidal behaviours in individuals with complex mental health needs across inpatient and community settings.A cohort study design of in-depth written medical notes (n = 80) for people who were known to have complex mental health needs across inpatient and community settings. Data were extracted from medical records onto a coproduced questionnaire. As well as demographic data, information was collated about previous self-harm, suicide planning, suicide attempts, and support seeking regarding suicidal thoughts. The study will include a quantitative in-depth description and inferential analysis of the demographic clinical characteristics of the patient group.Medical case notes were reviewed for 80 service users with complex mental health needs. Across both groups, approximately three-quarters of participants had previously self-harmed (76%), or planned suicide (n = 73%), and/or attempted suicide (63%). Self-harm (83% vs. 70%) and suicide attempts (72% vs. 65%) were more prevalent in the inpatient group compared to the community group. Social support was received more by community patients than inpatients (70% vs. 50%), even though inpatients were more likely to sough help when experiencing suicidal thoughts compared to people cared for in the community (38% vs. 30%). In both groups, there were often multiple suicide plans and attempts made over their timeline of contact with services.Self-harm, suicide planning, and suicide attempts were prevalent for people treated across both inpatient and community settings. Self-harm and suicide planning was indicative of a later suicide attempt within both settings. In those experiencing suicidal thoughts, few had sought help, suggesting the importance in staff training to enable then to recognise and identify patterns of self-harming and suicidal behaviours in individuals with complex mental health needs. Social support needs for inpatients should be increased, particularly when they sought help for suicidal thoughts. This may help to reduce length of stays in hospital or future readmissions to hospital; thus, reducing the cost implications for the NHS mental health services.