Vaccination remains one of the most effective means of reducing the burden of infectious diseases globally. Improving our understanding of the molecular basis for effective vaccine response is of paramount importance if we are to ensure the success of future vaccine development efforts.We applied cutting edge multi-omics approaches to extensively characterize temporal molecular responses following vaccination with hepatitis B virus (HBV) vaccine. Data were integrated across cellular, epigenomic, transcriptomic, proteomic, and fecal microbiome profiles, and correlated to final HBV antibody titres.Using both an unsupervised molecular-interaction network integration method  and a data-driven integration approach (DIABLO), we uncovered baseline molecular patterns and pathways associated with more effective vaccine responses to HBV. Biological associations were unravelled, with signalling pathways such as JAK-STAT and interleukin signalling, Toll-like receptor cascades, interferon signalling, and Th17 cell differentiation emerging as important pre-vaccination modulators of response.This study provides further evidence that baseline cellular and molecular characteristics of an individual’s immune system influence vaccine responses, and highlights the utility of integrating information across many parallel molecular datasets. Hepatitis B is a viral infection that primarily affects the liver of infected individuals, and can cause both acute and chronic disease. The WHO estimates that 257 million people had a chronic hepatitis B infection in 2015  can we identify baseline immune signatures that predict vaccine responses and differentiate between responders and non-responders, and (2) what temporal molecular changes occur following HBV vaccination? Baseline differences correlating with final HBV vaccine response could be identified in this small (n = 15) cohort of adults by using a multi-omics integration approach. This general concept of specific baseline immune signatures predicting vaccine responses has been demonstrated in large cohort studies in the context of HBV, influenza, and malaria vaccines (®-B) was undertaken, with recruitment occurring at the Vaccine Evaluation Center (VEC), BC Children’s Hospital Research Institute in Vancouver, Canada. Participants were recruited by e-mail, mail and telephone. All participants enrolled in the study provided written informed consent under a research protocol (H17-00175) approved by the University of British Columbia Women’s and Children’s research ethics board. All initial sample processing was undertaken at the VEC laboratory. Participants were healthy adults aged 44–73 years who were seronegative to HBV and with no prior history to HBV infection or vaccination, with demographics shown in Systems biology methods applied to blood and tissue for a comprehensive analysis of immune response to Hepatitis B vaccine in adults . At each visit, a number of molecular and clinical tests were performed on the collected biospecimens  of immune responses to the HBV vaccine  within subjects separately from the biological variation that existed between subjects  from subjects , 10. BasTo identify baseline differences between participants who responded to vaccine and those who did not, we used the HBV-specific antibody titre levels from Day 180 to divide the participants into either responders or non-responders, based on the well-established correlate of protection of 10 mIU/ml . This deSigora  from baseline omics profiles, we used two complementary data integration strategies: NetworkAnalyst and DIABLO.www.innatedb.com) removed. To highlight novel nodes in the combined networks, networks were constructed individually for the different omics methods and their node tables downloaded to enable comparison to the node table from the combined network. This allowed identification of nodes that were present in the combined network, but absent when examining each omics network separately. Node tables downloaded from NetworkAnalyst were tested for enriched Reactome or KEGG pathways using the R package Sigora as previously described . Ensemble gene IDs and UniProt protein IDs were mapped using the Biomart service from Ensembl  gene symbols. Methylated CpG dimers were mapped using the annotation provided by Illumina  using only the pre-vaccine transcriptomic data, 40 differentially expressed (DE) genes were identified, and used to construct a minimally-connected first-order PPI network (a first order network in which the interconnecting grey nodes that connect to only a single DE gene are removed), as shown in For the proteomic analysis, we were able to identify 267 unique peptides that changed in expression when comparing responders and non-responders at the pre-vaccine baseline  Cascade”. In addition, we identified some signatures of adaptive immune responses such as “IL17 signaling” and “Th17 cell differentiation”, providing further insights into immune differences between responders and non-responders. In particular, JAK-STAT is a major anti-viral pathway that when activated can lead to inhibition of HBV infections  predictors of vaccine responses based on multiple high-throughput datasets . To determine whether integrating the data in this manner resulted in models with better predictive performance, we fit DIABLO models of varying complexity , and compared them to sparse partial least squares regression [sPLS ] models Based on this rigorous statistical assessment, we chose to characterize the variables selected by the DIABLO  model thAdditionally, to assess the biological function of DIABLO selected features , we used NetworkAnalyst to construct PPI networks to determine whether these genes and proteins formed an interconnected biological network ; IL18 p, 39; IKB was more highly expressed in non-responders  demonstr.Butyricicoccus and Phascolarctobacterium, which were positively associated with anti-HB antibody titre response   .A better understanding of the complex regulatory interplay involved in the immune response to vaccination is a necessary step in the development of precision vaccinology. Leveraging systems biology approaches and collections of high-dimensional molecular immune readouts obtained from clinical cohorts may yield important insights. While these datasets are complex to synthesize and analyze, the complementary information they encode may strengthen biological findings and improve the accuracy of predictive models derived from them. Here we performed extensive molecular profiling of individuals receiving HBV vaccine to investigate vaccine response in adults. To our knowledge, this study constitutes the most comprehensive set of molecular immune readouts on a common set of individuals before and after HBV vaccination. Fifteen healthy HBV-seronegative adults received three doses of HBV vaccine to assess the correlate(s) of protection. We profiled the blood of participants before and after vaccination and defined both temporal changes in the various omics following vaccination, as well as baseline characteristics associated with a robust vaccine response Figure 1Using multi-omics integration strategies described herein, we were able to identify significant biological features and pathways from a small sample size of 15 participants. Where possible, we leveraged resampling strategies  to ensure the robustness of our findings, though we acknowledge the limits of doing so in so few samples. While larger studies will be able to provide more robust results, ours was designed to further demonstrate the feasibility of a multi-omics approach to studying vaccine response, even when applied to a relatively small cohort. As omics-based vaccine studies with large numbers of participants are prohibitively expensive to conduct, our integrative multi-omics strategy on a smaller cohort will help ensure these larger studies are conducted in a manner which extracts as much biological meaning as possible. Select findings could then be targeted for further validation in larger cohorts, using cost-effective platforms with more well-defined paths to clinical implementation.When analyzing patterns of change over time following vaccination, we were able to detect certain differences between pre- and post-vaccination samples 3. In paGiven that baseline immune profiles are known to predict vaccine responses to many agents , 45, 50,n i.e. few  biological samples, and a substantially larger p i.e. number of molecules or variables . This is sometimes referred to as the “n big psmall ” (or p>>>n) problem and can result in poor reproducibility and/or models that fail to generalize well to new data. DIABLO implements a number of strategies to tackle these challenges. First, it reduces the influence of noisy variables by means of dimensionality reduction techniques that summarise and/or identify useful and robust information from the data, leveraging penalisation (lasso) to carry out variable selection  and identify baseline features that could predict vaccine response. A particular concern with these methods is overfitting, particularly in studies with a relatively small election , 19. SecWe used DIABLO to derive a multi-omics model capable of predicting vaccine response from baseline molecular profiles. Critically, this DIABLO model outperformed models of comparable complexity derived from the individual omics analyses, highlighting the utility of multi-omics integration Figure 5®-B), suggesting that individuals who respond to vaccination may have greater intrinsic responses to adjuvant compared to non-responders (via inflammasomes (such as the NLRP3)  , 58.In this study, two participants did not reach the minimum titre threshold to be considered protected against HBV infection, even after receiving all three vaccine doses. These individuals were 63 and 72 years of age, placing them in the upper end of the range within our cohort of 15 adults. This implies that immunosenescence –61, and between data sets , and can be extracted with a statistical model that will appropriately aggregate independent information to increase statistical power. Such a requirement may seem to go against the key biological assumption that molecular data are inherently interrelated, i.e. it is believed that they act in unison within biological pathways. However, it remains unclear whether interrelatedness between molecules of different types directly implies statistical correlation , and is available under GSE155198. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020474. Microbiome data is available at the NCBI Short Read Archive (SRA) under accession number PRJNA658597. Flow cytometry data is available at flowRepository with the ID FR-FCM-Z2R9. Epigenetic data has been submitted to GEO and is available under GSE161020.The studies involving human participants were reviewed and approved by the University of British Columbia Clinical Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.CS advised on all omics analyses and carried out DIABLO integrative analysis. TB performed RNA-Seq and proteomics bioinformatics analyses. RB-O, TK, and MS recruited patients, collected samples, and coordinated data acquisition. NG and RE performed epigenomic analyses. NA performed microbiome sample processing and analyses. SD performed immune cell phenotyping bioinformatics analysis. QC performed initial screening and processing of proteomics samples. MK performed HBV serology for all participants. LF supervised proteomics sample collection, processing, and analysis. MS supervised epigenomics sample collection, processing, and analysis. WWM supervised microbiome sample collection, processing, and analysis. RB supervised immune cell phenotyping bioinformatics analysis. KL conceived and supervised the DIABLO integrative analysis. RHC supervised data deposition and contributed to conception of the manuscript. ST supervised the DIABLO integrative analysis. RH coordinated RNA-Seq sample collection and processing and supervised the RNA-Seq analysis. WK conceived the study and secured funding. TK and MS conceived the study and secured funding. CS, ST, RH, TK, MS and AL contributed to the conception of the manuscript. AL guided and advised on all omics and integrative analyses. CS, TB, RB-O, NG, NA, SD, LF, WWM, KLC, RH, TK, MS, and AL all contributed to manuscript writing. All authors contributed to the article and approved the submitted version.via salary awards from the BC Children’s Hospital Foundation, the Canadian Child Health Clinician Scientist Program and the Michael Smith Foundation for Health Research. MS has been an investigator on projects funded by GlaxoSmithKline, Merck, Pfizer, Sanofi-Pasteur, Seqirus, Symvivo and VBI Vaccines. All funds have been paid to his institute, and he has not received any personal payments. KL was supported in part by the National Health and Medical Research Council (NHMRC) Career Development fellowship (GNT1159458). TB, CS, and ST were supported by the National Institute of Health/National Institute of Allergy & Infectious Diseases Human Immunology Project Consortium Grant 5U19AI118608. AL is supported by Simon Fraser University New Faculty Start-up Grant.We acknowledge funding from the Human Vaccines Project. REWH was the recipient of a UBC Killam Professorship and a Canada Research Chair in Health and Genomics. REWH acknowledges funding from the Canadian Institutes of Health Research (CIHR) [funding reference number FDN-154287]. Mass spectrometry infrastructure used here was funded by the Canada Foundation for Innovation and the BC Knowledge Development Fund. Its operation is supported by Genome Canada and Genome BC (214PRO). NA and WWM were supported by a Project Grant (148781) from the Canadian Institutes for Health Research. MS is supported The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Indy (I’m not dead yet) gene encodes a plasma membrane citrate transporter in Drosophila. INDY reduction affects metabolism and extends longevity of flies and worms. In flies, INDY is predominantly expressed in the midgut, fat body and oenocytes, tissues with a key role in metabolism. We hypothesize that INDY reduction in the midgut regulates citrate levels leading to metabolic changes that preserve intestinal stem cell (ISC) homeostasis and slows aging by modifying Insulin/Insulin-like signaling (IIS), which is a key nutrient sensing pathway. Our second goal was to examine the role of JAK/STAT signaling pathway, which activates epithelial renewal in the gut, in response to aging-related stressors. We hypothesize that Indy reduction has effects on the microbiome, preventing bacterial overgrowth and altering community diversity, leading to extended longevity in a JAK/STAT-mediated fashion. Our data suggest that effects of Indy reduction is mediated by reduced IIS and JAK/STAT pathways


Child and adolescent obesity constitute one of the greatest contemporary public health menaces. The enduring disproportion between calorie intake and energy consumption, determined by a complex interaction of genetic, epigenetic, and environmental factors, finally leads to the development of overweight and obesity. Child and adolescent overweight/obesity promotes smoldering systemic inflammation (“para-inflammation”) and increases the likelihood of later metabolic and cardiovascular complications, including metabolic syndrome and its components, which progressively deteriorate during adulthood. Exosomes are endosome-derived extracellular vesicles that are secreted by a variety of cells, are naturally taken-up by target cells, and may be involved in many physiological and pathological processes. Over the last decade, intensive research has been conducted regarding the special role of exosomes and the non-coding (nc) RNAs they contain (primarily micro (mi) RNAs, long (l) non-coding RNAs, messenger (m) RNAs and other molecules) in inter-cellular communications. Through their action as communication mediators, exosomes may contribute to the pathogenesis of obesity and associated disorders. There is increasing evidence that exosomal miRNAs and lncRNAs are involved in pivotal processes of adipocyte biology and that, possibly, play important roles in gene regulation linked to human obesity. This review aims to improve our understanding of the roles of exosomes and their cargo in the development of obesity and related metabolic and inflammatory disorders. We examined their potential roles in adipose tissue physiology and reviewed the scarce data regarding the altered patterns of circulating miRNAs and lncRNAs observed in obese children and adolescents, compared them to the equivalent, more abundant existing findings of adult studies, and speculated on their proposed mechanisms of action. Exosomal miRNAs and lncRNAs could be applied as cardiometabolic risk biomarkers, useful in the early diagnosis and prevention of obesity. Furthermore, the targeting of crucial circulating exosomal cargo to tissues involved in the pathogenesis and maintenance of obesity could provide a novel therapeutic approach to this devastating and management-resistant pandemic. As defined by the World Health Organization (WHO), overweight and obesity are characterized by potentially detrimental excessive fat accumulation. Since 1975, worldwide obesity has almost tripled, while in 2016, more than 25% of the global population was overweight or obese, including 1.9 billion adults, with over 650 million of them being frankly obese , renderiBasic and applied research on obesity has been extensive lately, and mounting evidence has emerged from the promising field of extracellular vesicles (EVs) and their cargo. EVs are surrounded by a lipid bi-layer membrane and may be subdivided size-wise into exosomes (30–120 nm), microvesicles (100 nm–1 μm) and apoptotic bodies (2–3 μm). The latter originate from the blebbing of dying cells and have the size of platelets . DiffereAdipose tissue, apart from being an energy storage depot, acts also as an endocrine gland, secreting many bioactive peptides (adipokines) and metabolites that travel to distant cells and tissues and influence their function. Interestingly, adipocytes also secrete exosomes that can modulate the activity of cells within or outside adipose tissue in an autocrine, paracrine or endocrine manner. Thus, preadipocytes, endothelial cells, immune cells and other cells within adipose tissue or other distant tissues may be affected by exosomes . ExosomeThe scope of this review includes the role of exosomes and their cargo in adipose tissue physiology, as well as their probable dysregulation and contribution in the multifactorial obese state in children and adolescents, incorporating data from human clinical studies and animal models. Therefore, we systematically researched PubMed using various combinations of the subsequent words: “exosomes,” “obesity,” “children,” “adolescents,” “miRNAs,” “lncRNAs,” “adipose tissue,” “adipogenesis,” “epigenetics” and “stress”. The most recent, complete and relevant reviews and original articles on the topic, published up to November 2020, were selected.Exosomes are vesicles enclosed by a lipid membrane and have a diameter ranging from 30 to 120 nm, and an internal structure similar to that of a simple microbial cell . The bioVarious proteins modulate MVB fusion with the plasma membrane and subsequent exosome release. More specifically, Rab GTPases act on exosome traffic and fusion with the cell membrane in a cell-specific manner, meaning that different cells can use distinct Rab proteins to modulate exosome release . FurtherAfter release, exosomes interact with their target cells by transferring their bioactive cargo into them, thus, triggering phenotypic changes in the recipient cells . The tarGenerally, exosomes play a role in regulating metabolic homeostasis by transporting their specific cargos between different cell types. Exosomes transfer various types of biomolecules, such as proteins, DNA, mRNA and non-coding RNAs (ncRNAs). ncRNAs lack protein-coding capacity and they better reflect life complexity than the protein-coding genes, as they represent the majority of transcripts in the human genome that participate in dynamic gene regulation, mediated by a complex interaction with DNA, other transcripts and/or proteins . ncRNAs miRNAs. One exosomal cargo that has attracted particular interest lately for its possible diagnostic and therapeutic applications is miRNAs, a cohort of endogenous ncRNA transcripts with an approximate length of 20–22 nt, that are involved in the regulation of gene expression, primarily post-transcriptionally ,18. In glncRNAs. These have a length of more than 200 nt and bear minimal to no protein coding capacity ,23, contlncRNAs influence nuclear and cytoplasmic epigenetic functions (chromatin rearrangement and miRNA sponging), as well as translation and degradation of mRNA, and are involved in orchestrating gene expression at multiple levels . Lately,Intensive research conducted over the last decade has revealed that the functions of exosomes are determined by their originating cells or tissues and reflect their condition at the time of exosome biogenesis . ExosomeAcute stress exposure may lead to alterations in both exosomal membrane proteins and cargo, and these changes may cause a shift of immunomodulatory properties. Under pathological circumstances, exosomes may potentiate cellular stress causing cell damage . ExosomeAdipose tissue is a functionally pleiotropic tissue, considered to be of mesodermal origin ,35. AdipWAT. This tissue constitutes the main site of energy storage . After aObesity, especially the early-onset childhood type, is characterized by increased numbers of preadipocyte precursors and adipocytes with an impaired metabolism and reduced proliferative potential . As obesMetabolically unhealthy obesity is dominated by intra- and extra-abdominal WAT characterized by insulin resistance and inflammation and correlates more to visceral abdominal WAT (vWAT) than to subcutaneous WAT (sWAT) . AccordiBAT. This is a richly vascularized tissue that correlates negatively with risk