Multiple myeloma is a type of blood cancer caused by the uncontrolled growth of antibody producing B cells (known as plasma cells) that reside in the bone marrow. It is classed as a largely incurable cancer as whilst patients respond well to initial chemotherapy treatments, unfortunately after periods of disease remission, relapse usually occurs with the emergence of chemotherapy resistance. Therefore, there is a need for new approaches that not only reduce tumour load but also prevent tumour relapse. Oncolytic viruses (OVs) (tumour killing viruses) are being explored as a therapy for various cancers, including multiple myeloma. This review discusses the use of OVs in myeloma in preclinical model systems and early phase clinical trials, and discusses some of the hurdles involved in the translation to myeloma patients.Multiple myeloma accounts for 1% of all new cancers worldwide. It is the second most common haematological malignancy and has a low five-year survival rate (53.2%). Myeloma remains an incurable disease and is caused by the growth of malignant plasma cells in the bone marrow. Current anti-myeloma therapies  initially substantially debulk tumour burden, but after a period of remission ‘plateau phase’ disease invariably relapses due to tumour recrudescence from foci of minimal residual disease (MRD) and accumulating drug resistance. Therefore, there is a compelling clinical need for the development of novel treatment regimens to target MRD and effectively eliminate all remaining tumour cells. This review will discuss the potential use of oncolytic virus (OV) therapies in the treatment of myeloma. Specifically, it will focus on preclinical studies using DNA viruses (adenovirus (Ad), vaccinia virus (VV), myxoma virus (MYXV), and herpes simplex virus (HSV)), RNA viruses (reovirus (reo), coxsackie virus, measles virus (MV) and bovine viral diarrhoea virus (BVDV), and vesicular stomatitis virus (VSV)), and on four types of viruses  that have been assessed clinically in a small number of myeloma patients. Multiple myeloma accounts for 1% of all new cancers worldwide and it is the second most common haematological malignancy . In addiOver the last 20 years there have been significant changes in the landscape of anti-myeloma therapies due to a better understanding of its pathobiology. This has led to the introduction of more effective therapies which include immunomodulatory drugs (IMiDs), e.g., lenalidomide ; proteasNovel therapies that do not rely on the use of generic chemotherapies or targeted small molecule therapies either with their inevitable toxicities and/or drug resistance are highly desirable for the treatment of myeloma. This is because two of the major factors contributing to therapeutic failure in myeloma are tumour heterogeneity and plasticity leading to the emergence of resistant clones . OncolytOVs can infect both normal and malignant cells, but malignant cells provide a superior environment due to the presence of aberrant signalling pathways, abnormal homeostasis, and responses to stress, which are advantageous for viral replication, such as overexpressed surface attachment receptors, activated RAS or Akt, or defective IFN pathways. ,17. ThesOVs have been exploited for their anti-tumor effects across a range of solid and haematological malignancies. Specifically, in myeloma, a single case report from 1987 suggested that VV could (at least transiently) reduce systemic tumour burden in a patient . Since tAds are non-enveloped dsDNA viruses that have been used widely for gene therapy. An early study demonstrated that transduction with Ad vectors, using tumour selective DF3 promoter to drive the expression of thymidine kinase (TK), efficiently transduced myeloma cell lines . Fernand5 integrin is the primary receptor for Ads in CAR-negative cells [Given that most haematological malignancies do not express high levels of coxsackievirus and adenovirus receptor (CAR), the main entry receptor for Ad5, it has not generally been considered as an oncolytic agent for malignancies such as myeloma. However, Senac et al.  showed tve cells  and thisEarly work showed that WT Ad5 infects myeloma cell lines, but the life cycle is delayed compared to that of permissive cells ,29 potenMost in vivo studies using oncolytic Ads have been performed in immunodeficient mice. To fully test the potential therapeutic activity of OVs, studies should be performed in immunocompetent animals that can support active viral infection . HoweverDespite the preclinical activity of Ads in myeloma cell lines and mouse models, clinical investigation has not yet been realised, and the efficacy and safety