Many RNA binding proteins, including FUS, contain moderately repetitive, low complexity, intrinsically disordered domains. These sequence motifs have recently been found to underpin reversible liquid: liquid phase separation and gelation of these proteins, permitting them to reversibly transition from a monodispersed state to liquid droplet- or hydrogel-like states. This function allows the proteins to serve as scaffolds for the formation of reversible membraneless intracellular organelles such as nucleoli, stress granules and neuronal transport granules. Using FUS as an example, this review examines the biophysics of this physiological process, and reports on how mutations and changes in post-translational state alter phase behaviour, and lead to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The discovery of missense mutations in multiple RNA binding proteins, such as fused in sarcoma (FUS) , transacA common neuropathological feature of ALS and FTLD associated with mutations in RNA binding proteins is the deposition of visible aggregates of the corresponding proteins in the nucleus and/or cytoplasm of neurons and glia . These preversibly transition between a dispersed (mixed) state; a phase-separated state as liquid protein droplets suspended within a liquid; or in a gelled state as soft hydrogels somewhat similar to jelly dessert. This review will also describe work that has shown that disease associated mutations and disease associated changes in the post-translational modifications (PTM) in some of these proteins dramatically alter this process, resulting in formation of stable condensed gelled states that are not easily reversible by the cell. The physics of separation of polymers into droplets and gels are well known in materials science, but were not widely thought about in biological science until very recently. As a result, the terminology used in this field is in a state of flux, although efforts are being made to assemble a uniform nomenclature for these processes. Currently, the biophysical condensation process of polymer phase separation from a dispersed state into liquid droplet suspended in a liquid has been termed “liquid: liquid phase separation” or “coacervation”. The phase separation from a dispersed or liquid droplet state into a gelled state has been termed “gelation”. A more general term that is often applied to these alternate biophysical states is “condensate”  turns out to be crucial for the formation of a subset of intracellular organelles which lack limiting membranes. These membranous organelles include nucleoli, stress granules, neuronal transport granules, and postsynaptic densities to name a few. Most intracellular organelles possess a limiting membrane which, because of their very different biophysical properties and ability to bind histological dyes, facilitated their visualisation by early investigators using conventional light microscopy. In contrast, intracellular organelles that lack limiting membranes, and that have biophysical properties very similar to those of the surrounding cytoplasm, were difficult to visualise by conventional light and electron microscopy. As a result, their authenticity was initially questioned. However, recent advances in live cell imaging methods including the use of fluorescent tags and tools such as optical tweezers, have made it clear that membraneless organelles such as nucleoli, stress granules and RNP granules are indeed authentic physiological structures .Generally, but with some exceptions, membraneless organelles have the biophysical characteristics expected of liquid droplets suspended in an immiscible liquid. They are often spherical. They fuse into larger droplets when they contact each other . They diConceptually, the simplest membraneless intracellular organelle would be a “homotypic” droplet composed of a single phase separating polymer. However, in practical terms, most cellular membraneless organelles are more complex. As an example of morphological complexity, stress granules have been described as having a semisolid gel-like cores  or multi2The biophysical properties of membraneless organelles, which allow reversible transition between dispersed, liquid droplet and gel-like states, confer the ability of these organelles to perform a wide range of biological functions such as transport, storage and physical colocalization of components of intracellular signalling or metabolic machinery. For instance, stress granules allow sequestration of cargo  during cellular stress . Neurona3Most soluble intracellular proteins  exist as uniformly mixed, dispersed solutions of either monomeric protein or higher order complexes. These proteins generally fold into a limited number of well-defined, well-ordered three-dimensional shapes (“folds”) that confer the functional properties of these proteins. However, recent work has identified an increasingly large group of soluble proteins that can exist in a soluble dispersed state, but that can also transition into a separate physical state in which the protein exists in liquid droplets of concentrated protein suspended within the liquid solvent (i.e. the cytoplasm/nucleoplasm). Some of these proteins can also condense into jelly-like states, which are referred to as “hydrogels”. This ability to phase separate is also held by other biological polymers (e.g. some RNAs)  and by mA common feature of these phase-separating proteins is the presence of at least one domain composed of repetitive stretches of amino acids with polar side chains , nonpolar side chains (proline), positive side chains , negative side chains  or aromatic side chains . Hydrophobic residues on the other hand, are typically underrepresented. These amino acids are often arranged in imperfect, short repetitive motifs (e.g. enrichment of glutamine glycine serine tyrosine (“QGSY”) repeats in the N-terminus of FUS) A. Becaus4In the last several years the biophysics of phase-separated intracellular structures has been probed using both theoretical and experimental approaches . The phaThe forces (or “preferred interactions”) that drive liquid–liquid phase separation include both long-range forces that might initiate the phase separation, and intermediate-range forces that might subsequently stabilise the assembly once polymer molecules have begun to form a network with each other. The longer-range forces are typically charged interactions (e.g. glutamate – arginine), whereas the intermediate-range forces include directional interactions between positively charged residues and aromatic residues . Other intermediate-range interactions include dipole interactions between glycine, glutamine, asparagine and serine residues, as well as π-π interactions formed by stacking of aromatic rings or between the guanidino moiety of arginines and the rings of aromatic amino acids . Most loCrucially, the interactions driving phase separation are unlike the lock-and-key interactions of well-folded proteins, which rely on precise three-dimensionally specific point-to-point interactions. In phase separating proteins, the interactions build upon the disordered state and the repetitive sequence motifs that are characteristic of the LC domains of these proteins. Residues within these repetitive sequences can be considered as interaction points . These residues/nodes permit the formation of networks of intra- and inter-molecular interactions within and between phase-separating polymers . The strmultiple inter- and intra-polymer interactions that are not highly restricted to specific three-dimensional orientations, this allows phase separation to be tuned simply by manipulating number of available interactions/nodes . Thus, as described below, the number of available interactions/nodes in FUS can be changed by altering the post-translational state of the key residues (e.g. arginine methylation or serine phosphorylation) or by shielding the key residues/nodes by cloaking them with interacting chaperones (e.g. transportin 1 – TNPO1) (Because liquid: liquid phase separation is primarily driven by – TNPO1) .37-SYSGYS-42 and 54-SYSSYGQS-61 in the putative amyloid-core domain of FUS FUS) have recently been termed “low-complexity aromatic-rich kinked segments” or LARKS (Condensation into hydrogels and into stable fibrillar condensates (i.e. “gelation”) likely follows somewhat similar rules, but in a time- and concentration-dependent manner in which intermolecular hydrogen bonding of β-sheet domains becomes increasingly important, especially in fibrillar condensates. However, the specific details of this process are currently the topic of intense scrutiny. Work on fragments of FUS using electron microscopy, solid state nuclear magnetic resonance and x-ray diffraction studies of fibrillar condensates reveals several important differences from classical amyloids including: i) short β-sheet domains (often <5 residues); ii) a paucity of hydrophobic residues; iii) high prevalence of hydrophilic residues; iv) the presence of “kinks” at glycine, proline or aromatic residues which preclude long β-sheets and thus minimize the ability to make stable, steric zippers characteristic of conventional amyloids . These mor LARKS   , or moreIn these more physiological complex coacervation systems, liquid: liquid phase separation is probably driven both by homotypic interactions between a single type of polymer, and by heterotypic interactions (e.g. protein #1: protein #2 or protein: RNA interactions). The existence of such complex systems, allows for: i) the cooperative interaction of the different polymers in forming the phase separation at lower polymer concentrations; and ii) the existence of membraneless organelles with internal structures comprised of shells, cores, or multiple sub-droplets.As discussed below, a simple and intuitive example of cooperative interaction is where the introduction of small quantities of RNA can bind multiple FUS proteins through their RNA binding motifs, thereby bringing the protein molecules together at much higher effective/local concentrations than would occur in the absence of the “nucleating” RNA .The molecular diversity of membraneless organelles also allows for the co-existence of multiple phase states in the same condensate. The content of each sub-phase  is then driven by: i) the relative differences in the component polymers to partition into the different sub-phases based on their relative propensities to form homotypic or heterotypic interactions with each other; and ii) the resulting differences in surface tensions of the component droplets.Clearly these purely biophysical considerations underpin the structure and function of a diverse group of highly dynamic intracellular organelles. However, as described below using FUS as an example, the precarious balancing act between i) mixed/dispersed; ii) reversible liquid droplet; and iii) stable fibrillar states that may be irreversible in biological systems, and thus places them at risk of causing disease states.6yet been found of mutations in clinically relevant phase separating proteins that cause defective formation or defective cargo selection. However, it is now clear that disease-causing mutations and disease associated post-translational modifications of several known RNA binding scaffold proteins  can accelerate conversion of these proteins into stable, β-sheet rich, intermolecular hydrogen bonded assemblies  failure of formation of the granule; ii) abnormal partitioning and binding of clients into the granule; iii) abnormal transport of condensed granules; and/or iv) dysregulated release of clients. No examples have semblies . The res6.1FUS, Ewing sarcoma Breakpoint region 1 (EWS/EWSR1) and TATA box binding protein 15 (TAF15) (FET) family of RNA binding proteins (FUS is a 526 amino acid heterologous nuclear ribonucleoprotein (hnRNP), and a member of the proteins  . The