of metabolic disease and the body-mass index (BMI) . It funcBeige AT. It has been observed that following various stimuli, including cold exposure, beta-adrenergic stimulation, diet, exercise, as well as adipokines, adipocytes referred to as “beige”, that resemble brown adipocytes, arise within WAT and gain thermoregulatory functions, in a process called “WAT browning” or “beiging” . Even thAs previously mentioned, apart from substantially regulating fatty acid metabolism, both WAT and BAT produce and secrete many adipokines, as well as regulatory lipids, called lipokines, that transduce signals in an autocrine, paracrine or endocrine manner ,45. AmonLately, the up-and-coming research field of adipose-derived exosomes has been attracting interest as a novel type of complex biomolecules mediating inter-cellular and inter-organ communication. The adipose tissue origin of exosomes is ascertained by the identification of adipocyte-specific protein markers, such as fatty acid binding protein 4 (FABP4), adiponectin, and perilipin A . AccumulIt has been proposed that exosomes regulate metabolic processes by also modifying immune function. In one study, adipocyte-derived exosomes promoted monocyte differentiation into active macrophages and elevated levels of IL-1β and TNF-α, well-known pro-inflammatory cytokines , while imicroRNAs in adipocytes. Adipose-derived miRNAs represent a big part of circulating miRNAs and are mainly contained in exosomes secreted both from WAT and BAT . They plSpecifically, transplantation-induced improvement of glucose tolerance in ADicerKO mice correlated with a lower level of hepatic and systemic fibroblast growth-factor 21 (FGF21) . BAT-derLately, the role of circulating miRNA in adipocyte biology has attracted researchers’ interest. At WAT, miRNAs have been implicated in adipogenesis, endocrine function of AT, glucose and lipid metabolism and WAT hypoxia and browning . A grouplncRNAs in adipocytes. Several studies aiming to construct a lncRNA database in adipose tissue have proposed various lncRNAs as significant regulators of adipocyte biology, white adipocyte differentiation and function, and brown and beige adipose tissue thermogenesis . RecentlIn BAT, both brown fat lncRNA 1 (Blnc1) and lncRNA BAT enriched (lncBATE-1) induced brown and beige adipogenesis through the formation of a ribonucleoprotein complex with key transcriptional factors of thermogenesis . Brown pGrowth charts developed by the Centers for Disease Control and Prevention (CDC), based on data from national surveys, are used for the classification of overweight and obese status in children and adolescents, according to the age- and gender-specific percentiles of body mass index (BMI). Overweight is indicated by measurements over the 85th percentile and under the 95th percentile on age- and gender-specific body curves, while obesity is indicated by measurements over the 95th percentile . A BMI eIt is predicted that children and adolescents defined as overweight or obese will have a progressively higher BMI during adulthood . SimilarCommonly, obesity is primarily caused when endogenous and exogenous factors result in a discrepancy between energy intake and energy consumption, finally leading to excess weight gain. These include genetic and epigenetic, dietary, physical, psychosocial, and other environmental factors, which could all impact negatively on overall energy homeostasis . FurtherGenetic types of obesity present with a common clinical spectrum and can be classified into mendelian (monogenic) syndromic obesity, mendelian non-syndromic obesity, and polygenic obesity, which is most common . MonogenIt has been postulated that the difficulty encountered by many people in their attempt to lose weight and maintain weight loss could be attributed to powerful biologic systems protecting our body weight during our evolution, thus ensuring survival and adequate reproduction in a food-deficient environment, but contributing to obesity development in an obesogenic environment where food is abundant . The comEnvironmental contributors to obesity are complex and deep-rooted in modern society. These include a recent shift to plentiful, highly palatable processed food in combination with the adoption of a sedentary lifestyle and a disruption in normal circadian rhythms . FurtherEpigenetic contribution to obesity has attracted great interest, as it helps understand the interaction between genes and environment. There are rapid, dynamic and reversible or long-term less reversible adaptive alterations of genome activity without concurrent changes in the DNA sequence, following environmental stimuli . EnvironSignificantly, maternal under- or over-nutrition, as well as maternal insulin resistance and weight gain during pregnancy are markedly associated with offspring obesity . Even thExosomal miRNAs in obesity. Obesity has been associated with increased circulating EVs, including exosomes . The proCirculating miRNAs in child and adolescent obesity. Up to now, most studies have been conducted by identifying circulating miRNAs in blood samples rather than exosomal or even adipose-derived exosomal miRNAs and have been mostly focused in adult obesity, with a general scarcity of data regarding miRNA involvement in childhood obesity . HoweverIn a study by Thompson et al., obese children displayed a prominent increase in circulating miR-199a compared to lean controls . FunctioExosomal lncRNAs in obesity. Most studies investigating exosomal lncRNAs have mainly evaluated the function of cancer cell secreted exosomal lncRNAs. Nevertheless, one study demonstrated that adipose tissue secretes exosomes containing the lncRNA HOTAIR, that subsequently communicate with intestinal cells and promote their proliferation, thus linking sedentary lifestyle and obesity with colorectal cancer . AnotherCirculating lncRNAs in obesity. There is mounting evidence that lncRNAs could provide insight into obesity-related gene regulation. In a recent study, 249 lncRNAs and 392 mRNAs were abnormally expressed in obese adults compared to controls with a normal BMI . In partApart from genetic predisposition to obesity associated with functional polymorphisms affecting LEP and LEPR gene expression, epigenetic alterations in the transcriptional LEP gene regulation could lead to obesity by decreasing leptin secretion by adipocytes, suggesting that individuals with obesity and low leptin levels could remain sensitive to leptin and benefit from leptin therapy. Accordingly, Dallner et al. identified lncOb, that appears to increase LEP gene transcription by stabilizing the promoter complex loop; conversely, lncOb expression defects led to a form of obesity characterized by low leptin levels, that responded to leptin therapy . In lineDespite the fact that numerous lncRNAs have been identified as key regulators of adipocyte biology, the understanding of their role in childhood and adolescent obesity remains limited. A recent genome-wide association study (GWAS) analysis revealed an SNP in the lncRNA rhabdomyosarcoma 2-associated transcript (RMST) in children with severe obesity . FurtherIn a study by Chen et al., lncRNA HLA complex P5 (HCP5) and LINC00839 were considerably increased in obese children compared to controls . HCP5 poExosomes represent a novel promising class of microvesicles with potential applications as biomarkers for various diseases and as therapeutic biomolecule carriers . The abiThe establishment of circulating miRNAs as biomarkers of childhood obesity could provide a tool for evaluating the degree of obesity and risk factors for metabolic syndrome and T2DM prediction, which is of great importance for personalizing medical practice and preventing the future consequences of childhood obesity and associated comorbidities. For example, it has been suggested that miR-122 could potentially apply as a future biomarker for evaluating disease severity and monitoring disease progression in pediatric NAFLD . Such anRegarding the use of exosomes as therapeutic biomaterials, naturally derived exosomes may bear therapeutic properties proportionate to the ones of their cell of origin or could be used as active biomolecule carriers , with poDuring the last decade, miRNAs have become promising therapeutic agents for many disorders such as metabolic syndrome, neurological and autoimmune disorders, and cancer ,18. MoreAnother promising therapeutic strategy would be to target exosomal lncRNAs. This perspective has been evaluated mainly in cancer; however, obesity could be next. Pharmacological compounds could target lncRNAs that regulate white adipocyte differentiation, exploiting the recently uncovered structure and function of many lncRNAs. Alternative strategies could include stimulation of brown adipocytes by lncRNAs that possess such properties, such as Blnc1, or regulation of combined lncRNAs-miRNAs that interact during adipocyte differentiation, such as NEAT1 and miR-140 . The estAdipose tissue releases exosomes with distinct cargoes that constitute a novel way of metabolic organ inter-communication. These exosomes and their cargoes are altered in obesity and accompanying disorders, rendering them potential biomarkers for diagnosis and monitoring, as well as prospective therapeutic targets. Research on exosomes and metabolism keeps expanding and adipose-derived exosomes have been established as crucial components of inter-cellular communication networks, however, we are currently only beginning to unveil the complex mechanisms of exosome functions in adipose tissue and obesity pathogenesis.Some challenges encountered include technical difficulties in exosome isolation, quantification, characterization of the cell of origin, as well as a high heterogeneity between findings, possibly attributable to sample differences and different isolation techniques. It is speculated that a combination of circulating miRNAs, rather than a single miRNA, orchestrate metabolic processes, setting the objective of elaborating a full profile of circulating miRNAs and elucidating the mechanisms of their involvement in obesity. Furthermore, as already mentioned, studies evaluating exosomal, especially adipose-derived exosomal miRNAs and lncRNAs, are rare, as most studies investigate circulating mRNAs and lncRNAs in blood samples, pointing out the need to identify the tissue origin of circulating miRNAs and lncRNAs in the future, and for further conducting studies regarding exosomal miRNAs and lncRNAs.The roles of exosomes in obesity have been more extensively studied in adults than in children and adolescents. Therefore, a larger number of studies with more participants is required to draw safe conclusions regarding their participation and importance in childhood and adolescent obesity. We suggest that studies in the young age group could provide remarkable findings that will deepen our understanding of obesity, as children and adolescents have fewer comorbidities that could overshadow the mechanisms involved in obesity pathogenesis and progression.