profile need to be defined in phase I/II clinical trials.SIRT1).VV is a large dsDNA virus that belongs to the Poxviridae family, derived from the original cowpox or horsepox virus with the historical use as a smallpox vaccine with proven clinical safety in humans. However, to better target myeloma cells, VVs have been genetically modified. TK and vaccinia growth factor (VGF) genes were double-deleted in a highly attenuated VV (VVDD) . VVDD prIn the late 1980s, a single case study of a 67-year-old Japanese male patient with IgA myeloma was reported. Intravenous injection of VV (AS strain) markedly reduced IgA levels from 1309 mg/dL to 432 mg/dL after 96 days of treatment with no adverse effects reported . HoweverATF4 expression during the unfolded protein response [+ T cell response which resulted in a significant overall survival. Due to MYXV rapid induction of oncolysis, it has been proposed that MYXV may be an effective purging strategy for autologous stem cell transplants (ASCTs) [+ myeloma cells from patient bone marrow samples, but whether the same effects are seen across a wider range of heterogeneous myeloma patients need to be explored. More recently, autologous murine bone marrow carrier leukocytes, pre-armed with MYXV, were therapeutically superior to MYXV armed PBMCs or free virus [MYXV is a non-segmented dsDNA with a strict tropism for rabbits and hares. Whilst MYXV does not infect healthy non-malignant human cells, MYXV has been shown to infect a variety of cancer cell types. In myeloma cells, MYXV induced significant rapid oncolysis that was dependent upon caspase-8 mediated apoptosis and inhibited response ,39. In a (ASCTs) . Arming ee virus . Additio+ primary cells and caused cell death independent of HSV-1 replication, due to apoptosis. In a subcutaneous xenograft myeloma model, HSV-1 treatment decreased tumour volume after intratumoral injection [HSV is dsDNA virus belonging to Herpesviridae family and although HSV has shown success in melanoma , it has njection . Anothernjection . Co-cult+ stem cells, and demonstrated complete eradication of myeloma cells, preventing relapse and improved survival in mice [Reo is a dsRNA virus of the Reoviridae family of viruses, with the human type 3 Dearing strain being developed for oncolytic virotherapy. Reo has shown efficacy against myeloma cell lines and ex vivo tumour samples ,45, wher in mice . However in mice . Interes in mice .+ T cells, NK cells, PD-L1 expression and decreased T-regs and tumour associated macrophages (TAMS) [+ T cell numbers and activation, and upregulated effector-memory CD8+ T cells. Additionally, the study found that co-culture of myeloma cells with bone marrow stromal cells (BMSCs) was able to induce resistance to MPC reo oncolysis and bystander cytokine killing, but the BMSCs were not able to protect the MPCs from reo-activated NK cells and myeloma-specific cytotoxic T cells [More recently, reo has been shown to increase PD-L1 expression in MPC lines in vitro and in vivo, this increase was not observed when UV-inactivated reo was used . When res (TAMS) . Reo hass (TAMS) . In the  T cells .Twelve patients with symptomatic relapsed and refractory myeloma, with previous bortezomib and lenalidomide treatment with or without previous ASCT, were administered Reoylsin intravenously in a dose-escalating, single centre phase I clinical trial . Reolysi+ cells in myeloma patient bone marrow biopsies, whilst PBMCs expressed low levels of ICAM-1. CVA21 caused cytopathic effects in myeloma cells, but these were minimal in PBMCs compared to untreated controls, thought to be due to differences in viral replication. CVA21 purging of myeloma bone marrow biopsies varied, but bone marrow progenitor cells were not significantly affected [Coxsackie virus is a non-enveloped positive-sense ssRNA virus, with the most studied virus being CVA21, but limited work has been done in myeloma. Au et al.  showed maffected . CurrentMV is an enveloped negative-sense ssRNA virus in the family Paramyxoviridae. The most studied strain is the attenuated Edmonston strain (MV-Edm), which has mutations in two accessory proteins (C and V) leading to tumour selectivity, genetic stability and is non-transmissible . In vitrA phase I clinical trial with systemically delivered MV (MV-NIS) in patients with recurrent or refractory myeloma was published in 2017 . The tri+ primary myeloma cells from patient bone marrow aspirates and did not affect CD3+, CD19+, or CD56+ cell populations, suggesting that the BVDV oncolytic effect was limited to CD138+ myeloma cells. In vivo, in a subcutaneous xenograft myeloma mouse model, mice were treated with BVDV