resulting FUS/CHOP fusion protein likely phase separates in the nucleus, and then recruits chromatin remodelling or transcription factors in the same way that the EWS-FLI1 fusion protein does in Ewing sarcoma ; frontotemporal dementia (FTLD-FUS) and essential tremor type 4 . The latA small proportion (approximately 1–4%) of familial ALS (fALS) cases arise from missense or frameshift mutations in FUS (>50 mutations have been described to date). Most of these missense mutations and virtually all of the frameshift mutations cluster in the C-terminal region of FUS between residues 495 and 526, near the atypical nuclear localisation sequence (NLS) domain. A much smaller proportion of ALS associated mutations have been identified in the central and N-terminal region, with two mutations (Ser96Del and Gly156Asp) occurring within the LC domain itself .The clinicopathological characteristics of these fALS-FUS cases have been summarised in many recent reviews and will therefore not be discussed further here beyond noting that the pathognomonic neuropathological features of fALS-FUS are the presence of: profound cytoplasmic FUS aggregates; significant depletion of FUS from the nucleus; and occasional intranuclear FUS aggregates. Crucially, these pathological FUS assemblies typically contain normally methylated  FUS, and the aggregates do not contain other FET proteins or transportin (TNPO1) .Extensive cytoplasmic and some nuclear deposits of FUS are also present in neurons and glia in a significant proportion (approximately 10%) of apparently sporadic FTLD cases . In thesThe mechanism by which the FUS aggregates induce either fALS-FUS or FTLD-FUS has been extensively discussed in recent reviews, with three non-mutually exclusive ideas currently being considered, namely: 1) impairment of the nuclear transcriptional and splicing roles of FUS due to either its presence in the nucleus as pathological aggregates or due to its depletion from the nucleus; 2) loss of the normal cytoplasmic functions of FUS due to its sequestration in pathological assemblies; and 3) toxic gain of function effects arising from the pathological effects of the FUS assemblies such as retention of RNA templates inside pathological aggregates. Recent work using a variety of approaches has begun to allow these possibilities to be examined in finer detail. Thus, recent studies comparing the phenotype of conditional FUS knockout mice with that of mice expressing fALS-FUS mutant FUS suggest that a gain of toxic function is more probable . Similar7At physiological concentration range of FUS and salt, and at physiological temperature, FUS undergoes phase separation and gelation in a FUS protein concentration-dependent and salt concentration-dependent manner . These cThe first biologically important feature is the presence of multiple positively charged residues  and multiple aromatic residues  that could participate in cation-π interactions. This feature creates the basis for scalable, multivalent, location-independent interactions that drive phase separation, as discussed earlier. The current model for the role of these electrostatic cation-π interactions is that they initiate the phase separation process by promoting FUS: FUS interactions that recruit multiple FUS molecules into restricted volumes wherein there is a much higher local FUS concentrations than in the overall solute . This prThe second biologically important feature is that both arginine and lysine residues are post-translationally modifiable by methylation. Arginine can also be enzymatically deiminated to form citrulline . These pThe third notable feature is that the initial phase separation of FUS that is induced by cation-π interactions is unstable . These uThe fourth notable feature of FUS phase separation is that the arginine rich C-terminus  bind molecular chaperones. One such chaperone is transportin 1 , which binds to the atypical proline tyrosine nuclear localisation sequence (PY-NLS) B Dorman. Binding7.1As noted earlier, pathological deposits of FUS are prominent neuropathological features of both familial ALS (fALS FUS) and of sporadic FTLD (FTLD-FUS).7.2The above considerations provide a partial insight into how missense and C-terminal truncating frameshift mutations in FUS might promote assembly of FUS into stable fibrillar assemblies that impede FUS function in the ways described earlier .Only a very small proportion of mutations associated with fALS-FUS occur within the FUS LC domain (FUS Ser96Del and Gly156Asp). While the precise biophysics has not yet been fully worked out, these mutations directly increase the propensity of the LC domain to phase separate, and then form hydrogels . Thus, wMost fALS-FUS mutations, however, occur outside the LC domain . MutatioThe mutations outside the LC domain, but not near the PY-NLS sequence have not been studied in detail. However, they probably also affect FUS phase behaviour either by changing the intrinsic phase separation propensity of FUS and/or by altering the interaction of FUS with nucleating ligands such as RNA and other ribonucleoproteins that bind to the central zinc finger and RRM domains.7.3Glial and neuronal cytoplasmic and nuclear FUS aggregates are also a pathological feature of some cases of FTLD. Intriguingly, most cases, perhaps even all cases of FTLD-FUS are associated with wild-type FUS gene sequences. Moreover, in contrast to FUS inclusions in fALS-FUS, the FUS inclusions in FTLD-FUS coexist with EWS, TAF15 and TNPO1 . Importantly, while arginine residues in fALS-FUS-associated mutant FUS appears to be normally asymmetrically dimethylated (ADMA FUS), the arginine residues in FUS within the FTLD-FUS inclusions are significantly demethylated (HYPO FUS) .The underlying mechanisms leading to demethylation of FUS are not presently clear. However, the hypomethylation of arginines on FUS has two direct effects.First, hypomethylated or unmethylated FUS has dramatically increased binding affinity for the chaperone TNPO1 , which mSecond, FUS with unmethylated or hypomethylated arginines has an increased intrinsic propensity to both phase separate and gelate . This pr8The exciting work reviewed here describes a rapidly emerging area of cell biology related to the function of membraneless organelles such as nucleoli, P bodies, stress granules, neuronal transport granules and other RNP granules. The basic biophysics of how these phase separation and gelation events occur and the effect of disease-causing mutations/post-translational modifications on these events is now becoming clearer.The next phase of work will need to focus on understanding how normal assembly and relaxation/disassembly of condensates is physiologically regulated in response to cellular metabolic state.This future work may also provide some tractable molecular targets for novel treatments for diseases associated with abnormal phase separation of intrinsically disordered proteins, such as FUS, TDP-43 and other RNA binding proteins.


Zea mays L.). In this study, a degradome library constructed from Sugarcane mosaic virus (SCMV)-inoculated maize plants was analyzed to identify the cleavage sites in viral and host transcripts mainly produced by vsiRNAs. The results showed that 42 maize transcripts were possibly cleaved by vsiRNAs, among which several were involved in chloroplast functions and in biotic and abiotic stresses. In addition, more than 3000 cleavage sites possibly produced by vsiRNAs were identified in positive-strand RNAs of SCMV, while there were only four cleavage sites in the negative-strand RNAs. To determine the roles of vsiRNAs in targeting viral RNAs, six vsiRNAs were expressed in maize protoplast based on artificial microRNAs (amiRNAs), of which four could efficiently inhibit the accumulations of SCMV RNAs. These results provide new insights into the genetic manipulation of maize with resistance against virus infection by using amiRNA as a more predictable and useful approach.RNA silencing is a conserved surveillance mechanism against invading viruses in plants, which involves the production of virus-derived small interfering RNAs (vsiRNAs) that play essential roles in the silencing of viral RNAs and/or specific host transcripts. However, how vsiRNAs function to target viral and/or host transcripts is poorly studied, especially in maize ( In plants, RNA silencing is a conserved mechanism against virus infections, which involves the production of virus-derived small interfering RNAs (vsiRNAs) ,2. TheseTo understand the potential regulatory and biological functions, it is essential to find the target sites for si/miRNAs . In planArtificial miRNA (amiRNA) is an artificial small RNA (sRNA) engineered to silence a specific transcript, which is produced by the expression of a native miRNA precursor with customized miRNA/miRNA* sequence . Like miSugarcane mosaic virus (SCMV), belonging to the genus Potyvirus in the family Potyviridae, can infect various crops, such as maize (Zea mays L.), sugarcane (Saccharum spp) and sorghum (Sorghum vulgare), leads to typical symptoms including mosaic, chlorosis and dwarfing, and causes considerable losses in different field crops worldwide [orldwide . SCMV isorldwide ,25. Our orldwide ,27. HoweSCMV Beijing isolate (GenBank accession number AY042184) was from previously published sources . Maize iAt least 15 maize seedlings inoculated with SCMV at 8 dpi were pooled for RNA extraction using TRIzol reagent . Degradome sequencing and analysis was performed as described previously . CleaveLXhoI/BamHI  and inserted into the same digested pGD vector [Arabidopsis miR319a precursor or rice miR528 precursor as the template was used to assemble the precursors for individual amiRNAs by a two-step overlapping PCR method as described previously [SalI/BamHI  and inserted into the same digested pGD vector. For the plasmids expressing polycistronic amiRNAs, the second and the third pre-amiRNAs were obtained by digesting the first pre-amiRNAs by XbaI/BamHI  and sequentially inserted into SpeI/BamHI  digested sites downstream the first pre-amiRNA within the pGD vector.All plasmids used in this work were listed in the D vector  for traneviously . PCR proTotal RNA extracted as described above was used for detection of vsiRNAs by Northern blotting and reverse transcription after DNase I  treatment . The pro5 maize protoplasts. The transfected protoplasts were harvested at 16 h post transfection and used for further analysis. Three independent experiments were conducted and at least 10 Eppendorf tubes of protoplasts were used for each treatment within each experiment, which were pooled for RNA isolation followed by qRT-PCR and Northern blotting analysis. For pGD-GFP/mGFP, another 10 tubes of protoplasts from the same treatment were pooled and used for protein extraction and Western blot assay as described previously [Maize inbred line Zheng58 was used for protoplasts isolation and transfection as described previously . Approxieviously .The fluorescence signals of GFP and mGFP were visualized under a Nikon Eclipse TE 2000-E laser-scanning confocal microscope equipped with Nikon EZ-C1 FreeViewer 3.00  as described previously . The fluPreviously, we used a bioinformatics tool to integrate miRNAs from a small RNA library with a genome-wide map of uncapped transcripts (degradome) from the same SCMV-infected maize plants. Indeed, a number of maize transcripts targeted by conserved and novel maize miRNAs were identified . In thisGRMZM2G009443_T01 that encodes a putative chloroplastic chaperone protein ClpC1 [GRMZM2G032628_T01, GRMZM5G806622_T01, GRMZM2G066234_T01, GRMZM2G130121_T01 and GRMZM2G132847_T01, also encode putative chloroplastic proteins  with low reads  from the antisense strand. The uncapped sequences were almost continuously but heterogeneously distributed along the sense strand of the SCMV genome, and mainly concentrated in