The nanomaterial industry generates gigantic quantities of metal-based nanomaterials for various technological and biomedical applications; however, concomitantly, it places a massive burden on the environment by utilizing toxic chemicals for the production process and leaving hazardous waste materials behind. Moreover, the employed, often unpleasant chemicals can affect the biocompatibility of the generated particles and severely restrict their application possibilities. On these grounds, green synthetic approaches have emerged, offering eco-friendly, sustainable, nature-derived alternative production methods, thus attenuating the ecological footprint of the nanomaterial industry. In the last decade, a plethora of biological materials has been tested to probe their suitability for nanomaterial synthesis. Although most of these approaches were successful, a large body of evidence indicates that the green material or entity used for the production would substantially define the physical and chemical properties and as a consequence, the biological activities of the obtained nanomaterials. The present review provides a comprehensive collection of the most recent green methodologies, surveys the major nanoparticle characterization techniques and screens the effects triggered by the obtained nanomaterials in various living systems to give an impression on the biomedical potential of green synthesized silver and gold nanoparticles. Owing to a number of revolutionary developments in nanobiotechnology, the synthesis methods of various nanomaterials seem uncomplicated and straightforward and enable the construction of literally any type and structured nanoparticle designed and tailored to essentially every possible application let it be in industry, technology or medicine. Metal nanoparticles represent a major class of nanomaterials, where singular physicochemical characteristics yield an ideal platform for the exploitation of such nanomaterials (mainly of silver and gold nanoparticles) for electronics, optics, household items, catalysis, and for various biomedical applications as well . TogetheSince traditional physical or chemical methods of metal nanoparticle synthesis have obvious limitations and disadvantages, green chemical processes have emerged as a new direction in the chemical industry about two decades ago . Ever siRecently, various microorganisms, mainly bacteria and fungi, have been engaged to produce different metal nanoparticles, such as silver, gold, silver–gold alloy, iron, copper, zinc, palladium and titanium nanomaterials ,32. The Pseudomonas stutzeri ..Padina gNumerous yeasts, fungal and actinomycete strains, and even viruses, have been utilized to assemble gold nanoparticles to form microstructures . NeverthDespite the huge potential of using microorganisms for nanomaterial production, there are some limitations which should be considered before use. In fact, using biological agents for the synthesis of silver and gold NPs is preferable over chemical methods due to the simple, eco-friendly approach and also for minimizing the application of harmful chemical solvents and reagents. However, after carefully examining the above-presented microbe-assisted syntheses and the applied biological entities, it can be concluded that despite these approaches being relatively straightforward and favorable, they require specific and rather tedious preparations and multistep processes such as culture isolation, maintenance or growth and inoculum standardization. Moreover, on the surface of the obtained particles, multicomponent residuals from microorganisms can accumulate, which would not only define the physical, chemical and biological characteristics of the obtained nanomaterials and their fate in the presence of living systems, but would also trigger potential immunological reactions after entering the organism. For this reason, the synthesis of metal particles using plants or any part of plants came forth and were later more and more prioritized. These reactions tend to be faster than those performed by microorganisms, are more cost effective and are relatively easy to scale-up for the generation of larger amounts of nanoparticles.As discussed above, nanoparticle synthesis using microorganisms is often rather slow, as the availability and maintenance of the various species used in the process is difficult and expensive; moreover, their application on a large scale is fairly restricted . On the The possible mechanisms of nanoparticle formation using plant extracts have been examined by several authors . Two mai+-dihydromyricetin ,18162,18he cells .Nocardiopsis alba-synthesized AgNPs inhibited the action of Newcastle Disease Virus (NDV) [Andrographis paniculata, Phyllanthus niruri and Tinospora cordifolia) were used, decreased the infection caused by chikungunya virus. In this study, however, it is not clear that the observed antiviral activity is owing to the reducing agents or to the AgNP itself, since the antiviral properties of these plants were previously known [Gold nanoparticles prepared using garlic extract were tested and proven to be effective against measles virus probably for similar reasons as described above in Haggag’s work: via binding to the viral surface proteins, AuNPs are able to inhibit the attachment of the virus particles to their receptors on the surface of the cells . Avilalaus (NDV) . Antivirly known . UnfortuOn the contrary to antiviral features, the antibacterial and/or the antifungal activity of biologically synthesized NPs have been tested by many research groups that are actively working on green nanoparticle synthesis and their application possibilities ,36,62. TPseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. Interestingly, the susceptibility of Gram-positive and Gram-negative bacteria to silver nanoparticles differs somewhat; Gram-negative bacteria are more susceptible than Gram-positive counterparts. This difference is related to the distinct structure of their cell wall. Gram-negative bacteria have a thin cell wall consisting of a peptidoglycan layer; therefore, AgNPs penetrate more easily across this structure than across the thick cell wall of the Gram-positive species [The antibacterial efficiency of various green synthesized silver nanoparticles has been studied intensively see . These s species .Another rather valuable property of AgNPs is their biofilm-eradicating capability ,219. BioThe antibacterial and antifungal effect of biologically formed gold nanoparticles is ambiguous. In some relatively extensive studies, no effect could be attributed to AuNPs ,221; howOne of the most important advantages of green synthesized AgNPs and AuNPs is their potentially enhanced biocompatibility compared to nanoparticles of the same chemical element synthesized in a classical chemical procedure. Similarly to their non-green generated counterparts, these particles could be exploited in the future in cancer therapy; however, their effects on human cells, not only on cancerous ones, needs to be evaluated. As potential therapeutic molecules, they come into contact with numerous cells of the body; thus, it is essential to investigate thoroughly the cytotoxic activity of these nanoparticles.in vitro and in vivo as well. Green synthesis with plants or other organic materials gives the opportunity to prepare nanoparticle solutions carrying biologically active compounds coming from the applied natural extract, which might result in a modulated anticancer activity, rendering the particle more or even less potent, or toxic towards human cancer and non-cancerous cells. In fact, a large number of green synthesized AgNPs and AuNPs were proven to exhibit anticancer activity  than in human primary neonatal skin stromal cells (hSSCs) [Herbal medicinal plants are often preferred as biological entities for nanoparticle green synthesis. te cells . In a sier cells . Alsalhi (hSSCs) .Trichosanthes kirilowii caused cell cycle arrest in G0/G1 phase and induced apoptosis via activating several apoptotic pathways, involving bid, bax/bcl2 and caspases in colon cancer cells [Marsdenia tenacissima plant extract [Cordyceps militaris mushroom extract [Nerium oleander extract also decreased the viability of MCF-7 cells. In this case, a significant cancer cell selective activity was observed, since these green AuNPs did not affect the cell viability of primary non-cancerous lymphocytes. As the N. oleander plant extract itself caused significantly lower toxicity on both MCF-7 and non-cancerous primary lymphocytes, as compared to the AuNPs, the authors concluded that the natural extract applied during the synthesis of nanoparticles is only partly responsible for the observed biological effects of these AuNPs [Despite numerous studies suggesting the biocompatibility of AuNPs, the anticancer activity of some green synthesized AuNPs was confirmed. As an example, AuNPs synthesized with er cells . Similar extract  and AuNP extract , respectse AuNPs .Gelidium pusillum induced apoptosis in MDA-MB-231 cancerous cells; in contrast, fewer apoptotic cells were observed in the non-cancerous HEK-293 samples, despite some DNA fragmentation occurring at a nanoparticle concentration of 150 μg/mL [Tribulus terrestris, but in this case, the effects of two AuNPs with different sizes were examined (7 and 55 nm diameters). The study confirmed that cultures of AGS cells treated with larger sized AuNPs contained a lower number of apoptotic cells than cell cultures treated with the smaller AuNPs [AuNPs synthesized by 50 μg/mL . A very er AuNPs .Peltophorum pterocarpum leaf extract-mediated AuNPs were tested on human normal endothelial cells . No inhibition of cell proliferation was measured, confirming the biocompatibility of these AuNPs in an in vitro system. This is one of the few studies where the nanoparticle-induced toxicity was investigated in an in vivo model as well. Nanoparticle exposure did not induce any adverse clinical signs or weight changes in C57BL6/J mice. No significant changes were detected in the levels of total glucose, urea nitrogen, transaminases and uric acid in the serum compared to the control group of mice except for triglycerides and cholesterol levels in the AuNP treated group. Moreover, no major histopathological differences were observed among the treated and untreated groups [The cytotoxic effect of aqueous d groups .Anemarrhena asphodeloides showed anticancer activity on several tested tumor cells, while neither of them affected the viability of normal pre-adipocyte cells. Furthermore, when the effect of green synthesized AgNPs were compared to commercially available AgNPs, the green AgNPs showed higher cytotoxicity on cancer cells than commercial AgNPs. The reason behind this difference was not established, since ROS production upon the various treatments was not consistent [Artemisia turcomanica leaf extract) and commercial AgNPs using cancer and non-cancerous cells as both AgNPs induced apoptosis in cancer cells to the same extent [Oftentimes, when a natural extract is utilized for nanoparticle production, AgNPs as well as AuNPs are concomitantly synthesized. It is generally accepted that mostly AgNPs show high anticancer activity compared to AuNPs; however, this feature can depend enormously on the natural extract used for the nanoparticle synthesis and nonetheless on the tested cell lines. A few studies have attempted to compare the biological performance of the green synthesized metal particles and commercially available, chemically generated counterparts. Both AuNPs and AgNPs