intratumorally, which led to a reduction in tumour volume, and an increase in caspase-3 activity. Additionally, the authors showed pretreatment with bortezomib increased BVDV efficacy in myeloma cell lines in vitro and suggested this was due to the activation of caspase-3-meidated apoptosis [BVDV is a ss-RNA virus belonging to the Flaviviridae family, and is a major viral pathogen of cattle . Myelomapoptosis . Due to VSV is a member of the family Rhabdoviridae and is an enveloped negative-sense ssRNA virus that commonly infects livestock animals, but it can infect humans causing flu-like illnesses. Preclinically, VSV has demonstrated effectiveness against myeloma in vitro and in vivo . VSV wasPreclinical results with VSV have led to the establishment of an early phase clinical trial (NCT03017820) with VSV-IFNβ-NIS in patients with haematological malignancies, including relapsed myeloma. The trial is currently active and recruiting.n = 31) [Multiple myeloma poses considerable therapeutic challenges to be overcome if oncolytic virotherapy is to translate to an effective treatment strategy. The field suffers from several obvious issues, which must be addressed. Firstly, the impact of disease heterogeneity needs to be examined in the context of oncolytic virotherapy. A review of the literature showed that preclinical results derive more from tests on myeloma cell lines than on patient-derived primary myeloma cells . Howevern = 31) . The fieSecondly, most of the literature studying OVs in myeloma administer OVs intratumorally in immunodeficient models of a single subcutaneous plasmacytoma. Although these studies represent an easy starting point, they are not clinically relevant myeloma models, and fail to recapitulate the clinical realities in two ways. First, due to the nature of OV, there are different treatment challenges associated with systemic versus localised disease. In particular, delivery of a virus through the circulation to sites of systemic disease is a major translational hurdle . Since mLastly, combining OVs with more typical myeloma standards of care therapies, such as chemotherapy, radiation or mAbs is lacking but may be the best approach to increase efficacy. Results from the first completed clinical trial utilising oncolytic reo in myeloma support this notion . EnhanceMultiple myeloma remains a predominantly incurable disease, despite current therapies debulking 90% or more of the initial tumour burden. Patients usually succumb to cycles of disease relapse due to disease recrudescence from foci of MRD and the emergence of chemo-refractory clones of MPCs . Therefo


The novel scaffolds retainthe biological relevance of natural products but are not obtainablethrough known biosynthetic pathways which can lead to new chemotypesthat may have unexpected or unprecedented bioactivities. Herein, wecover the workflow of pseudo-natural product design and development,highlight recent examples, and discuss a cheminformatic analysis inwhich a significant portion of biologically active synthetic compoundswere found to be pseudo-natural products. We compare the concept tonatural evolution and discuss pseudo-natural products as the human-madeequivalent, i.e. the chemical evolution of natural product structure.Natural productsare the result of Nature’s explorationof biologically relevant chemical space through evolution and an invaluablesource of bioactive small molecules for chemical biology and medicinalchemistry. Novel concepts for the discovery of new bioactive compoundclasses based on natural product structure may enable explorationof wider biologically relevant chemical space. The pseudo-naturalproduct concept merges the relevance of natural product structurewith efficient exploration of chemical space by means of fragment-basedcompound development to inspire the discovery of new bioactive chemicalmatter through  This is a challenging endeavorsince the possible number of compounds that may qualify as bioactivesmall molecules is so vast that the time and resources needed to synthesize,let alone biologically evaluate, exceed reality.3 Therefore, there is a need for methods and sources of inspirationthat can help navigate through chemical space and focus on areas thatare biologically relevant.4The importance of smallmolecules that can perturb biological systemsin a controlled fashion is underpinned by their successful applicationsin chemical biologyThroughevolution, Nature has been exploring chemical space whichis reflected in secondary metabolites or natural products (NPs). Viaenzymatic cascades, organisms produce NPs that can carry out specificbiological functions either within