the 3′-proximal region. To identify the putative vsiRNA-mediated cleavage sites, we used the CleaveLand pipeline to search for vsiRNAs (both of 21 and 22 nucleotides) correlated with the datasets of SCMV degradomes. Almost all of cleavage sites could be explained by vsiRNAs, while the reads between cleavage sites and vsiRNAs were not consistent; despite the presence of many vsiRNA hotspots, only a few were associated with cleavage sites.In this study, the precursor of ath-miR319a was used as the backbone of amiRNAs to express miR-GFP and miR168 in maize protoplasts as previously reported [GFP carried a synonymously mutated version of the relevant miR-GFP target sites  of which might be involved in chloroplast development and function [GRMZM2G032628_T01  were inefficient in guiding the formed RISC and only a distinct number rather than a broad variety of cleavage products were obtained, revealing that only some specific vsiRNAs might be highly effective [It has been revealed that vsiRNAs produced in the host cell can be incorporated into AGO-containing RISC to silence viral genome . Howeverffective . In anotffective . In our ffective . It was ffective .It has been reported that amiRNAs highly accessible to targets confer efficient virus resistance in several plant species ,19,20. TRNA silencing as a plant immune system against viruses is triggered by the production of vsiRNAs that efficiently inhibit viral accumulation . Howeverin vivo. Further work will be needed to confirm the roles of vsiRNAs in targeting viral and host transcripts.Virus infection triggers the production of vsiRNAs that can silence viral RNAs and specific host transcripts. However, which and how vsiRNAs function is poorly studied. Our previous studies have preliminarily characterized the vsiRNAs derived from SCMV and identified the target genes of maize miRNAs through degradome analysis. In this study, a degradome library was constructed and analyzed to identify the cleavage sites in viral and host transcripts mainly produced by vsiRNAs. We identified 42 maize transcripts that were possibly targeted by vsiRNAs, amang which several were involved in chloroplast functions and in biotic and abiotic stresses. Moreover, we obtained more than 3000 possible cleavage sites in positive-strand RNAs of SCMV, while there were only four cleavage sites in negative-strand RNAs. amiRNAs were designed and expressed in maize protoplasts to explore the roles of vsiRNAs in targeting SCMV RNAs. Four of the six vsiRNAs expressed by amiRNA technology could efficiently inhibit the accumulation of SCMV RNAs, suggesting that the transient expression system could effectively assess amiRNA activity of silencing viral RNAs


The plasma level of TMAO is determined by the genetic variation, diet and composition of gut microbiota. Multiple studies have demonstrated an association between TMAO plasma levels and the risk of atherothrombotic cardiovascular disease (CVD). We aimed to review the molecular pathways by which TMAO production and FMO3 exert their proatherogenic effects. TMAO may promote foam cell formation by upregulating macrophage scavenger receptors, deregulating enterohepatic cholesterol and bile acid metabolism and impairing macrophage reverse cholesterol transport (RCT). Furthermore, FMO3 may promote dyslipidemia by regulating multiple genes involved in hepatic lipogenesis and gluconeogenesis. FMO3 also impairs multiple aspects of cholesterol homeostasis, including transintestinal cholesterol export and macrophage-specific RCT. At least part of these FMO3-mediated effects on lipid metabolism and atherogenesis seem to be independent of the TMA/TMAO formation. Overall, these findings have the potential to open a new era for the therapeutic manipulation of the gut microbiota to improve CVD risk.Recent evidence, including massive gene-expression analysis and a wide-variety of other multi-omics approaches, demonstrates an interplay between gut microbiota and the regulation of plasma lipids. Gut microbial metabolism of choline and  Cardiovascular disease (CVD) accounts for approximately 17 million deaths worldwide each year and remains the main cause of mortality in the United States . EnvironN-oxide (TMAO) from trimethylamine (TMA) by host’s flavin monooxygenase 3 (FMO3) [Several studies have identified novel microbial and mammalian cometabolic pathways where the microbiota could promote CVD pathogenesis via the formation of trimethylamine-3 (FMO3) ,15. Thes3 (FMO3) ,15. HoweMethods to measure TMA and TMAO in plasma and urine include liquid chromatography-mass spectrometry, proton nuclear magnetic resonance spectrometry, headspace gas chromatography, electrospray ionization tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . ConcernIn the present review, we aimed to describe and update the potential mechanisms by which TMAO production and FMO3 alter enterohepatic cholesterol and bile acid metabolism and, in consequence, impair a major HDL-mediated cardioprotective function.l-carnitine [l-carnitine to TMAO [TMAO was first described as an osmolyte of marine organism tissue . TMAO isarnitine . TMA is  to TMAO . TMAO isAmong the five members of the FMO family, only FMO1 and FMO3 have the ability to oxidize TMA to TMAO—where FMO3 is the main isoform expressed in the human liver . For FMOIn addition to its expression in the liver, FMO3 is also expressed in the lungs, adrenals and aorta. Interestingly, a sexually dimorphic expression pattern has been observed both in mice and humans, with females showing higher expression than males ,25. ThisFasting plasma concentrations of TMAO exhibit a wide inter- and intra-individual variation . The levAnother important cause of plasma TMAO variability is diet. Food containing TMAO or its precursors increases blood and urine TMAO levels. Red meat, eggs and dairy products are all rich in TMA precursors, and therefore, a potential source of TMAO . In addiConcerning the gut microbiota, several families of bacteria from the Firmicutes and Proteobacteria phyla isolated from commensal bacteria in the human intestine have been identified as choline and carnitine consumers and, therefore, potential TMA producers ,37. PathFMO3 gene. In addition to mutations causing trimethylaminuria, single nucleotide polymorphisms such as E158K and E308G have been reported to reduce FMO3 activity [A great effort to find a relationship between gene expression and plasma TMAO levels was exerted over the last years. Various studies have focused on TMAO and CVD to determine the genetics that possibly underlie their relationship. Functional differences in FMO3 activity can occur in humans, secondary to variations within the activity . Howeveractivity ,42,43.FMO3, genome-wide association studies (GWAS) in male mice have allowed the identification of other genes related to TMAO metabolism. For instance, the Slc30a7 gene has been found to be related to TMAO metabolism and is associated with a transporter of dietary zinc absorption (ZNT7) located on chromosome 3 [Apart from mosome 3 . Nonethemosome 3 .A role for genetic methylation in TMAO homeostasis has recently been proposed by a study that reported an inversed association between plasma TMAO and the methylation capacity in humans . HoweverTMAO came recently under the spotlight due to its reported association with human CVD. In 2011, metabolomic studies identified three candidate molecules that were significantly correlated—after adjusting for traditional cardiac risk factors and medication usage—with CVD, which included the fasting TMAO, choline and phosphatidylcholine plasma levels . SeveralTo better understand TMAO’s role in CVD, large population studies were performed with subjects undergoing elective diagnostic coronary angiography ,51. A diStudies that focus on specific pathologies  have also demonstrated a higher concentration of plasma TMAO compared to their respective controls. Furthermore, this higher concentration was associated with poor prognosis for heart failure patients and, consistently, plasma TMAO levels correlated positively with other cardiac biomarkers such as B-type natriuretic peptide .An increase in the all-cause mortality and an improvement in risk estimation were also observed in patients with peripheral artery disease in relation to their TMAO plasma levels . IncreasIt should be noted that not all studies have demonstrated an association between TMAO and vascular diseases. For instance, a recent study reported a reduction in TMAO plasma levels and dysbiosis of the gut microbiota in patients that had a stroke or transient ischaemic attack . The autThe underlying mechanisms whereby TMAO contributes to CVD are not fully understood. The first hypothesis that explained the atherosclerotic role of TMAO stated a decrease in HDL levels. This was based on the inverse association between TMAO and HDL, which had been found in mice concomitant to a reduction in HDL-mediated reverse cholesterol transport  A-I—the main HDL protein—into circulation . The intClostridiales colonisation at the expense of Roseburia [Studies of gut microbiota composition and obesity have shown a direct association between Firmicutes phyla and obesity in both mice  and humaoseburia . A metagoseburia .Collinsella than the control group, which showed enrichment in Bacteroides, Eubacterium and Roseburia—these three are all genera involved in anti-inflammatory and antioxidant processes [The role of gut microbiota in lipid metabolism and in the pathogenesis of CVD has been widely investigated. A study performed on symptomatic atherosclerotic patients revealed a greater abundance of rocesses . Richnesrocesses . A comparocesses .Beyond the effects of intestinal microbiota on triglycerides and HDL cholesterol levels, the suppression of the intestinal microbiota was correlated with enhanced macrophage-to-feces RCT . This chFirmicutes (relative to Bacteroidetes) were detected among healthy young men who exhibited a greater postprandial increase in circulating TMAO after consuming eggs and beef [Since the bile acid pool size and its composition seem to regulate the gut microbial community structure , the comand beef .Several research groups have attempted to provide a mechanistic basis for TMAO-mediated atherosclerosis, where the variation in the gut microbiota could be a direct contributor to the pathogenesis and progression of the disease. Transferring choline diet-induced TMAO production via fecal transplantation in apoE-deficient (−/−) mice resulted in an increased atherosclerosis risk . In the cyp7a1 and cyp27a1 -treated mice, but neither cholesterol balance nor gene expression in the liver . This inKnocking down liver FMO3 in LDL receptor-deficient mice reduced the hepatic and plasma lipid levels, bile acid pool size, liver triglyceride secretion, ketone bodies and glucose and insulin levels, and prevented atherosclerosis . Global As stated above, elevated systemic levels of TMAO have been associated with type 2 diabetes . Moreoveabca1 gene in J774.A1 murine macrophages treated with TMAO for 24 h [As stated above, several studies have demonstrated that TMAO can promote macrophage cholesterol accumulation in a microbiota-dependent manner by increasing cell surface expression of two scavenger receptors . Howeverfor 24 h  but thisfor 24 h .l-carnitine-enriched diet showed an impaired macrophage-to-feces RCT in vivo [Beyond the potential effect of TMAO on macrophage cholesterol efflux, mice fed with a choline- or  in vivo . This pr in vivo . Consist in vivo . As discA liver microarray analysis was conducted in two independent mouse models with an enhanced TICE to identify potential regulators of macrophage RCT. Less than 100 differentially expressed genes were detected within each array data set . From thl-carnitine or TMAO in animals with an intact gut microbiota enhances the macrophage scavenger receptors and promotes foam cell formation. However, the diets enriched in TMAO precursors have not always been found to be proatherogenic in experimental studies. This dietary administration impairs the macrophage-to-feces RCT pathway. Furthermore, TMAO impairs the FXR-target genes cyp7a1 and cyp27a1, which affect liver cholesterol, bile acid production and biliary excretion. FMO3 may also promote atherosclerosis by enhancing hepatic lipogenesis and gluconeogenesis as well as by impairing the TICE. At least a part of the FMO3-mediated effects on lipid metabolism seems to be independent of TMA/TMAO formation, suggesting multiple distinct mechanistic links between the TMAO-producing diet, FMO3 and atherogenesis (see There is significant evidence supporting the hypothesis that gut microbiota-derived production of TMAO increases the risk of atherosclerotic CVD. The concentration of circulating TMAO is determined by several main factors, including the dietary habits, gut microbiota, FMO3 activity and kidney function. Several important published works in TMAO research have used omics-techniques, mainly focusing on its involvement in CVD (see