produced with rhizome of nsistent . In anote extent . A simile extent . Black te extent . These rin vitro experiments, which is fairly understandable, there are occasionally publications revealing the impacts of green synthesized nanoparticles on in vivo model systems. Yan He et al. performed in vitro experiments first to screen the anticancer effects of Dimocarpus longan-mediated AgNPs on lung, pancreas and prostate cancer cells. AgNPs showed great inhibitory effects on the H1299 lung cancer, but they were less effective on the VCaP prostate cancer and BxPC-3 pancreas cancer cells. Treatment of H1299 cells with these green AgNPs induced apoptosis with dose-dependent decreases of NF-κB transcriptional activities. This finding is relevant because activated NF-κB is a key regulator of programmed cell death and is associated with lung cancer progression by the transcriptional regulation of responsive genes [in vitro examinations, the effect of green AgNPs was investigated in vivo on mice carrying H1299 lung tumors. AgNPs could inhibit tumor growth in mice, as significant differences in the tumor size between control and AgNP-treated group was detected. These results indicate that green AgNPs could be effective candidates for the treatment of lung cancer in vivo as a complementary to classical chemotherapy [Although the majority of the toxicity studies are based on ve genes . The impve genes ,226. Folotherapy .Ficus religiosa-synthesized AgNPs, cancer cell viability decreased in a time- and AgNP dose-dependent manner in vitro. AgNPs synthesized by F. religiosa brought about cell death in A549 and Hep2 cells through the induction of apoptosis by increased generation of ROS and decreased levels of antioxidants. Furthermore, both the extrinsic and the mitochondrial apoptotic pathways were activated in AgNP-treated tumor cells. The following in vivo studies performed on rats revealed significant increases in the serum levels of AST, ALT, LDH, TNF-α and IL-6 after oral administration of F. religiosa-mediated AgNPs and showed accumulation of silver in liver, brain and lungs. However, the levels of these serum parameters reverted back to normal and the complete elimination of AgNPs was also observed by the end of the wash out period [In case of t period .In recent years, hybrid/composite or core-shell bimetallic or trimetallic nanoparticle systems of gold and silver were also developed, which shifted somewhat the focus of investigations. Green synthesized Ag-Au composites give the opportunity to exploit the anticancer effects of both AgNPs and AuNPs and fine-tune the biological activities of the obtained nanomaterials. In one of these studies, the anticancer effects of starch-mediated bimetallic Ag-AuNPs were investigated. Ag-AuNPs were not toxic to human dermal fibroblasts, while they significantly decreased the viability of melanoma cells. In comparison, monometallic AuNPs synthesized with starch were not toxic to either fibroblasts or melanoma cells .Trapa natans peel extract were investigated on p53 wild type and p53 knockout cells. Ag-AuNPs induced oxidative stress in cancer cells, and caused apoptotic cell death in a ROS-mediated p53-independent way via mitochondrial damage and through activation of caspase-3. It was emphasized that ROS production must be an important factor in the mechanism of action of this green Ag-AuNP composite, because apoptosis was attenuated upon decreasing ROS levels [The molecular mechanisms behind the anticancer effect of Ag-AuNP composites synthesized with S levels .Escherichia fergusonii exhibit a toxic effect on MCF-7 breast cancer cells by inducing ROS generation, leading to cellular apoptosis. This study indicated that these bacteria-mediated nanoparticles could have antiproliferative effects as well [Pseudomonas aeruginosa-synthesized AgNPs and their proliferation capacity decreased with increasing dose of AgNPs [Pseudomonas species was also proven to be an adequate candidate for nanoparticle synthesis. Gopinath et al. investigated the cytotoxic effects of P. putida-generated AgNPs on HEp-2 cells, which revealed that AgNPs did not affect significantly the viability of these cells. It is important to mention that the AgNP concentration used on human cells was lethal for the bacteria tested in parallel. These results indicate that the biogenic, P. putida-synthesized AgNPs are capable of displaying antibacterial activity without being harmful to tumor cells at this concentration [Bacillus funiculus-mediated AgNPs were also investigated by assessing cell viability, metabolic activity and oxidative stress on MDA-MB-231 breast cancer cells. Dose-dependent cytotoxicity, activation of caspase-3 and generation of ROS were demonstrated for these AgNPs against MDA-MB-231 cells. The resulting apoptosis was further confirmed by detecting nuclear fragmentation [Paracoccus haeundaensis BC74171T bacteria were biologically inert on normal cells and showed slight toxicity on cancerous cells in the highest applied concentrations [Obviously, besides plant-mediated nanoparticle synthesis methods, there are other green, cost-effective and rapid techniques utilizing fungi and bacteria for this purpose ,228. In  as well . Interesntration . The celentation . AuNPs strations .Oscillatoria limnetica, were also utilized to produce metal nanoparticles, and the cytotoxic potential of the obtained nanomaterials were tested against human cancer cell lines. In case of HCT-116 cells, cyanobacterium-synthesized AgNPs decreased the cell viability more than in the case of MCF-7 cells via inducing apoptosis, which was represented by the morphological changes in tumor cells [Cyanobacteria, such as or cells . Since ior cells .In vitro AgNPs synthesized by Agaricus bisporus showed dose-dependent toxicity on MCF-7 human breast cancer cells. In in vivo experiments, it was also demonstrated that the combination of these AgNPs and gamma radiation could induce apoptosis in Ehrlich solid tumor cells in mice via a mechanism involving caspase-3 activation. In Ehrlich solid tumor cells, this treatment combination mitigated significant superoxide dismutase and catalase activities and reduced glutathione levels, whereas it increased malondialdehyde and nitric oxide levels [Ganoderma neo-japonicum-mediated AgNPs reduced the cell viability and induced membrane leakage in a dose-dependent manner. Tumor cells exposed to these nanoparticles showed increased amounts of ROS and triggered hydroxyl radical production. In fact, the apoptotic effects of AgNPs were confirmed by activation of caspase-3 and DNA nuclear fragmentation [The application of fungi as reducing and stabilizing agents in the biologically synthesized AgNPs is engaging due to the production of large quantities of proteins, high yields, easy handling and low toxicity of the residues . In vitre levels . In anotentation .In the last few decades, research on efficient metal nanoparticle-based medical approaches for drug-delivery, regenerative medicine, imaging and biosensing have gathered impetus. Provided by this stimulus, alternative, mainly green synthetic methods for silver and gold nanoparticles destined intentionally for such specific purposes have received scientific attention.Tricholoma crassum were found to be promising candidates for gene delivery, since green fluorescent protein (GFP) was successfully carried into mouse sarcoma cancer cells using a plasmid DNA-AuNP complex. Moreover, these AuNPs showed low hemolytic activity towards human erythrocytes, which confirms their biocompatible nature. These results indicated that green nanoparticles could be considered as potential drug delivery platforms of cancer therapeutics [in vitro systems and an in vivo mouse model, that Peltophorum pterocarpum-synthesized AuNPs are suitable and effective anticancer drug-carriers. They designed a biosynthesized AuNP-based drug delivery system, in which these particles were conjugated with Doxorubicin (Dox). In vitro Dox conjugated-AuNPs showed high antiproliferative activity against A549 lung cancer and B16F10 melanoma cells. They obtained similar results in vivo, as a significant reduction in tumor growth was observed compared to the untreated and the unconjugated-AuNP treated group. Significant amounts of conjugated AuNPs accumulated in the spleen 2 h after the treatment; however, 24 h post-injection, the tumors showed a strong tendency to accumulate high levels of Dox conjugated-AuNP. The biodistribution of the drug conjugated-AuNPs reflected the selectivity of this drug-carrier system [Butea monosperma synthesized AuNPs and AgNPs. In vitro the drug conjugated-AuNPs and AgNPs showed significant cell proliferation inhibitory activity towards B16F10 cells in a dose-dependent manner compared to free Doxorubicin applied at the same concentration. The enhanced anticancer effects of Dox conjugated-nanoparticles were verified by apoptosis detection [Ever since, several articles describing the carrier function of biological synthesized nanoparticles—especially gold nanoparticles—have been published. In one study, protein-coated AuNPs synthesized by apeutics . Mukherjr system . Similaretection . Gellan etection .Delftia sp. strain) was tested on A549 human lung cancer and on MCR-5 normal fibroblast cells. Resveratrol conjugated-AuNPs showed significantly higher cytotoxic activity towards A549 cells than resveratrol alone. However, no cytotoxicity was observed on non-cancerous MRC-5 fibroblasts [The toxic effect of resveratrol conjugated-biosynthesized AuNPs (generated by roblasts .Punica granatum synthesized AuNPs were conjugated with the chemotherapeutic agent 5-Fluorouracil (5-Fu). The problem in using this drug is its toxicity to bone marrow and to the gastrointestinal tract. To tackle this problem, Ganeshkumar et al. have developed a method to biosynthesize AuNPs functionalized with folic acid (FA) for targeted 5-Fu delivery. The rationale to functionalize nanoparticles with FA is that folic acid receptors on the cell membrane can be targeted for tumor selective drug delivery. Several liver and breast cancer cell lines are known to overexpress folate receptors; thus, the in vitro cytotoxicity of this drug delivery system was investigated on MCF-7 breast cancer cells. Higher cytotoxic effect was measured in case of 5-Fu@nanogold-FA treatment compared to 5-Fu alone or 5-Fu@AuNPs in the same concentrations. The authors found that these drug-conjugated AuNPs functionalized with FA could induce the expression of both p53 and p21 in a concentration-dependent manner in MCF-7 cells. These findings suggest that the JNK/ERK signaling pathway could be involved in p21WAF1-mediated G1-phase cell cycle arrest and growth inhibition in 5-Fu@nanogold-FA treated breast cancer cells [In vitro cytotoxicity assays on HepG2 hepatocarcinoma cells revealed that 5-Fu@AuNPs-FA exhibit higher toxic activity than 5-Fu alone or 5-Fu@AuNPs, which again pointed to a conclusion that 5-Fu@AuNPs-FA could be a promising alternative carrier for targeting liver cancer [Mappia foetida to examine their applicability in targeted cancer therapy. They described that Doxorubicin-loaded AuNPs conjugated with n-hydroxysuccinamide (NHS) activated folic acid (FA) showed low toxicity against Vero normal epithelial cells and high cytotoxic activity against human cancer cells  [er cells . A furthr cancer . Yallapp Hep-G2) .in vitro and in vivo imaging. These nanoparticles are not only biocompatible, but their colloid solution is pure enough for in vivo applications. Cinnamon-AuNPs are capable of labeling cancer