and/or between organisms to givethe producing organism a selective advantage. Based on the demandsof the environment, i.e. selectivity pressures, NPs with differentmolecular scaffolds can be produced that modulate different targetsdirectly or indirectly to provide the organism with a higher levelof fitness. Therefore, NPs reflect organisms’ adaptations totheir environments through evolution and Nature’s explorationof biologically relevant chemical space. Accordingly, NP structurecan be considered biologically prevalidated.5 however, NPs havebeen subjected to evolutionary constraints. Natural evolution itselfis very slow and, in tandem with selection pressures, has resultedin NPs occupying only a fraction of theoretical NP-like chemical space.6 These constraints may have led to many knownNP scaffolds being highly conserved and therefore the diversity ofscaffolds readily accessible from Nature to be limited. Additionally,there are undoubtably numerous biologically intriguing NPs that havenot yet been identified. Therefore, solely relying on Nature as asource of biologically active molecules brings about limitations.NPs have been andcontinue to be a reservoir for successful moleculardiscovery programs;7Attempts to override the limitations of Nature and expand beyondthe chemical space occupied by NPs to provide new small moleculeswith unexpected or unprecedented bioactivities need to face the questionhow biologically relevant chemical space can be explored efficiently?8 Diversity-oriented synthesis (DOS) provides chemically diverse librariesenriched in variations of scaffolds, stereochemistry, and appendagesto quickly probe larger amounts of chemical space.9 A combinatorial approach utilizing DNA encoded libraries(DEL) has emerged as a method for the generation of relatively largelibraries with chemical diversity.10 WhileDOS and DEL can generate expansive libraries that can quickly explorediverse chemical space, more recently in tandem,11 and have provided biologically valuable compounds,13 much of the space explored may not necessarily be biologically relevant.Several chemical design strategies havebeen developed for thesearch of biologically active compounds.15 have reduced complexity relative to theparent NP while those from dynamic retrosynthetic analysis proposedby Shenvi et al.17 retain the intrinsic complexityof the parent NP while minimizing synthetic complexity. While bothmethods can yield compounds with similar or enhanced biological properties,the chemical and biological space explored is narrow and usually similarto the parent NP’s.Several approaches prioritize exploring more focused areas of chemicalspace that are occupied by NPs of biological interest. Access to thisspace is facilitated by the design of more synthetically tractablederivatives relative to the parent NP. Compounds resulting from function-orientedsynthesis19 Biology-oriented synthesis (BIOS)is based on the principle that proteins and NPs have coevolved toencompass only a small portion of chemical space.22 While appendagesof NPs can be highly diverse, the core scaffolds of NPs are conservedas are the corresponding binding pockets of proteins. Using this logic,BIOS employs hierarchical classification to identify simplified NPcore structures that may retain biologically relevant characteristics.23 Decoration of these scaffolds has led to severalbiologically active compound collections that retain relevance toNPs but are more synthetically tractable .ructures 1b. This 44 In contrast, the new arrangements of NP fragmentsin the resulting pseudo-NPs are intended to explore new areas of biologicallyrelevant chemical space and have biological targets that are unrelatedto the NP fragments and NPs from which they are derived.Notably, the biological goal of pseudo-NPdesign differs from thesynthetic NP-hybridization strategy. The NP-hybridization combinespharmacophores of two NPs that have different but synergistic biologicaleffects. The resulting chimeric molecules can have enhanced therapeuticvalue but most likely retain the mode of actions and/or biologicaltargets of the two parent NPs.Inthis perspective, we discuss the design principles and biologicalevaluation of pseudo-NP collections as well as highlight recent examples.We discuss that, through cheminformatic analysis, pseudo-NPs havebeen made unintentionally for decades and comprise a significant portionof synthetically made bioactive small molecules. We then tie our studiesof pseudo-NPs to the concept of chemical evolution, and argue thatthe method can be considered the manmade equivalent of NP structureevolution. Taken all together, we argue that the pseudo-NP