Claudins are a multigene transmembrane protein family comprising at least 27 members. In gastrointestinal tract, claudins are mainly located in the intestinal epithelia; many types of claudins form a network of strands in tight junction plaques within the intercellular space of neighboring epithelial cells and build paracellular selective channels, while others act as signaling proteins and mediates cell behaviors. Claudin dysfunction may contribute to epithelial permeation disorder and multiple intestinal diseases. Over recent years, the importance of claudins in the pathogenesis of inflammatory bowel diseases (IBD) has gained focus and is being investigated. This review analyzes the expression pattern and regulatory mechanism of claudins based on existing evidence and elucidates the fact that claudin dysregulation correlates with increased intestinal permeability, sustained activation of inflammation, epithelial-to-mesenchymal transition (EMT), and tumor progression in IBD as well as consequent colitis-associated colorectal cancer (CAC), possibly shedding new light on further etiologic research and clinical treatments. Claudins, a multigene transmembrane protein family comprising at least 27 members , reporteIBD are a series of chronic gastrointestinal inflammatory disorders, among which CD and UC are the most prevalent and well-studied . The sopNOD2 mutations engender anti-biotic α-defensin reduction and altered composition of TJ proteins , resulting in higher susceptibility to luminal bacterial infection in CD patients (SHANK3 mutations downregulate ZO-1 by inhibiting protein kinase Cε (PKCε), thus engendering bacterial paracellular influx and increasing propensity to inflammation in CD patients  and IL-10 to induce immunotolerance  and upregulating TJ proteins  in colitis mice models -induced myosin light chain kinase (MLCK) activation  turns IL-1β precursor into the active form  and generates EpIC, a co-transcription factor that collaborates with β-catenin in cancer initiating cells, contributing to EMT and cancer metastasis /prostaglandin-E pathway , where does the dysregulation exactly occur among various types of gastrointestinal cells and the relevant modulating mechanisms remain obscure. Though claudins dysregulation predominantly occurs in epithelial cells, whether it influences other cell types  awaits further analysis. In addition, the sophisticated modulating mechanism of claudins makes it difficult to determine which signaling pathway is primary in IBD and CAC, especially for different cancer subtypes. Therefore, conclusions from different signaling studies may be contradictory, which has become an obstacle for claudins to be therapeutic targets for IBD and CAC. The potential of claudins in promoting mucosal healing by restoring TJ structures as TJ-forming proteins and regulating inflammation as well as tumor behaviors as signaling proteins awaits further analysis.LZ and JH searched for and analyzed referenced articles. LZ and JH wrote the manuscript. LZ, JH, LL, YW, and YL summarized relevant information and designed the tables and figures in the review. SZ designed the study and revised the review. All authors agreed to be responsible for the content of the review.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.


Over 257 million people live with chronic hepatitis B virus (HBV) infection and there is no known cure. The effective preventative vaccine has no impact on existing infection. Despite the existence of drugs which efficiently suppress viral replication, treatment is usually life-long and finite therapies that cure HBV infection are urgently required. However, even if such therapies were available today, it is unlikely they would reach all of those who need it most, due to chronic hepatitis B (CHB) being largely undiagnosed across the globe and to the dire need for health systems promoting access to therapy. Considerable challenges to developing and implementing an effective HBV cure remain. Nonetheless, important advances towards a cure are being made, both in the development of a multitude of new therapeutic agents currently undergoing clinical trials, and through the establishment of a new global initiative dedicated to an HBV cure, ICE-HBV, that is working together with existing organisations to fast-track an HBV cure available to all. Today, over 1.3 billion people show evidence of past hepatitis B virus (HBV) infection, with 257 million people having progressed to chronic hepatitis B (CHB), which directly contributes to the deaths of over 880,000 people annually . Also, tThe inability of current therapies to induce a functional cure means that patients are invariably committed to life-long therapy. With the presence of a highly effective prophylactic vaccine, if finite curative regimens were to be developed and effectively distributed to all persons living with CHB, CHB could rapidly be eliminated as a global public health challenge. This review discusses some of the obstacles associated with meeting this admirable goal as well as describing a new collaborative approach to fast-track an HBV cure, recently instigated by the international HBV research community.https://ice-hbv.org/) was formed in 2016 following a call to promote global collaboration in HBV cure research [The International Coalition to Eliminate Hepatitis B (ICE-HBV) (research  and otheresearch . These sHBV virions enter hepatocytes via the sodium-taurocholate co-transporting polypeptide (NTCP) receptor , where HDifficulties in targeting the HBV cccDNA reservoir means that much of the current emphasis from pharmaceutical companies and academia in identifying new targets and developing CHB curative regimens lies in developing agents which target other steps of the HBV replication cycle. These include siRNA molecules which target HBV mRNAs ,23, capsThere are also numerous immunological barriers to hepatitis B cure. These include weak innate immune responses ,38, T-ceIt is likely that an eventual HBV curative regimen will require the combination of two or more of these approaches, either DAAs alone or in combination with approaches that stimulate innate and/or adaptive immune responses. For an up-to-date description of antivirals at different stages of clinical development, the reader is encouraged to regularly consult the Hepatitis B Foundation’s drug watch website . A summaIn addition, Bristol Myers Squib recently concluded a Phase I/II trial on the checkpoint inhibitor Nivolumab in advanced hepatocellular carcinoma . A numbeChronic hepatitis B is not a “one size fits all” disease, with striking differences in HBV natural history, disease progression and even treatment response across the globe ,46,47,48We endorse the “Find the Missing Millions” campaign recently initiated by The World Hepatitis Alliance (WHA)  to identThere has never been more interest amongst the HBV research community, public health organisations, pharmaceutical companies and the HBV-affected community in the development of an effective HBV cure. Collaborative approaches such as those fostered by ICE-HBV in partnership with the International HBV Meeting , WHO 6161, WHA , The Coa


Many conditions affecting the heart, brain, and even the eyes have their origins in blood vessel pathology, underscoring the role of vascular regulation. In age-related macular degeneration (AMD), there is excessive growth of abnormal blood vessels in the eye , eventually leading to vision loss due to detachment of retinal pigmented epithelium. As the advanced stage of this disease involves loss of retinal pigmented epithelium, much less attention has been given to early vascular events such as endothelial dysfunction. Although current gold standard therapy using inhibitors of vascular endothelial growth factor (VEGF) have achieved initial successes, some drawbacks include the lack of long-term restoration of visual acuity, as well as a subset of the patients being refractory to existing treatment, alluding us and others to hypothesize upon VEGF-independent mechanisms. Against this backdrop, we present here a nonexhaustive review on the vascular underpinnings of AMD, implications with genetic and systemic factors, experimental models for studying choroidal neovascularization, and interestingly, on both endothelial-centric pathways and noncell autonomous mechanisms. We hope to shed light on future research directions in improving vascular function in ocular disorders. Endothelial dysfunction underlies the crux of many conditions, which may implicate comorbidities. One example is choroidal neovascularization, a process in wet or exudative age-related macular degeneration (AMD), characterized by the abnormal intravasation of choroidal vasculature into the retinal epithelium or subretinal tissue. This often involves dysfunctional and leaky vessels, which then lead to the accumulation of fluid and blood in the macula . AMD is Endothelial dysfunction plays a role in many human diseases. Patients with early vascular abnormalities have been found to acquire AMD and associated cardiovascular and cerebrovascular diseases later in their lives . ResearcWe recognize that emerging studies are discovering a significant involvement of endothelial pathology in choroidal neovascularization. Here, we provide a nonexhaustive review to address the vascular underpinnings of AMD, provide information on state-of-the-art experimental models of choroidal neovascularization, and interpret existing knowledge on endothelial mechanisms with heterotypic interplay of different cell types and environmental factors.While the pathogenesis of wet AMD is still poorly understood, several reports suggest a vascular etiology for the disease. The choroidal endothelial cells that form choriocapillaris vessel walls are lost even before the occurrence of retinal pigmented epithelium dysfunction, suggesting that vascular dysfunction could be the first trigger of wet AMD . HistoloIn addition to vascular degeneration in the early stages of AMD, vascular dysfunction is manifested in late stage neovascular outcomes, such as polypoidal choroidal vasculopathy (PCV). PCV is a subtype of wet AMD that is most prevalent in Asians . It is cARMS2 A69S (rs10490924) and CFH (rs1329428) (1329428) , and cho1329428)   .ARMS2-HTRA1 rs10490924, CFH rs10737680, CETP rs3764261, ADAMTS9 rs6795735, C2-CFB rs429608, and CFI rs4698775 were the most significantly associated with wet AMD. In European and Asian populations, the most common SNPs seem to converge on the gene HTRA1 and complement pathway-related genes. Advances in GWAS have generated more targets than biological interpretation can translate them into new therapeutics. Emerging functional studies are primarily focused on how those SNPs impact on