cells in vitro, and can be detected by photoacoustic methods; furthermore, with the help of these green synthesized AuNPs, circulating tumor cells can potentially be detected in vivo as well. Moreover, biodistribution studies revealed that cinnamon-AuNPs are mostly accumulated in the lungs, indicating their use as contrast agents targeting the lung [Olax scandens leaf extract were aimed for both therapeutic and diagnostic purposes. The phytochemicals of Olax scandens leaf yield anti-cancer properties to the as-prepared AuNPs, and owing to fluorescent proteins provided by the green extract, these fluorescent nanoparticles enable the detection of the cancer cells [For diagnostic purposes, biocompatible AuNPs have to be designed and tested. Albeit green synthesized nanoparticles could hold great potential in diagnostics, their biocompatibility is strongly dependent on the natural extract applied upon synthesis. Green magnetite-gold nanohybrids (Fe(3)O(4)/Au) produced with grape seed proanthocyanidin can be suitable for MRI and CT imaging as contrast agents. The magnetite part gives superparamagnetism in MRI, while the gold part of the hybrid provides high X-ray contrast in CT. The nanocomposites are biocompatible and suitable for labeling and imaging stem cells, since nanocomposites could be internalized and accumulated in the cytoplasm of these cells . Cinnamothe lung . Fluoreser cells .Sanghuangporus sanghuang polysaccharide synthesized AgNPs with chitosan, forming a porous sponge structured matrix, increased wound healing via inducing wound contraction and internal tissue regeneration in damaged skin of animals and disinfected the skin surface to inhibit the growth of Escherichia coli and Staphylococcus aureus [Coleus forskohlii root extract could enhance the wound closure, suppress the inflammation and induce the re-epithelization of excision in Wistar rats [Another application purpose of green synthesized AgNPs could be regenerative medicine. Nanoparticles are mostly applied in such studies because of their wound healing-inducing activity. s aureus . Green Atar rats .Citrus lemon fruit extract, supporting the applicability of green synthesized noble metal nanoparticles as biosensors [Green synthesized metal nanoparticles are potential biosensor candidates. The shift of the surface plasmon resonance (SPR) peak in the spectra of green synthesized nanoparticles is usually followed, which can also vary depending on the natural extract applied upon synthesis ,242. Forosensors .in vivo studies and extending the technical repertoire with additional assays. We cannot stress enough the relevance of performing the outlined characterization route; otherwise, the chemical and biological profile of the obtained green nanomaterial may not be confidently trusted and adverse effects will be observed upon its application.Based on the numerous experimental data accumulated in the literature and summarized here in this review, it is evident that the field of green synthesized metal nanoparticles is expanding continuously, and every new prospective emerging on the horizon offers the possibility of finding other, more innovative ways and means to produce silver or gold nanoparticles with the exact properties needed for a specific purpose. Since the biological entities potentially applicable for green synthesis are practically endless, research has to continue to prepare, test and experiment with nanoparticles—synthesized in an eco-friendly approach, using green and renewable materials directly from nature—which exhibit unique properties and behave in the desired manner upon encountering living systems, such as human or fungal cells, bacteria or even viruses. Nevertheless, careful considerations in the selection of the green material for nanomaterial production, and more importantly, a comprehensive screening protocol of these green particles, are obligatory to predict the attitude and performance of NPs on living cells. First of all, the chemical composition of the applicable green material should be considered to estimate which biomolecules have the capacity to act as reducing or capping agents and which of them can be potentially adsorbed on the nanoparticle surface, creating a bioactive coating to interact with living cells upon action. The examples itemized above in this review clearly show that the green materials employed for the synthesis will define or at least fine-tune the chemical and physical properties and surface chemistry and thereby the biological activity of the obtained nanoparticles. After the green material is selected, and its chemical composition and its active ingredients have been regarded, all other chemicals required for nanoparticle synthesis should be attentively picked to preferentially utilize biocompatible substances and to avoid toxic chemicals, leaving only nonirritating, innocuous waste materials behind. When the nanomaterial is readily obtained, a meticulous examination of its structure and physicochemical properties has to be completed to reveal the average size, morphology, surface chemistry and other critical factors. This step is just as important as the synthesis approach, since these findings either promote the nanomaterials for biological tests or advise further optimization of the preparation protocol in case nanoparticles with undesired properties are formed. Finally, a comprehensive biological screening has to be carried out by inspecting the toxicity of the green nanoparticles on various human cell types, on Gram-negative and -positive bacteria, on a number of fungal strains; eventually, the antiviral propensity can be assessed as well. Depending on the original purpose of nanoparticle synthesis, each of these biological characterizations should be broadened by further implementing cell types and strains or even by


Epileptogenicity following brain insult depends on various factors including severity of the resulting lesion and extent of brain damage. We report a 54-year-old female patient who developed medically refractory epilepsy resulting from the interplay of pre-existing and post-insult pathologies. She presented with subarachnoid hemorrhage (SAH) due to a ruptured aneurysm and underwent clipping surgery. Seizures started 3 months post-operatively. MRI revealed cerebral ischemia and hemosiderin deposits in the left temporal lobes, and left hippocampal atrophy was suspected. As anti-seizure medications and vagus nerve stimulation failed to control her seizures, she underwent left temporal lobe resection and placement of a ventriculoperitoneal shunt for the post-operative complication of hydrocephalus. She remains seizure-free to date. Neuropathology revealed a previously undiagnosed focal cortical dysplasia (FCD) type 1a. Brain insult likely had a second hit effect in the late onset of epilepsy in this patient with pre-existing mild MCD, in whom secondary epilepsy can be attributed to the interplay of multiple underlying pathologies. EpilIn neuropathology of temporal lobe epilepsy, epileptogenicity has been attributed to various lesions including hippocampal sclerosis, malformation of cortical development , as well low-grade tumors , 9.In daily practice, acute or remote damage resulting from a previous insult is considered as the sole cause of epileptogenicity. As many patients do not require surgery, information on etiology derives solely from magnetic resonance imaging (MRI). However, many pre-existing and yet undiagnosed factors as well as complications from an acute insult can impact the establishment of an epilepsy network by affecting the neurotransmitter system and intracellular and extracellular homeostasis. Indeed, it is common clinical experience that some patients develop epilepsy following an insult and others do not, even though they have the same degree of brain damage.We have previously identified the presence of an underlying FCD as a significant factor associated with epilepsy in patients who developed post-traumatic epilepsy following severe head trauma . We postHere, we describe the management of a patient who developed epilepsy and who initially presented for medical care due to a ruptured aneurysmal SAH. In addition to multiple complications due to her SAH , her seizures became medically refractory and warranted resection surgery, leading to the discovery of a previously unknown area suggestive of FCD type 1a.A 54-year-old right-handed female was evaluated at the Comprehensive Epilepsy Center, Seirei Hamamatsu General Hospital  due to medically refractory weekly epileptic seizures.Past medical history was relevant for a SAH due to a ruptured left middle cerebral artery aneurysm and status post-aneurysm clipping 11 months prior. Her clinical evolution during acute care was complicated by a hemorrhagic cerebral infarction in the left temporal lobes due to symptomatic cerebral vasospasm.Focal aware and unaware seizures started 3 months after the surgery. Seizure semiology consisted of an epigastric sensation followed by aphasia and then loss of awareness, with occasional focal to bilateral tonic-clonic seizures. The electroencephalogram (EEG) showed interictal epileptiform discharges over the left fronto-temporal region, in keeping with topography of the bleeding and complications.Levetiracetam, Zonisamide, Valproic acid, and Lamotrigine did not control her seizures, thus fulfilling criteria for drug-resistant seizures , 12. The18F]fluoro-2-deoxy-D-glucose (18FDG)-positron emission tomography (PET), and video EEG monitoring (VEEG). MRI showed T2 signal hyperintensity lesions in the frontal and temporal lobes, with hemosiderin deposits in the temporal area, as well as severe left hippocampal atrophy. 18FDG-PET showed hypometabolism in the left frontal and temporal lobe   , 14 at aNeuropathology revealed a small number of ectopic neurons in the white matter of the temporal lobe, as well as satellite oligogenesis growth, suggestive of FCD type 1a . DespiteIn this patient, her late-onset epilepsy was at first attributed to prior SAH complicated with left frontal and temporal ischemic stroke with hemorrhagic transformation due to severe vasospasm. However, upon development of drug resistance and partial response to VNS, a full work-up for possible resection surgery, including resection of a generator in the mesial temporal structures, was postulated based on MRI signs that indicated hippocampal atrophy. Although hippocampal pathology did not confirm the presence of hippocampal sclerosis, the decision for resection surgery as the next step in this patient's management revealed FCD 1a in the temporal neocortex. This pre-existing and not yet diagnosed epileptogenic lesion could play a role in the development of epilepsy in this patient, as not all patients with a prior vascular insult will present with chronic unprovoked seizures.Many patients, like ours, will present with recurrent seizures that develop following the diagnosis of an insult, such as stroke, head trauma, or tumors –21. The An imbalance between excitatory and inhibitory neurotransmission causing epileptic seizures –24 is a 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 Submission of this case report was approved by the ethics review board at Seirei Hamamatsu General Hospital, and written informed consent was obtained from the patient. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.All authors made substantial contributions to the conception, validation, design, acquisition of data, or analysis and interpretation of data.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.