conceptis a valid principle for the wider exploration of biologically relevantchemical space.3 atoms and abundant stereogenicityobserved in NP structure.46 On the other hand,pseudo-NPs should have a degree of chemical distinguishability sincefragment combinations and/or orientations in pseudo-NPs are distinctfrom NPs.The general design principle of pseudo-NPsrevolves around thecombination of NP fragments to arrive at scaffolds that resemble NPsbut are not possible through known biosynthetic pathways. Pseudo-NPsshould share some chemical similarities with NPs in that they areboth comprised of NP fragments and should therefore resemble the relativelyhigh fraction of sp47 for fragments may not be entirely valid forNP fragments.48 Therefore, NP-like fragmentscan be considered to have an AlogP < 3.5, a molecular weight between120 and 350 Da, ≤ 3 hydrogen bond donors, ≤ 6 hydrogenbond acceptors, and ≤6 rotatable bonds.35 Within these criteria, NP fragments can include fully syntheticfragments, NPs that are themselves fragment-sized, and products resultingfrom the distortion or fragmentation of NPs  to ensurethe exploration of new chemical space. It should be considered thatcriteria typically employed by the “rule of three”n of NPs 2a.49 Sui33 A further degree of scaffold diversificationis possible by combining similar fragments with similar connectivitypatterns but having different regioisomeric arrangements. Taken together,the combination of different NP fragments with different connectiontypes and/or different regioisomeric arrangements may lead to chemicallyand biologically diverse pseudo-NP collections.Chemically diverse pseudo-NP collections can be designedby usingdifferent combination strategies 2b. Novel51 We have successfully employed different cell-basedphenotypic assays to identify pseudo-NPs that affect important cellularprocesses or signaling cascades, including monitoring glucose uptake,autophagy, Wnt and Hedgehog signaling, T-cell differentiation, andinduction of reactive oxygen species.Since their scaffoldsare novel, pseudo-NPs may lead to new chemotypesfor known biological targets or to the discovery of unknown biologicaltargets. To efficiently probe broad areas of biological space, biologicalevaluation of pseudo-NPs should employ unbiased, target-agnostic assays.53 In the Cell PaintingAssay, images of treated cells are acquired through fluorescent microscopyfollowed by the extraction of several hundred features to providea characteristic “fingerprint” that encompasses thecells’ morphological changes. Compound-induced morphologicalperturbation can be quantified as an “induction value”by calculating the number of significantly changed features relativeto a DMSO control. Compounds that have suitable induction values areconsidered bioactive. The fingerprints can also be compared to referencecompounds with known bioactivities to generate target/modes of actionhypotheses or compared within and between compound collections forbiological performance.Beyond phenotypic assays,morphological profiling via the “CellPainting Assay” can be used to evaluate compound-induced morphologicalchanges in the entire cell.43 Dimerized NPs are typicallycombined via a monopodal connection and retain resemblance to thecore scaffolds of the monomeric NPs, such as thiomarinol and vincristine.The structure of thiomarinol resembles the combination of biosyntheticderivatives of the antibiotics pseudomonic acid C and holothin ,scaffold 3c.61−64 patterns 3d.65Where these two approaches diverge is in the preparationof newmolecules. Nature synthesizes NP hybrids through enzymatic reactionsthat are summarized as biosynthetic pathways. Through evolution, newbiosynthetic pathways can arise that may yield new NP hybrids. Onthe other hand, pseudo-NPs are designed by deconstruction of NPs toNP fragments through cheminformatic analyses. De novo recombinationsare envisioned and synthesized via chemical reactions that may notoccur in Nature.Both methods may produce biologically relevantchemical matter;however, evolution of biosynthetic pathways in Nature to provide newmolecules is slow while synthesis allows this process to be acceleratedand more flexible. By employing chemical synthesis, reaction pathwayscan be employed that are not possible through biosynthesis resultingin new combinations and connectivities of NP fragments. Therefore,the pseudo-NP concept does not necessarily compete with NP hybridizationbut rather complements it and explores areas of biologically relevantchemical space that are not possible through existing biosyntheticpathways.H-pyranoindoleand the fragment-sized NP griseofulin to arrive at