retinal pigmented epithelium. We will further discuss how HTRA1 and complement factors could lead to aberrant vascular outcomes in a later section on Vascular Mechanisms in Choroidal Neovascularization.In the past few decades, GWAS on AMD cohorts have revealed several disease-associated risk variants. The Genetics of AMD in Asians Consortium conducted a genome-wide and exome-wide association study to uncover the most common single nucleotide polymorphisms (SNPs) associated with wet AMD specifically in the East Asian population . ConsistHTRA1 seems to be associated with elevated levels of HTRA1 in the retinal pigmented epithelium. It has been postulated that HTRA1 upregulation could lead to Bruch’s membrane matrix breakdown, hence promoting choroidal vessel invasion , the choroid-restricted marker carbonic anhydrase IV, AMD-related proteins (CFH), and display functional endothelial characteristics  is a multi-functional serine protease expressed in endothelium, epidermis, and neurons that regulates vascular growth and maintenance and is required for the normal development of vasculature in the brain and eye . In 2006D donors . In lineD donors . ElevateD donors . The oveD donors . FurtherD donors . SeveralD donors . In vivorane ECM . It was rane ECM . Secondlrane ECM . Mathurarane ECM . TherefoAntioxidants have been found to slow progression of progression from early AMD to advanced stages of AMD (wet AMD or severe geographic atrophy), thus highlighting the role of oxidative stress in AMD progression . Oxidatiin vivo increased levels of C3a and C5a, and C3a and C5a induced increase in VEGF secretion by primary human RPE in vitro  is a DNA aptamer against PDGF that was recently assessed for its efficacy in combination therapy with the anti-VEGF treatment ranibizumab (Lucentis). The results from a phase 2b clinical trial showed that there was a 62% benefit from baseline with combination therapy compared with anti-VEGF monotherapy . HoweverIn vivo experiments have recently demonstrated ANG2 and VEGF combinatory inhibition led to reduced neovascular lesion formation in a spontaneous chronic choroidal neovascularization mouse model (JR5558 mice) (Angiopoietin-2 (ANG2) is a proangiogenic cytokine that plays a role in both angiogenesis and immune activation, both of which are integral processes in the pathogenesis of wet AMD . ANG2 le58 mice) . This ha58 mice) . The shiEndothelial cells produce a physiological balance of vasoactive substances to regulate vascular function, such as the vasoconstrictor endothelin-1 (ET-1) and the vasodilator nitric oxide (NO). ET-1 levels increase while NO availability decreases during aging, resulting in increased vasoconstriction and impaired vasodilation, which could lead to constriction of smaller vessels associated with ischemia of the choriocapillaris  seen in severe dry AMD . DecreasWith the great momentum in the study of choroidal neovascularization, there remains knowledge gaps which the scientific and clinical communities could address. We propose that further research on the following areas could be illuminating. (1) AMD subtypes could have different etiology, rendering it important to investigate subtype-specific mechanisms. Genomics distinguishing typical AMD and polypoidal choroidal vasculopathy may elucidate subtype-specific mechanisms. (2) Perturbations to choroidal vasculatures may have to be looked in the context of other influences. Existing experimental models could be adapted to recapitulate potential systemic/immune factors, as well as to study endothelial interplay with other cell types. (3) Finally, there is a need to explore both VEGF- and non-VEGF pathways to enhance the success of combinatorial treatment. We hope that vascular-targeting strategy will help advance therapy for early intervention.CC provided the strategic focus for this review paper and edited the manuscript. NY and EC wrote and prepared the manuscript.We are grateful for the funding support of an Academic Research Fund (AcRF) Tier 2 grant (MOE2018-T2-1-042) from the Ministry of Education, Singapore, as well as support from the SERI-IMCB Program in Retinal Angiogenic Diseases (SIPRAD) grant number SPF2014/002, a grant from A-STAR .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.


Mycobacterium tuberculosis. It may be difficult to distinguish IRIS from tuberculosis and other opportunistic infections radiologically; therefore, radiological findings must be interpreted with clinical and laboratory findings. In this review article, we describe the clinical and radiological manifestations of IRIS in children and provide illustrative radiological examples.Immune Reconstitution Inflammatory Syndrome (IRIS) refers to a collection of inflammatory disorders, predominantly related to infectious processes that manifest after the initiation of antiretroviral therapy (ART) and can be classified as unmasking or paradoxical. The prevalence of IRIS in children in sub-Saharan Africa is low. Approximately half of all cases are associated with  Compared to untreated children, life expectancy and quality of life are substantially better for children receiving ART.6IRIS refers to a collection of inflammatory disorders, predominantly related to infectious processes that manifest after initiation of ART. It is usually a self-limiting condition, but on occasion may be severe and rarely can be life-threatening. In most cases, ART can be continued and appropriate antimicrobial therapy is provided for the relevant opportunistic infection.8 but as the name implies, IRIS occurs when ART-associated improved immune function results in an excessive immune response.9 Interestingly, paradoxical worsening of symptoms following initiation of treatment has been described in HIV-uninfected patients and follows a similar improvement in immune function. Paradoxical tuberculosis (TB) reactions are the best known examples of this.11The immunological changes that occur following initiation of ART are complex,13 A recent South African study using the International Network for the Study of HIV-associated IRIS criteria reported a paradoxical TB-IRIS incidence of 4.8% among 104 children under 8 years of age.14 The incidence of IRIS varies according to the associated micro-organism or underlying condition, the definition of IRIS used, including whether paradoxical or unmasking forms of IRIS or both are included, the region of the world and local incidence of particular opportunistic infections and age of the children. Epidemiological studies on the overall incidence of IRIS and on specific forms of IRIS in children are limited in number and as a result of the factors mentioned above.Early, retrospective studies estimated IRIS incidence as high as 30%; however, more recent evidence suggests a much lower rate of < 10%.15IRIS is typically classified as either unmasking or paradoxical; however, this distinction may be difficult, as both forms may coexist in the same child and other explanations for the findings should be considered.17Unmasking IRIS refers to clinical features related to a previously undiagnosed, asymptomatic or subclinical infection, whereas paradoxical IRIS is worsening of a previously diagnosed or partially treated condition. Non-infectious forms of IRIS include autoimmune processes and malignancies.18Precise diagnostic criteria for IRIS have been suggested, but consensus on such criteria has not been reached. However, IRIS should be strongly considered when:ART has been recently initiated, usually within the last 2–6 weeks, but IRIS has been reported many months after initiation.There has been a substantial decline in HIV viral load, usually with a significant rise in CD4 count – however, IRIS may occur in the absence of either or both of these.Local or systemic inflammatory changes occur related to a previously diagnosed  or unrecognised (unmasking IRIS) infectious condition – however, IRIS has been reported for non-infectious conditions, including malignancies and autoimmune diseases.An alternative explanation for these changes is not identified. This can be very challenging as progression of HIV, new opportunistic infections, drug-resistant pathogens, and medication side effects may present similarly.13 Secondly, the virological and immunological responses to ART; the more rapid the decline in HIV viral load and the faster the rise in CD4 count, the greater the likelihood of developing IRIS.18 This likely explains the declining incidence in paediatric IRIS as children are being diagnosed earlier and are started on ART soon thereafter, usually before progression to profound immune suppression.19The risk for and severity of IRIS are largely informed by two factors. Firstly, the degree of CD4+ T-cell immune suppression; the lower the pre-treatment CD4 count, the higher the risk of IRIS.Clinical manifestations of IRIS are dependent on the implicated pathogen and frequently involve worsening of previously identified manifestations or the onset of new symptoms, similar to those encountered with non-IRIS–related infections with the same pathogen.Mycobacterium tuberculosis (TB) is implicated in approximately half of adults with IRIS.21 TB is less commonly implicated in paediatric IRIS, possibly related to the paucibacillary nature of paediatric TB.14 Other pathogens commonly implicated are Bacille Calmette–Guérin (BCG) vaccine,23 cytomegalovirus (CMV)24 and non-tuberculous mycobacteria (NTM).25 Less common causes of paediatric IRIS include herpes simplex, Kaposi sarcoma (caused by human herpesvirus 8),27Cryptococcus neoformans,28Pneumocystis jirovecii29 and parvovirus B19.30 This differs from adults, where cryptococcal IRIS28 is more likely and BCG-IRIS essentially does not occur. Systemic manifestations include fever, weight loss, fatigue and pain.20 Common clinical manifestations of IRIS in children are tabulated in 31Radiologically it is important to distinguish IRIS from TB or other opportunistic infections, including NTM and CMV. Drug-resistant TB is a clinical and laboratory diagnosis, not a radiological diagnosis and both drug-sensitive and drug-resistant TB can manifest IRIS reactions.31The most common radiological manifestations of TB-IRIS occur within the chest and include new or worsening hilar or mediastinal lymphadenopathy, which may cause tracheo-bronchial compression. Other common chest radiological findings include worsening or new air space consolidation, pleural effusions, reticular infiltrates or nodular infiltrates –3.14,16,16 Central nervous system (CNS) manifestations are uncommon and include TB meningitis, which may cause obstructive hydrocephalus, or associated tuberculomas.16Abdominal manifestations of TB-IRIS include ascites, abdominal lymphadenopathy, and splenic micro-abscesses.16 and in some settings is the most commonly identified cause.22 It usually manifests with inflammatory changes at the injection site and/or within the ipsilateral draining lymph nodes. Chest radiographic changes are uncommon and may be indistinguishable from TB.22 Unusual manifestations of BCG-IRIS have been reported (BCG-associated IRIS is well recognisedreported .Mycobacterium avium complex (MAC)–associated IRIS manifests radiologically as necrotic lymph nodes identified on ultrasound, computed tomography or magnetic resonance imaging. These may be indistinguishable from other mycobacterial infections. The diagnosis of MAC may be made from tissue culture;16 however, cultures are commonly negative. Therefore, a high level of clinical suspicion is required.31Radiologically, it may be difficult to distinguish IRIS from TB and other opportunistic infections so radiological findings must be interpreted in combination with clinical and laboratory findings.18 Of note, children with suspected local or regional BCG-IRIS can generally be diagnosed based on clinical manifestations and microbiological confirmation using lymph