Despite sound basis to suspect that aggressive and early administration of nutritional support may hold therapeutic benefits during sepsis, recommendations for nutritional support have been somewhat underwhelming. Current guidelines (ESPEN and ASPEN) recognise a lack of clear evidence demonstrating the beneficial effect of nutritional support during sepsis, raising the question: why, given the perceived low efficacy of nutritionals support, are there no high-quality clinical trials on the efficacy of permissive underfeeding in sepsis? Here, we review clinically relevant beneficial effects of permissive underfeeding, motivating the urgent need to investigate the clinical benefits of delaying nutritional support during sepsis. Despite sound basis to suspect that aggressive and early administration of nutritional support may hold therapeutic benefits during sepsis, recommendations for nutritional support have been somewhat underwhelming. The Surviving Sepsis Campaign recommends against early parenteral nutrition, based on studies of low to moderate quality, yet the early initiation of progressive enteral nutrition was encouraged . The latThis observation raises a question: why, given the “paucity of studies”, are there no high-quality clinical trials on the efficacy of permissive underfeeding in sepsis? Indeed, initiating early parenteral nutrition has even been found to solicit detrimental effects in at least some large clinical trials , 5, and Gastrointestinal (GI) complications are common in critical care patients . HoweverThere is also evidence that inflammatory mediators may alter pancreatic exocrine function. In patients with sepsis, exocrine dysfunction seems to mirror disease severity , implicaEarlier studies have shown that both Il-1β and TNF inhibited gastric acid secretion by rabbit parietal cells . More reInflammatory mediators thus do not only supress appetite (i.e. SAA), but play a much more involved role in antagonising digestion. Such a comprehensive inhibition of gastric function may well represent a strategy to avoid investing resources into a system that is unlikely to be utilised during an infection. However, this observation also raises two further questions. Firstly, if inflammation inhibits GI function on numerous levels, what is the effect of enteral nutritional support? Feeding during a severe inflammatory response may represent an underappreciated cause of complications through the forced engagement of physiological processes that are not intended to be operational during an infection. Secondly, we speculate that the suppression of digestive machinery is more than just conservation of energy. Specifically, we hypothesise that the decommissioning of the GI tract is an extension of SAA and forms part of an immunological strategy to augment systemic catabolism in non-immune tissue.Inflammatory mediators are potent inducers of catabolism. Indeed, cytokines have a well-appreciated role in inducing the breakdown of proteins in muscle, promoting bone resorption and also driving lipolysis in adipocytes . In turnMacro-autophagy (hereafter simply autophagy) is an evolutionarily conserved catabolic process that plays an essential role in promoting cell survival . ConceptMisfolded proteins represent a major cellular danger, as these disorganised proteins are prone to form toxic protein aggregates. Proteins may be misfolded, either because of a denaturing environment or because of direct protein damage . Interestingly, studies in mice show that febrile range increases in body temperature resulted in an increased expression of heat shock proteins : this obThough overt cell death is actually rare in sepsis, there is evidence of increased mitochondrial dysfunction , suggestMycobacterium tuberculosis surface protein such as Rv1468c can be targeted for ubiquitination, followed by p62 recruitment, and the subsequent delivery of the Rv1468c-ubiquitin-p62 complex to LC3-decorated autophagosomes for selective degradation [We have previously pointed out that autophagy may play a critical role in the removal of inflammogens, such as LPS, by the lysosomal enzyme, acyloxyacyl hydrolase . Moreoveradation . Importaradation . For exaradation . Howeverradation : since dFinally, autophagy contributes to the processing and presentation of peptides on both MHC I and II. As an example, autophagy can also act as a proteasome-independent alternative pathway for the processing and loading of viral-derived peptides onto MHC I . SpecifiSince fasting is a potent inducer of autophagy, we have previously argued that SAA ensures adequate levels of autophagic activity during an infection . In thisThe preceding discussion also highlights that other aspect of nutritional support besides nutrient content and suppression of catabolic processes may be impacted by nutritional support. BA release following nutritional support may also impact on immune cell function, inhibiting autophagy and vascular tone (e.g. increase splanchnic blood flow) . SimilarThe loss of appetite as part of sickness behaviour, coupled with the comprehensive shutdown of the gastric system, is likely an adaptive response aimed at sustaining elevated levels of catabolism and, specifically, autophagy. Remarkably, it is also worth noting that several pro-inflammatory cytokines  have been shown to regulate autophagy (reviewed ). SimilaFor clinicians, the term “catabolism” usually carries strong negative connotations. Remarkably, however, it is worth noting that across the animal kingdom, organisms in a non-anabolic state (e.g. spores or a dauer and pupa phase) display greater resilience to various stressors. Similarly, mice in a fasted state are far more tolerant of the toxic effects of both radiation and chemotherapy: this observation forms the basis for a number of clinical trials which evaluate the efficacy of fasting prior to receiving chemotherapy. Here, we have outlined a number of key physiological processes invoked by catabolism and provide a rational justification for the evaluation of the efficacy of permissive underfeeding during sepsis in otherwise well-nourished individuals.


Insects have evolved an extraordinary range of nutritional adaptations to exploit other animals, plants, bacteria, fungi and soils as resources in terrestrial and aquatic environments. This special issue provides some new insights into the mechanisms underlying these adaptations. Contributions comprise lab and field studies investigating the chemical, physiological, cognitive and behavioral mechanisms that enable resource exploitation and nutrient intake regulation in insects. The collection of papers highlights the need for more studies on the comparative sensory ecology, underlying nutritional quality assessment, cue perception and decision making to fully understand how insects adjust resource selection and exploitation in response to environmental heterogeneity and variability. Knautia arvensis) plants growing in the same and different plant communities. Russo and colleagues [Carduus nutans when plants were grown at higher temperatures. Increased lipid contents strongly decrease pollen collection, survival and reproductive fitness in bumble bees (Bombus terrestris) [Insects need to acquire specific amounts and ratios of nutrients to develop, survive and raise offspring . Findinglleagues  show tharestris) , and mayrestris)  who docuApis mellifera), dietary restriction shortens lifespan in colony-living workers, because their maturation is accelerated, and they start foraging comparatively earlier when food deprived, as reviewed by Ihle and colleagues [insulin receptor substrate (irs) gene in two honey bee lines. Treated bees were then brought back into their mother colonies, and the onset of foraging and lifespan were recorded. Individuals with suppressed irs showed an earlier foraging onset and did not live as long, suggesting that the IIS pathway links nutritional regulation with behavioral maturation and life span.In eusocial insects, such as ants and several bee species, the challenge of coping with variation in resource chemistry is further amplified by often largely different caste- or symbiont-specific nutritional needs among colony members, which all need to be met simultaneously, as discussed by Crumière and colleagues . Moreovelleagues . Similarlleagues . As insulleagues  postulatTaraxacum (Asteraceae), which are known for their specialized pollinator interactions. The authors show that a Taraxacum diet strongly reduces offspring production and health in a generalist bumble bee (B. terrestris). This effect is unlikely to be caused by pollen mechanical properties , but rather a consequence of lacking essential dietary components or of the presence of toxic compounds that interfere with physiological processes .Insects have evolved sophisticated traits and strategies to locate, memorize, select and exploit valuable nutritional resources, in complex and variable environments. The exploitation and use of pollen in the plant-pollinator mutualism are particularly interesting, because pollen has an intriguing dual role. It carries the male gametes and thus ensures plant fertilization, and it can (in some plant species) serve as a reward to pollinators, as reviewed by Ruedenauer and colleagues . Many plWhile most solitary bees and several primitively eusocial bumble bees provision their offspring with pollen and nectar without  modifying it, the eusocial stingless bees and honey bees convert the collected nectar into honey . For honInsects that cannot alter the nutritional composition of allocated food resources may compensate for inter- and intra-specific variation in resource chemistry by relying on resource diversity. For example, floral diversity is known to ensure a continuous resource supply which allows for a continuously high food intake and an increase in the quantity of stored food resources . Floral B. terrestris). Other nutrients (such as lipids and micro-nutrients) are required for physiological maintenance. Moreover, several studies indicate that species-specific target ratios of micro- and macro-nutrients are critical for the health and fitness of animals in general [It has long been known that insects primarily choose their food based on qualitative nutrient contents. For example, Grund-Mueller and colleagues  show tha general , and ins general ,24. Howe general  and othe general ,26,27).  general  and Crum general  highligh general , see des general  call for general . StudyinB. terrestris) can perceive all those amino acids that have a polarized functional group, in addition to the amino acid-specific amino- and carboxy-group [B. impatiens preferentially foraged on plants with pollen of high protein to fat content. The prioritized perception of a critical dietary component not only increases the chances of survival and thus reproduction, but also reduces the costs associated with cue perception. It may represent a sophisticated strategy of generalist consumers to efficiently exploit diverse resources through rapid quality assessment at low cost. This would allow them to maximally benefit from resource diversity, through selecting the most beneficial resources or resource mixture.The observed variation in resource chemistry requires that insects consuming these resources are able to assess  their chemical/nutritional profiles, in order to make appropriate foraging decisions. Numerous behavioral studies indicate that they are , but thexy-group . Moreovexy-group . Interesxy-group , indicatxy-group . This fixy-group , who shoThe studies published in this Special Issue highlight the need for more detailed and comparative studies on the sensory ecology of insects to investigate the factors (e.g., dietary specialization, the level of sociality  or the a