indofulvins (66 A collection of 19 indofulvins was obtained by employingan iso-oxa-Pictet Spengler (IOPS) reaction as the key step to combinethe two fragments with a fusion spiro connection pattern. While thePictet Spengler reaction is present in biosynthetic pathways, itsiso-version is not currently in the biosynthetic repertoire, resultingin a novel arrangement of the 4H-pyranoindole fragment.Arecent example of the pseudo-NP conceptcombined the indole-containing fragment 4ofulvins 4a.66 A cindofulvin 1 being the most potent compound (IC50 = 0.82 μM). While indofulvin 1 inhibitedautophagy and was validated at a protein level, neither griseofulvinnor the 4H-pyranoindole fragment of indofulvin1 affected autophagy , representing NPs, and synthetic,non-natural compounds in the ChEMBL database, representing syntheticbioactive compounds, were cheminformatically analyzed and compared.Analysis of curated DNP andChEMBL databases revealed that bothhave a similarly high percentage of compounds that contain two ormore NP fragments . Furthermore, 344,394(21%) compounds in the curated ChEMBL data set fit the definitionof pseudo-NPs. These pseudo-NPs are predominantly comprised of 2–4NP fragments that are combined by 5 different connectivity patterns.Overall, the ChEMBL pseudo-NPs are highly diverse and have 117,000unique scaffolds with <100 members per scaffold.Since thereis a high proportion of pseudo-NPs in the ChEMBL database,a literature search was conducted to see their historical trends.It was found that pseudo-NPs have been made continuously for over40 years, and that the percentage of pseudo-NPs in published structuresper year has been increasing over time. These studies indicate thata significant portion of bioactive synthetic compounds are pseudo-NPsand have been synthesized without the pseudo-NP concept as a guidingprinciple, further adding validation to the overall concept.70 On the other hand, the DNPset is enriched in alicyclic and oxygen-containing ring systems withonly one aromatic fragment in the top 10  NP fragments observed in the ChEMBL pseudo-NPsare nitrogen- and sulfur-containing heterocycles, with more than halfof them being aromatic 6a.70 On e top 10 6b. Trende top 10 6c.SI Figure 1). NPs are more enriched in oxygencontent while ChEMBL pseudo-NPs have a higher abundance of nitrogenatoms. Furthermore, the fraction of sp3 hybridized atomsin NPs is significantly higher than ChEMBL pseudo-NPs. NP-likenessscores71 of the two data sets were calculatedin which a higher value indicates the set is more NP-like. Even thoughthe ChEMBL pseudo-NPs are comprised of NP fragments, the NP-likenessscore was significantly lower than that for the DNP data set. Thedifferences in molecular features and NP-likeness scores may reflectthe frequency with which fragments are used and how they are combined.The realization of these trends may aid in the rational design offuture bioactivity-enriched pseudo-NP collections and is expandedupon in the The molecular featuresalso varied significantly between data sets. The totalityof genetic information or hereditary makeup of an organism is called genotype. It is translated into a totality of characteristics,called phenotype, that results after expression ofgenetic information in the form of proteins. These enable a wide repertoireof functions because of their ability to fold into three-dimensionalstructures and having a greater spectrum of specific chemical propertiesthan nucleic acids.Natural evolution relieson chemicalinformation, and molecular structures are depicted (or encoded) by two-dimensional representations and sometimesby additionalcodes such as SMILES. Additionally, a molecule can be described bya number of variables which include the microenvironment of everysingle atom such as the number of its valence electrons, bonds, non-Hatoms, and specific symmetry properties. The chemical informationon this molecule then results from the sum of these variables . The term “information”,however, is of value only with the assignment of an information contentor a meaning. In Nature, the meaning of a gene becomesevident after translation into a protein with the ability to performa defined task, for example, as an enzyme. A protein will affect thequality of life, or fitness, of an organism whichis continually evaluated by natural evolution. For small molecules,two-dimensional projections correspond to structures with spatialand electronic properties; these express a meaning based on the factthat they can interact  and reactwith other molecules. These functions may be observede.g. with pharmacological or biochemical properties such as inhibitoryactivity (Ki) or