node biopsy or fine-needle aspiration is seldom warranted unless alternative diagnoses are under consideration. If samples are submitted from patients with suspected BCG-related disease, it is vital to indicate this on the laboratory request form so that appropriate testing to differentiate TB from BCG can be performed.Investigation is largely informed by the particular infection under consideration. Measurement of viral load and CD4 counts are helpful, particularly when poor adherence to therapy is suspected. A significant decline in viral load from baseline, as well as a substantial increase in CD4 count, is suggestive of IRIS, but not essential for making the diagnosis.6 In patients with severe or life-threatening IRIS, including airway compression by hilar or mediastinal lymph nodes or expansile CNS mass lesions, it may rarely be appropriate to interrupt ART until their condition stabilises, whereafter ART can be safely restarted.6 Stopping ART is not to be taken lightly and when being considered should involve discussion with a specialist or subspecialist.The most important components of effective management are to treat the IRIS-related infection using the same modalities as for non-IRIS–related infections with that pathogen. Because IRIS is frequently mild and self-limiting, in most patients continuation of ART is to be encouraged.22 In addition to continuation of ART and treating the implicated pathogen, patients with IRIS may benefit from anti-inflammatory medicines. In mild and moderate cases, non-steroidal anti-inflammatory drugs (NSAIDs) have been used to provide symptomatic relief and may hasten IRIS resolution. In more severe cases, corticosteroids may be required for a number of weeks, whereafter they can be tapered and stopped.6 The additional immune suppression related to corticosteroid use is a concern in such patients and specialist input is advised before their commencement.In children with pain and discomfort from BCG-IRIS lymphadenitis, consideration should be given to needle aspiration of fluctuant areas to relieve symptoms. Such aspiration may need to be repeated.23 Recent improvements in early infant diagnosis of HIV followed by almost immediate initiation of ART have resulted in IRIS becoming uncommon.23If HIV is diagnosed early and ART initiated soon thereafter, IRIS is unlikely.32 In most patients with TB, ART can be safely initiated about 2 weeks after TB treatment has commenced; however, extrapolating from experience in adults with HIV-associated cryptococcal meningitis, ART initiation should be delayed by at least 6–8 weeks.33 Routine prophylactic use of NSAIDs or corticosteroids to reduce the risk of IRIS is not recommended, rather patients at high risk for IRIS should be closely monitored for new or worsening symptoms so that IRIS can be timeously identified and treated.Furthermore, it is important to consider opportunistic infection prior to ART initiation as initiating treatment for them first may reduce the risk of IRIS developing. This is particularly true for CNS TB and cryptococcal meningitis where ART initiation should be delayed during the initial weeks of treatment in order to reduce the risk of IRIS. Tuberculous mass lesions (tuberculomas) have been associated with life-threatening CNS-IRIS.M. tuberculosis. The commonest radiological manifestations of TB-IRIS include new or worsening hilar or mediastinal lymphadenopathy, worsening or new air space consolidation, pleural effusions, reticular infiltrates or nodular infiltrates. It may be difficult radiologically to distinguish IRIS in children from TB and other opportunistic infections so radiological findings must be interpreted in combination with clinical and laboratory findings.While ART has revolutionised the care of HIV-infected children, ART has side effects and potential adverse consequences which include IRIS. IRIS is typically classified as either unmasking or paradoxical; however, this distinction may be difficult, as both forms may coexist in the same child and other explanations for the clinical findings should be considered. Approximately half of all IRIS (adult and paediatric) cases are associated with


Obesity is associated with the development of metabolic diseases such as type 2 diabetes and non-alcoholic fatty liver disease. The presence of chronic, low-grade inflammation appears to be an important mechanistic link between excess nutrients and clinical disease. The onset of these metabolic disorders coincides with changes in the number and phenotype of macrophages in peripheral organs, particularly in the liver and adipose tissue. Macrophage accumulation in these tissues has been implicated in tissue inflammation and fibrosis, contributing to metabolic disease progression. Recently, the concept has emerged that changes in macrophage metabolism affects their functional phenotype, possibly triggered by distinct environmental metabolic cues. This may be of particular importance in the setting of obesity, where both liver and adipose tissue are faced with a high metabolic burden. In the first part of this review we will discuss current knowledge regarding macrophage dynamics in both adipose tissue and liver in obesity. Then in the second part, we will highlight data linking macrophage metabolism to functional phenotype with an emphasis on macrophage activation in metabolic disease. The importance of understanding how tissue niche influences macrophage function in obesity will be highlighted. In addition, we will identify important knowledge gaps and outstanding questions that are relevant for future research in this area and will facilitate the identification of novel targets for therapeutic intervention in associated metabolic diseases. Adipose tissue and liver contain tissue resident macrophages that are indispensable for tissue homeostasis. Tissue macrophages can either be derived from primitive hematopoiesis in the embryonic yolk-sac or from definitive hematopoiesis from the fetal liver or bone marrow-derived monocytes . Fate-maTraditionally, macrophage activation has been described by the two-dimensional M1 and M2 spectrum, i.e., classical or alternative activation, respectively. Recently, this paradigm has been replaced by the concept that there is a continuous spectrum of macrophage activation that can be shaped by tissue environment, cell programming, and activating stimuli. In large part, this notion has arisen from data using fate mapping and phenotyping techniques combined with systems biology . TherefoLean adipose tissue contains resident macrophages that reside between adipocytes and alongside vascular structures. These resident adipose tissue macrophages (ATMs) have various roles in adipose tissue homeostasis including efferocytosis , lipid buffering, and adipogenesis  8). In . In 8). hi monocytes to adipose tissue , in which macrophages appear to surround apoptotic adipocytes , 25. Int+, CD11b+, CD206+, CD301+ cells. In obese adipose tissue, CCR2+/Ly6Chi monocytes enter the tissue where they can differentiate to ATMs and/or proliferate , and C-type lectin domain family 4 member F (Clec4F) . KCs represent 35% of all liver non-parenchymal cells and are the largest tissue population of resident macrophages in the body. They are largely derived from erythromyeloid progenitors of the embryonic yolk sac and maintained by self-renewal , 44. The(Clec4F)  47, 48), 48hi, C(Clec4F) . MdMs arammation . However+ liver macrophages in the healthy human liver, with one population having an inflammatory gene expression pattern and the second population was characterized by an immunotolerance phenotype . Although it was initially tempting to speculate that similar inflammatory responses might occur in both adipose tissue and liver with obesity, it is now apparent that these organs respond in very different ways to lipid stress. NAFLD is characterized by excessive hepatic lipid accumulation, which can result in hepatocyte injury and cell death. This can trigger an inflammatory response and progression to non-alcoholic steatohepatitis (NASH), advanced fibrosis, cirrhosis, and hepatocellular carcinoma . Macroph+, Cx3cr1+, Ly6Chi monocytes infiltrate the liver in experimental models of NASH and appear to give rise to “pro-inflammatory” macrophages . Mo. Mohi, Ces NAMs, . Gene exFuture studies will be required to dissect the dynamic shifts that occur in macrophage subsets during NASH pathogenesis. In addition, the specific cues that induce macrophage polarization in NAFLD are not completely understood and the interplay between metabolic cues and macrophage function may be crucial for NASH progression.+ macrophage infiltration into the adipose tissue. These macrophages enhance lipolysis via modulation of epinephrine and norepinephrine levels. Interestingly, this wave of adipocyte death also causes acute secondary liver injury which is dependent upon CCR2+ macrophage infiltration into the liver  generation , 100. ThThe NLRP3 inflammasome has recently been recognized as an important driver of inflammation in many complications of obesity including atherosclerosis, insulin resistance, and NASH , 103. InUpregulation of glucose uptake also feeds substrate into the pentose phosphate pathway (PPP) which is also upregulated in inflammatory macrophages . The PPPAlthough increased glycolytic flux is a hallmark of inflammatory macrophage activation, glycolysis is also required for alternatively macrophage activation. Mechanistically, it has been shown that IL-4 and M-CSF activate mTORC2, which subsequently upregulates glycolysis via IRF4 . HoweverIn summary, although glucose metabolism is associated with classical inflammatory macrophage activation, it is also required for alternative macrophage activation. Thus, selective modulation of glucose metabolism is a more viable approach for disrupting macrophage inflammation. Further investigation will also be necessary to unravel the complex interplay between macrophage glucose handling and the hyperglycemia that frequently exists in states of obesity and insulin resistance.in vivo is not known.In contrast to the stimulation of glycolytic metabolism that occurs in inflammatory macrophages, increased lipid uptake and oxidation is a hallmark of alternative macrophage activation induced by IL-4. In this system, IL-4 increases mitochondrial fatty acid oxidation (FAO) and this metabolic response appears to be necessary for macrophages to acquire an M2 phenotype . This isIn vivo, loss of FATP1 in bone marrow derived cells was associated with increased HFD-induced weight gain, glucose intolerance, and adipose tissue inflammation. In contrast, deletion or inhibition of FABP4 or FABP5 attenuates lipid-induced inflammation and improves metabolic phenotypes in vivo  act as lipid sensors and modulate both lipid storage and utilization . Both PPIn vitro studies have shown that ligands of LXR receptors inhibit expression of inflammatory mediators as iNOS, COX-2, MMP-9, and IL-6 , CCAAT enhancer binding proteins (C/EBPs), and sterol regulatory element binding proteins (SREBPs). Of interest, KCs express high levels of LXRα, which acts as cholesterol sensor and regulates intracellular cholesterol levels by expression of cholesterol efflux transporters ABCA1 and ABCG1 , 127 as and IL-6  and can and IL-6 . SREBPs in vitro . Deletioin vitro . These fC/EBPα and C/EBPβ are also widely expressed in macrophages and have been implicated in regulating glucose and lipid metabolism . C/EBPβ Recently, the requirement of FAO for alternative macrophage activation has been questioned. Primary macrophages lacking CPT2 have reduced rates of FAO, but have no defect in IL-4-induced alternative activation . In addide novo lipogenesis, production of proinflammatory lipid mediators (prostaglandin E2) and synthesis of NO  has been postulated to be important for M1 polarization as it reduces entry of carbons