As the crucial non-cellular component of tissues, the extracellular matrix (ECM) provides both physical support and signaling regulation to cells. Some ECM molecules provide a fibrillar environment around cells, while others provide a sheet-like basement membrane scaffold beneath epithelial cells. In this Review, we focus on recent studies investigating the mechanical, biophysical and signaling cues provided to developing tissues by different types of ECM in a variety of developing organisms. In addition, we discuss how the ECM helps to regulate tissue morphology during embryonic development by governing key elements of cell shape, adhesion, migration and differentiation. Summary: This Review discusses our current understanding of how the extracellular matrix helps guide developing tissues by influencing cell adhesion, migration, shape and differentiation, emphasizing the biophysical cues it provides. The extracellular matrix (ECM) is essential for metazoan life; without it, we would be merely an amorphous mass of cells. The ECM is the non-cellular component of all tissues, forming the physical environment surrounding cells, and playing both structural and signaling roles . As summanoikis)  can provide regulatory information to cells . In addianoikis) .The importance of the ECM in normal mouse and human development is demonstrated by the many examples of embryonic lethality or functional disorders caused by deficiency or mutation, either experimentally or in a multitude of genetic diseases. Human genetic disorders can result from perturbed ECM structure, dynamics, components and/or interactions . In compDrosophila and Tribolium.This Review summarizes key roles of the ECM in cellular processes and tissue morphogenesis during embryonic development. After first introducing principles of cell-ECM interactions, we focus primarily on recently published examples to discuss how the ECM helps to direct developing tissues by influencing cell adhesion, migration, shape and differentiation A-D. We pThe ECM comprises predominantly protein and polysaccharide components , but theCell adhesions are the attachment structures between cells and the ECM, or between cells and other cells. They are essential for the organization of individual cells into three-dimensional tissues. The specific properties of cell-ECM adhesions, such as their distribution, quantity and stability/duration, can vary between organisms, tissues, developmental stages and even neighboring cells . Amidst Embryogenesis requires a coordinated balance between cell-ECM and cell-cell dynamics. Cell-cell adhesions mediate tissue cohesion and organization. Through cell-cell adhesions, the ECM can exert physical effects beyond only the first layer of ECM-attached cells into the interior of the tissue/organ. As with cell-ECM adhesions, a variety of cell-cell adhesive contacts are found in the developing embryo, for example various adherens junctions , desmosoIn the context of this Review, the ECM uses cellular adhesions to regulate or modulate tissue shaping by anchoring, signal/force transmission and cell migration. Owing to the importance of cell migration in embryogenesis, we devote a separate section to this topic below.Tribolium, Drosophila and other non-mammalian species  anchors distal epithelial cells of the Drosophila pupal wing to the surrounding chitinous cuticle in a patterned manner  to generate polarized protrusions that interact functionally with fibronectin, and undergo extensive directed cell migration during axis extension and head formation . Depleti defects . Furtherigration .Drosophila embryonic hemocytes require prolonged, stable cell-ECM interactions to migrate along the ventral nerve cord. Hemocytes achieve stability of lamellipodia and prolonged cell-ECM adhesion states through autocrine deposition of laminin, a major basement membrane structural glycoprotein, in a Rab8-regulated manner  and establish sites of tendon attachment during lateral transverse muscle development. Exploratory and sensory behavior of cellular protrusions, cell-substrate adhesion and cell traction-force generation involve a multitude of signaling mechanisms that contribute to cell migration during embryogenesis  of lateral epidermal cells  . The ECMIn addition to promoting stem cell support within a specific microenvironmental niche, the mechanical properties of the ECM can strongly influence a variety of stem cell behaviors including maintenance, self-renewal, proliferation and differentiation D Gattaz. For exaCompared with adult stem cells, the body of literature describing biophysical contributions of the ECM to pluripotent ESC differentiation for potential clinical application is currently strikingly sparse , with maDrosophila, C. elegans and Xenopus. Considerably less is known about the biophysical regulation of embryos developing in utero, although in ovo studies in avian embryos  and microrheology Laser ablation Force-sensing molecules Local force determination via analyzing droplet deformation Local application of force [e.g. by magnetic beads or optical tweezers ]Over the past decade, classical and innovative research approaches and techniques  have ide embryos  and new  embryos  are provin vitro bioengineered models recapitulating key milestones of mammalian development provide preliminary insights into the mechanisms of ECM mechanical regulation (Adding to the complexity of species differences in development, the ECM is not static during embryogenesis. Developing organs and tissues interact with similarly dynamically changing matrices throughout embryogenesis . To addrgulation . HoweverWhat controls the changing biophysical properties of the ECM at progressive developmental stages? What molecular mechanisms allow the ECM and the cells that synthesize ECM to sense and respond to cues from cells and tissues? Are these mechanisms consistent between different tissue types? Are such mechanisms conserved between different organisms? These and many other unanswered questions, combined with rapidly emerging new techniques to explore these topics, make this an exciting time for the field of ECM developmental biology. The field is likely to move toward increasingly quantitative approaches involving directly quantifying changing ECM composition and physical parameters as development proceeds, combined with mathematical modeling to characterize mechanisms and generate new testable hypotheses. ECM molecules continue to be identified as therapeutic and prognostic targets in disease . Approac


Posterior reversible encephalopathy syndrome (PRES) represents a unique clinical entity with non-specific clinical symptoms and unique neuroradiological findings. This syndrome may present with a broad range of clinical symptoms from headache and visual disturbances to seizure and altered mentation. Typical imaging findings include posterior-circulation predominant vasogenic edema. Although there are many well-documented diseases associated with PRES, the exact pathophysiologic mechanism has yet to be fully elucidated. Generally accepted theories revolve around disruption of the blood-brain barrier secondary to elevated intracranial pressures or endothelial injury. In this article, we will review the clinical, typical, and atypical radiological features of PRES, as well as the most common theories behind the pathophysiology of PRES. Additionally, we will discuss some of the treatment strategies for PRES related to the underlying disease state. Posterior reversible encephalopathy syndrome (PRES), first described by Hinchey et al. in 1996, represents a neurological disorder with varied clinical presentation and typical imaging findings of parieto-occipital predominant pattern of vasogenic edema . There aClinical manifestations are acute to subacute and range from headache and visual disturbances to altered levels of consciousness and seizure in more severe cases . TreatmeThe precise pathophysiologic mechanism(s) behind PRES have yet to be fully elucidated and remain controversial . There aThe second major theory addresses the fact that up to 30% of patients with PRES do not exhibit the elevated arterial pressures necessary to exceed the autoregulatory control of the cerebral vasculature , 19. ThiClinical manifestations depend on the involved region(s) of the brain; thus, the presentation may be broad. For example, primary involvement of the occipital lobes may result in visual disturbances/hallucinations. Focal neurological deficits corresponding to the location of focal lesions occurs in ~5–15% of patients with PRES . Rarely,As its name suggests, PRES typically manifests on imaging studies as posterior-predominant white matter vasogenic edema. The parietal and occipital lobes are almost universally involved and findings are typically symmetrical and bilateral . InvolveThe differential diagnosis for PRES is broad and includes entities with similar confluent T2 white matter hyperintensity. Examples include: ischemia/infarction (particularly posterior circulation), demyelinating diseases, infectious etiologies , progressive multifocal leukoencephalopathy (PML), vasculitis, and various metabolic disorders . A cliniPRES can typically be distinguished from acute ischemia because the latter invariably demonstrates cytotoxic edema and diffusion restriction. Restricted diffusion in acute ischemia can be easily detected on diffusion-weighted imaging (DWI) and apparent diffusion coefficient (ADC) as a hyperintense signal on DWI with corresponding decreased signal on ADC (due to the relatively decreased movement of intracellular water molecules). Vasogenic edema in the setting of PRES, on the other hand, may show hyperintense signal on DWI that is not accompanied by a corresponding decreased signal on ADC . Additio“Advanced” imaging techniques in PRES have recently been described as an adjunct tool in difficult or equivocal cases. These advanced imaging techniques include: CT/MR perfusion, MR Spectroscopy (MRS), Susceptibility weighted imaging (SWI), and nuclear medicine techniques, including single-photon emission tomography (SPECT) and positron emission tomography (PET) with varying radiotracers. Although a full discussion is beyond the scope of this review, a variety of imaging findings can be seen on these advanced techniques to help suggest a diagnosis of PRES. Hyperperfusion may be seen on CT/MR perfusion studies demonstrated by increased cerebral blood flow and blood volume with reduced time to perfusion and mean transit time  althoughAdditionally, PRES can be distinguished from other conditions such as autoimmune encephalitis in the setting of acute disseminated encephalomyelitis (ADEM) by the former's diffuse bilateral but asymmetric vasogenic edema . PML mayAtypical features of PRES include areas of contrast enhancement, hemorrhage, or diffusion restriction . AlthougAdditionally, an early finding of PRES, which may precede the typical parieto-occipital edema includes mild sulcal FLAIR hyperintensity and leptomeningeal enhancement on post-contrast T1 weighted images, as described by Nakagawa et al. . BenziadTreatment of PRES is typically aimed at controlling the primary etiology causing PRES . For exaIn cases of (pre)eclampsia, treatment is aimed at the timely delivery of the fetus as well as blood pressure management and magnesium sulfate for seizure prophylaxis . In the PRES is a unique entity with characteristic clinical and neuroradiological findings, in addition to myriad well-documented causes. Although the precise pathophysiologic mechanism(s) behind PRES has yet to be elucidated (and indeed may be due to a combination of interrelated processes), the generally accepted mechanism is dysfunction of the blood-brain barrier resulting in vasogenic edema with posterior-circulation predominance. Imaging features are best evaluated on fluid-sensitive MR sequences which reveal parieto-occipital predominant white matter T2 hyperintensities, although many atypical imaging features can be seen and should be kept in mind when evaluating challenging cases. Various advanced imaging tools are available to help in difficult or equivocal cases. Treatment is aimed at managing the underlying cause with specific attention to blood pressure monitoring and possible seizure prophylaxis.R-CA and JA: Contributions to the manuscript, figures, and tables. VP: Contributions to the manuscript and figures. NS-B, CL, AR, MS, PK, JG, and AL: Contributions to 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.