substrate quality (KM) and,similar to the function of a protein, be targeted by evolution—inNature or experiment.74 Evolution of smallmolecule structure and function during chemical synthesis, however, requires that thechemical information describing its properties and its potential tointeract can be inherited to derivatives and undergo mutation, i.e. by modification of its molecular symbols. In this sense, chemical information is geneticinformation. In congruence with definitions for “genotype”and “phenotype” that were originally derived for a moleculardescription of evolution by M. Eigen,75 the structure of a molecule can be viewed as its genotype, and the function it performs as its phenotype .As a result, 74 novel structures were identified of which more than75% had not been described previously. Analysis revealed that somecombinations of substructures emerged for the first time while othermolecules showed known substructure combinations but with new connectivities. It can be concluded that the recombinationof biosynthetic pathways, as shown in this study, leads to new linkagesof the symbols  and thereby to the encoding ofnew information by new structures. A similar statement has been madeby Shenvi for new encoding by chemical synthesis.73An example for accessing previously unknown NPs that combine knownfragments in ways not observed before was recently demonstrated ina landmark study that employed genetic material encoding enzymes andenzymatic cascades from a collection of natural sources 8.77 GeneNature’s potential to mutate, recombine, and select thatleads to hybrid NP structures as described above can be combined withinsights into the informational background and biosynthetic mimicryof evolutionary processes. To meet this goal, NP fragments can beconsidered as building blocks with inheritable genetic informationwhich can not only be combined according to “Nature’sstate-of-the-art” but also by use of a repertoire of syntheticstrategies. Thereby, new and unprecedented connectivities may be explored,which may not be possible solely based on natural (biosynthetic) reactions.This requires reaction mechanisms that reflect all possible electronicand geometric degrees of freedom of the respective fragments.The pseudo-NP strategy requires that NP fragments (= inheritablegenotypes) are varied by mutation  andrecombination (fragment assembly). In a combinatorial synthetic setting,the pool of fragments will be combined and result in a pool of PNPswith new three-dimensional structures that open up new possibilitiesto recognize, bind, and react (= new phenotypes). These can be testedfor novel or desired bioactivities using appropriate target moleculesor cellular systems then identified and categorized with regard tostructure–activity-relationships, mode-of-action, and putativetargets. The outcome of such a cycle of variation (mutation) and identification(selection) is, besides the new molecule(s), new chemical and biologicalinformation that can be used as input for another cycle 9. In con33 however, it may be possible to improve the iterativeoptimization strategy by greater involvement of evolutionary aspectsby (1) including the target molecule of interest for the directselection of active ligands or inhibitors and (2) allowingfor reversible and competitive product formation in compound libraries.Dynamic combinatorial chemistry (DCC) provides for libraries capableof responding to target-binding events by changing their composition,thus offering a chance to improve signals for detection. In the contextof pseudo-NP synthesis, DCC will start from pools of soluble, reasonablystable NP fragments (building blocks) that react in a single compartmentto form an adaptive library—provided that appropriate functionalgroups and reversible reaction types are available that give accessto continually interchanging adducts. In the presence of a target,such a dynamic process allows for the amplification of active molecules within the library, in the sense of a “self-screeningprocess” that was already suggested by J.