from glycolysis into the TCA cycle by inhibiting pyruvate dehydrogenase (PHD). Indeed, pharmacological inhibition of PDK2 and−4 blocks inflammatory activation of cultured macrophages . MyeloidCitrate can also serve as a precursor for itaconate, which is produced by the enzyme immunoresponsive gene 1 (Irg1). This metabolite subsequently inhibits succinate dehydrogenase (SDH) and elevates succinate levels , 149. SuGlutamine is one the most important amino acids to fuel macrophage activation. Interestingly, glutamine has different metabolic fates in M1 and M2-like macrophages. In inflammatory macrophages, glutamine-derived glutamate fuels in to the TCA cycle for succinate production via GABA production . Hence iThe amino acid L-arginine also has divergent roles in macrophage polarization. L-arginine is an essential precursor for the production of NO via iNOS, which is induced during inflammatory macrophage activation. On the other hand, metabolism of L-arginine by arginase is associated with alternative macrophage activation and appears important for tissue repair . It is lAs reviewed above, targeting important metabolic pathways in macrophages has the potential to alter not only macrophage function, but also systemic metabolism. However, the exact mechanisms by which tissue macrophages regulate whole-body metabolism are largely unknown and this remains an important knowledge gap in the field. As discussed above, it is clear that macrophages can interact with parenchymal cells in the adipose and liver in ways that modulate systemic glucose homeostasis  159). I. I159). Obesity exposes tissue macrophages to a microenvironment that consists of excess nutrients as well as stress signals from damaged and apoptotic parenchymal cells. These environmental cues can activate macrophages and rewire their cellular metabolism in ways that may promote maladaptive inflammation and macrophage dysfunction. Macrophage activation may initially be beneficial to facilitate the removal of apoptotic cells, clear excess lipids, and restore tissue homeostasis. However, aberrant metabolic reprogramming of macrophages in the setting of sustained lipid-induced tissue injury likely provokes pathologic macrophage phenotypes. When this occurs the macrophage secretome may act locally and/or systemically to drive metabolic disease development . This coin vitro models or non-selective myeloid knock-out systems. Ultimately, elucidating the heterogeneity of macrophage populations in different organs and their relation to metabolic disease will allow more specific therapeutic targeting. However, our understanding of this has been limited by the lack of tissue-specific macrophage markers to identify and manipulate resident macrophages in different tissues. As such, most studies have used generic myeloid knock-out models, such as LysM-Cre or bone-marrow transplantation. The identification of unique markers of tissue macrophages through the use of sophisticated unbiased approaches such as scRNA-seq, has the potential to open up new avenues to investigate the role of distinct macrophage subsets in tissue and whole-body metabolism. As an example, it has recently been shown that liver resident KCs uniquely express the surface receptors Tim4 and Clec4f. This discovery facilitated the development of Clec4f-Cre transgenic mice, which will enable the study KC specific functions in liver pathology and metabolic disease (Although it is established that perturbing macrophage cellular metabolism can improve metabolic disease phenotypes, the precise cellular and molecular details remain poorly understood. In part this is due to the fact that much of the data in this field comes from knockout or overexpression of key metabolic genes in the context of  disease .Another challenge to advancing this field relates to the impact of variations in genetic background, microbiota composition, and research environment on phenotypes in metabolic disease research , 163. AsThe pathogenesis of obesity-related metabolic disorders, such as insulin resistance is highly complex and involves metabolic perturbations in multiple organs. Tissue macrophages secrete a plethora of factors that may affect macrophage polarization and subsequently tissue function in distant organs. Evidence already exists to support the concept that adipose tissue macrophages can direct relevant pathological events in the liver in the setting of obesity. Conversely, it is attractive to hypothesize that signaling from liver macrophages to adipose tissue also occurs. It will be important to identify the key molecules involved in inter-organ crosstalk and to evaluate their effects on tissue macrophage biology.The objective of this review was to discuss our current understanding of macrophage diversity in metabolically relevant tissues and to consider the intersection of these principles with the regulation of macrophage cellular metabolism. Although significant progress has been in these areas, further investigation and integration of these fields will be necessary to harness the therapeutic potential of re-programming macrophage metabolism in obesity and diabetes. In addition, defining the molecular basis of crosstalk between macrophages and parenchymal cells in adipose tissue, liver, and the brain will also require more study. Another important issue to resolve in this area is whether targeting macrophage metabolism can attenuate pathologic inflammation during obesity without disabling the fundamental roles of these immune cells in tissue repair and the maintenance of homeostasis. Eventually, the use of targeted small molecules or nanoparticles are attractive modalities to modulate macrophage function. Although there is much yet to be learned, metabolic targeting of macrophage biology holds promise as therapeutic strategy to modulate metabolic disease.SD and JS conceptualized and wrote 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.


Enhanced recovery after surgery (ERAS) has rapidly gained popularity among hip or knee arthroplasty area which can decrease hospital length of stay (LOS). However, limited data exist regarding its safety and efficacy among intertrochanteric fracture patients. The purpose of this study was to determine if LOS associated with intertrochanteric fracture patients can be improved following an existing orthopedic ERAS procedure.We reviewed the outcomes of all patients who had been treated with the PFNA intramedullary fixation at our institution. Open fractures, metastatic pathological fractures, patients unable to walk independently before fracture and patients with Alzheimer's disease were excluded. A quasi-experimental study was adopted between patients treated in an ERAS after intramedullary fixation with those rehabilitated on a traditional pathway. Clinical and demographic data were collected among the two pathway cohorts including LOS, Harris hip scores (HHS), visual analog scale (VAS), and activity of daily living scale (ADL).p < 0.05).A total of 100 intertrochanteric fracture patients  were selected between January 2016 and December 2017 met the inclusion criteria. ERAS procedure was associated with shorter LOS, lower postoperative VAS scores, reduced opioid consumption, earlier mobilization, significant improvement in the mean HHS scores at 3 months postoperatively, lower risk of complications, lower rates of readmission, and reoperation and higher likelihood of being discharged home. The mean LOS decreased from 8.21 ± 0.83 days to 5.82 ± 0.64 days after implementation of the evidence-based orthopedic ERAS pathway (This series of intertrochanteric fracture patients treated with the orthopedic ERAS procedure demonstrated that the procedure is capable of reducing LOS and preserving hip function without compromising functional outcome. This improvement was possible without a concomitant increase in postoperative complications and readmission rates.Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence. Intertrochanteric fracture is a major public health issue which has a high mortality rate within one year –4. IntraLOS associated with intertrochanteric fracture is a major public health issue due to the aging population –14. SomeThe concept of ERAS originated from colorectal surgery in the late 20th century which have successfully demonstrated benefits to the surgical patient in colorectal and upper gastrointestinal , 12. PreIn summary, the potential of ERAS for patients undergoing intramedullary fixation is great, but the overall level of evidence is low. The purpose of this study was to evaluate the effect of an evidence-based ERAS clinical pathway on perioperative outcomes in intertrochanteric fracture patients undergoing intramedullary fixation.This was a quasi-experimental design study that compared a prospective cohort (ERAS: January–December 2017) with a historical standardized care pathway cohort (control: January–December 2016). Emergency admissions with a primary diagnosis of intertrochanteric fracture between January 2016 and December 2017 in our hospital were selectively enrolled. Ethical approval was obtained from the internal review board, and orthopedic departmental approval was gained to proceed with prospective data collection. All patients were treated with PFNA intramedullary fixation at our institution. Inclusion criteria consisted of patients who had undergone surgery with a standardized care pathway and those who had undergone surgery with the ERAS pathway. Radiological data were reviewed to identify the fractures type based on the AO classification. Patients undergoing PFNA intramedullary fixation surgery were eligible to participate in the prospective arm of the study. Written and verbal informed consent was obtained from each patient before inclusion in the study. Exclusion criteria comprised patients with open fractures, metastatic pathological fractures, patients unable to walk independently before fracture, and inability to follow verbal or written instructions , patients were enrolled consecutively. The consecutive data collected for both groups were compared for age, gender, body mass index (BMI), LOS, the pre-, and postoperative HHS. Preoperative comorbid status was assessed by means of the American Society of Anesthesiologists (ASA) score and the presence of diabetes mellitus, hypertension, hypercholesterolemia, chronic obstructive pulmonary disease (COPD), ischemic heart disease (IHD), and atrial fibrillation (AF) . Thirty-The primary outcome measure was LOS at discharge, and the secondary outcome measure was the patient’s early postoperative joint specific function (HHS score). The other secondary outcomes included VAS score, ADL scale, opioid consumption, opioid-related adverse effects, 30-day readmission rates, postoperative transfusion rates, discharge to home, and in-hospital mortality. The other secondary outcomes included VAS score, ADL scale, opioid consumption, opioid-related adverse effects, 30-day readmission rates, postoperative transfusion rates, discharge to home, and in-hospital mortality.2) on the day of surgery. Opioid-free spinal anesthesia was preferred at the discretion of the anesthetist. Intravenous (IV) tranexamic acid (1 g) was given preoperatively if not contra-indicated. Dexamethasone 8 mg (IV) and 40 mg of parecoxib sodium (IV) were given intraoperatively. Early mobilization was facilitated under a rehabilitation therapist. Oral postoperative opioid-sparing analgesia including non-steroidal anti-inflammatory drugs  provided on a regular basis (assuming no contraindications) and tramadol  were routinely prescribed when the patient returned to the ward.The basic components of the multi-disciplinary and multimodal ERAS pathway which we used are shown in Table t test or chi-square test as appropriate. Mean values for normally distributed continuous data such as LOS , HHS scores, and complication rates between the two groups were compared using two-tailed unpaired Student’s t tests or chi-squared test as appropriate. The mean and standard deviation, alongside the minimum and maximum values, were determined for all outcomes. A p value < 0.05 was considered statistically significant.Descriptive statistics were calculated using Statistical Package for Social Sciences (SPSS) . Demographic and baseline clinical characteristics were compared using two-tailed unpaired Student’s A total of 100 intertrochanteric fracture patients  were selected between January 2016 and December 2017 while following the ERAS and traditional care pathway. The average age of the intertrochanteric fracture patients in our study have no statistical differences in the ERAS and traditional pathway groups, respectively, and 68% were female. All of the patients underwent PFNA intramedullary fixation. There were no baseline statistical differences between groups in age, gender, BMI, ASA scores  compared to 7~9 days  in the traditional rehabilitation group  was achieved in all of the patients in the ERAS group. Eighty-two of those patients mobilizing early were successfully discharged within 4 days or less. This is in contrast to the control group, which demonstrated only 48% of patients mobilizing within 24 h of their operation. There was a significant improvement in the mean HHS scores in ERAS group at 3 months but no statistically significant differences between the groups at 6 months postoperatively. Besides, there were no statistical differences in the mean ADL scores both at 3 and 6 months postoperatively, see Table Summary of postoperative complications in 30 days is listed in Table This study shows that LOS after intramedullary fixation in the intertrochanteric fracture can be significantly reduced by using ERAS pathway, while maintaining better early functional outcomes to those rehabilitated in a traditional manner, and without increasing the rate of complications or mortality postoperatively.Intertrochanteric fractures commonly occurred in the elderly and caused high fatality rate due to loss of walking ability –3. In orIntramedullary fixation system has mechanical and biological advantages in intertrochanteric fracture –5. PFNA A long LOS for intertrochanteric fracture patients is not desirable, as they have an increased risk of developing complications and losing their independence , 13. FurSeveral clinical studies involving ERAS on arthroplasty have been described previously in the literature –20, but In the ERAS concept, pain management is one of the most important constituents. Postoperative pain not only prolongs the LOS, but also reduces the subjective willingness of patients to take early rehabilitation exercises, and therefore resulting in decreased joint function , 18. TheFor elderly patients, early rehabilitation exercise is very important, ERAS concept emphasizes early functional rehabilitation which increases intestinal peristalsis and lung capacity, promotes lower limb muscle strength recovery, maintains hip activity, accelerates the blood circulation at the incision site, promotes incision healing and lower limb venous reflux, and prevents the formation of deep venous thrombosis after operation –17. AlthThe limitations of this study are primarily the result of its retrospective design. In addition, the number of cases is small and the follow-up period is somewhat short. Nevertheless, the intraoperative and postoperative protocols were uniform. Besides, we have not reported on more detailed functional outcomes such as walking distance, ability to walk independently and climb stairs, return to work and driving after the discharge or blood loss during the procedure, which is also a limitation of our existing ERAS database. The results of this study need to be substantiated with adequately powered randomized control trials.In conclusion, an orthopedic-specific ERAS program implementation in intertrochanteric fracture independently reduces median LOS without compromising early functional outcome. The improvement methodology not only supports the implementation of ERAS protocols but also establishes change that is sustainable with minimal additional resource use. This has not previously been reported. Further studies should be undertaken to determine the relative importance of various individual measures and the longer-term functional outcome in patients treated on ERAS.


Lumen-apposing metal stents (LAMS) use in gastrointestinal endoscopy has been on the rise for various indications for the last few years. Currently, LAMS is a well-established treatment for post-pancreatitis peri-pancreatic fluid collections and walled-off necrosis (WON), but it is still not a standard of care in the treatment of post-surgical fluid collections (PSFC). Most of the earlier studies for treatment of PSFC utilized double pigtail plastic stents (DPS). We present a series of 3 cases where LAMS was successfully used for PSFC drainage. The cases include a patient with perigastric abscess after Whipple's procedure, a case of peri-pancreatic collection after distal pancreatectomy, and a patient with peri-pancreatic fluid collection after right partial hepatectomy and splenectomy due to lacerations from a motor vehicle accident. Post-surgical fluid collections (PSFCs) can be an important cause of morbidity and mortality depending upon the nature of the surgery and site of the collection. Over the last few decades, percutaneous radiologic drainage (PCD) has been considered the first-line treatment of PSFC with a success rate of 80-100% . Due to With advances in therapeutic endoscopy over the past few years, there has been increasing use of plastic and metal stents as an attractive alternative to PCD, especially in the treatment of pancreatic and peripancreatic fluid collections and walled-off necrosis (WON) , 4. ReceA 54-year-old male with a history of renal cell carcinoma, pancreatic adenocarcinoma stage II (T2 N1 3/5 lymph nodes positive), having previously received chemotherapy followed by stereotactic body radiation therapy (SBRT) presented 5 months after the Whipple's surgery with failure to thrive, fatigue, and nausea. Exam was unremarkable and laboratory investigations revealed albumin of 1.3 mg /dl, bilirubin of 2.8 mg/dl, mainly conjugated, serum alkaline phosphatase of 825 U/L, and CA 19-9 of 81.4 . Computed tomography scan (CT) of abdomen showed a perigastric abscess adjacent to the fundus . EndoscoThe patient improved clinically along with significant improvement in his bilirubin and alkaline phosphatase after the procedure. Repeat CT abdomen after one week of stent placement showed a near-complete resolution of the abscess , althoug54-year-old male underwent distal pancreatectomy with splenectomy for treatment of pancreatic neuroendocrine tumor. In the immediate postoperative period, patient developed a pancreatic fluid leak from the tail of pancreas and an intra-abdominal drain was placed that was removed after it stopped draining. CT scan showed interval increase in the size of the rim enhancing fluid collection around tail of pancreas 1 month after drain removal . ERCP waA 34-year-old male presented to hospital after a motor vehicle accident. Patient was hypotensive on arrival and underwent exploratory laparotomy, splenectomy, embolization of hepatic vessels, and right-sided partial hepatectomy due to grade V liver laceration. Postoperatively, the patient developed bilious drainage from an intra-abdominal drain and underwent ERCP for suspected bile leak. ERCP revealed a leak from the right biliary system; therefore a 10 Fr x 5 cm plastic biliary stent was placed. A week later, the patient continued to have high output of amylase-rich fluid from a separate intra-abdominal drain which was suspicious for a pancreatic duct leak. Repeat ERCP with pancreatogram revealed a leak from the pancreatic tail for which a 5 Fr x 13 cm pancreatic duct stent was placed and the biliary stent was upsized to a 10 mm x 4 cm covered self-expanding metallic stent (SEMS) due to a persistent biliary leak.After 1 month, the patient became septic and was found to have a peri-pancreatic abscess on CT abdomen. IR performed a percutaneous drainage of the peripancreatic abscess with minimal drainage through the drain, without any clinical improvement. EUS was then performed by one of the authors (JN) that revealed a 55 mm, oval, heterogeneous peri-pancreatic fluid collection which had hyperechoic material consistent with the solid debris. A 10mm x 10mm LAMS was then placed from the stomach into the fluid collection with subsequent drainage of pus and debris. The patient clinically improved and was discharged home with improvement of the fluid collection. A follow-up CT Abdomen 4 weeks later showed interval decrease in the size of the previous small fluid collection. The LAMS was uneventfully removed endoscopically at 8 weeks after the initial placement.Current treatment options for postoperative fluid collections include percutaneous drainage, EUS–guided drainage, and open surgical drainage. Of these, PCD is the current standard of care. Despite its success rate of more than 80%, PCD has numerous disadvantages. Although good results can be achieved with surgery, it is not preferred these days due to relatively higher morbidity and costs [Limitations of PCD include higher costs compared to EUS-GD due to need for regular monitoring by health care providers, tendency to get obstructed, repeated need for use of disposable health supplies, increased radiation from multiple CT scans, and longer median hospital stay –11. SignCurrently, there are a large number of studies supporting the use of stents in the treatment of pancreatic pseudocysts and WON , 13–15. There are multiple studies in the literature supporting successful use of LAMS for pancreatic fluid collections drainage, but the only study that is available at this time for the use of LAMS in PSFCs of all etiologies is a US multicentre, retrospective analysis of 47 patients by Mudireddy et al. . The autLAMS has many potential advantages compared to plastic stents in PSFC drainage including obviating the need for fluoroscopic guidance as the stent is well visualized under EUS. The possibility of performing the entire procedure under EUS guidance alone is a major advance in therapeutic endoscopy. LAMS are also technically easier to deploy and extract thereby reducing the procedure time and has higher technical success rates , 21. TheThe drawbacks of LAMS include paucity of experience in the use of these stents but that is changing quickly with the wider acceptance of this stent. Higher costs of LAMS at the time of placement is a deterrent compared to PCD . SecondlStent migration (both spontaneous and during direct necrosectomy) has also been noted with LAMS possibly due to rapid decompression of cysts with LAMS . MaldeplFinally, authors acknowledge that although the procedure was technically successful in all the cases, the final outcome of the patients did not improve in 2 out of the 3 cases, primarily due to the underlying diagnosis of malignancy that adversely affected their prognosis. Therefore, appropriate patient selection is very important in deciding who would benefit from the procedure.In conclusion, EUS-GD using LAMS is an equally effective but safer and cost-efficient alternative to PCD for treatment of PSFCs. With the increased availability of forward viewing echoendoscopes, the therapeutic possibilities have increased including accessing perisplenic collections and collections around modified anatomy such as Roux-en–Y gastric bypass. With availability of 20 mm diameter LAMS, the efficacy of drainage of PSFCs may even improve further. Further studies for PSFC treatment with EUS-GD using LAMS are warranted for understanding their full scope of use and possibly becoming an established standard of care.