-M. Lehn synthesis which enables the generation and selection of large-scaledynamic libraries on the basis of DNA-mediated assembly, and facilitatesidentification of individual compounds by deciphering of a DNA tagwhich encodes building blocks and reaction steps. Substantial progressin this field81 may also provide for a basisto dynamic combinatorial libraries of future pseudo-NPs.Although DCC has alreadybecome important for biomedical research,it is limited by low library diversity and missing tools for the analysisof large libraries.Thepseudo-NP concept is intended to overcome limitations that are inherentto other NP-inspired molecular design principles and explore new areasof biologically relevant chemical space. The concept merges the biologicalrelevance of NPs with the facile exploration of chemical space offragment-based discovery by combining NP fragments to obtain scaffoldsthat retain relevance to NPs but are not possible through currentbiosynthetic pathways.39 as well as combined the same two NP fragmentsbut with different connectivity patterns and/or fragment orientations.40 Evaluation of the pseudo-NPs by cheminformaticsand the Cell Painting Assay revealed that the compound sets were indeedchemically and biologically diverse and supports the hypothesis thatdifferent fragment combination strategies can lead to diverse compoundcollections.The pseudo-NP scaffolds can be made upof the combination of NP fragments that are synthetically made, fragment-sizedNPs, or derived from NPs by ring distortion or degradation. Scaffolddiversity can be achieved through combining different NP fragments,fusing fragments together with different connectivity patterns, and/orhaving fragments in different arrangements. We recently investigatedwhether different pseudo-NP fragment combination principles  areactually pseudo-NPs.Froma conceptual perspective, the pseudo-NP workflow representsa chemical evolution of NP structures. Genotypes encoded in biologicallyrelevant NP fragments can undergo “mutations” by chemicalalteration and recombination in unprecedented manners that may affordnew phenotypes in the form of pseudo-NP scaffolds that can interactwith biological systems. “Selection” of pseudo-NPs viabiological assays may then lead to further iterations of the cycleto provide more optimal or “evolved” chemical and biologicalinformation.34 the pseudo-NPconcept has combined fragment-based discovery with the biologicalrelevance of NPs to guide the design of biologically unprecedentedcompounds. The logic of the concept represents a human-made equivalentto natural evolution and can be viewed as the chemical evolution ofNP structure. Taken all together, the pseudo-NP approach representsa validated design principle for the wider exploration of biologicallyrelevant chemical space.As highlighted here and elsewhere,3 and nitrogen-rich).89 Accordingly, it is recommended that future pseudo-NP collectionsshould not focus on combining solely nitrogen-rich and/or aromaticNP fragments, but deviate to incorporate a balance of saturated oxygen-containingand/or aliphatic NP fragments with a maximum total of four NP fragments.Furthermore, NP fragment connectivity patterns should not be so heavilyenriched by monopodal connections . This isclear from the large number of bioactive molecules that contain orare exclusively composed of purely synthetic fragments.94 These man-made fragments in bioactive compounds should thereforealso be considered biologically relevant. The evolutionary algorithmof the pseudo-NP concept (The initial design of the pseudo-NP concept is to employNPs asstarting points since they are biologically prevalidated compoundsselected through natural evolution, as are their fragments. concept 9 may theThe pseudo-NP design criteriaprovide numerous opportunities formolecular discovery programs. Nevertheless, multidisciplinary investigationsand advancements are needed to facilitate the exploration of the biologicallyrelevant chemical space that can be accessed by pseudo-NP structure.From a cheminformatic perspective, a further understanding of thedifferences between how Nature and chemists have selected which NPfragments to combine and how to combine them may aid in the designof future compound collections. At the core of accessing new pseudo-NPsare novel fragment combinations through organic synthesis which willheavily rely on the use and development of complexity-generating methodologiesenvisioned by organic chemists. Furthermore, the identification ofbiological relevance of future pseudo-NPs will be underpinned by thecontinuous development of broad, high content assays and new techniquesfor target identification by biologists and chemical biologists. Throughmultidisciplinary collaboration and advancements, the design, synthesis,and biological evaluation of new pseudo-NPs may help navigate biologicallyrelevant chemical space to provide compounds that will be beneficialto chemical biology and medicinal chemistry programs.


In fetal aortic stenosis (AS), it remains challenging to predict left ventricular development over the course of pregnancy. Myocardial organization, differentiation and fibrosis could be potential biomarkers relevant for biventricular outcome. We present four cases of fetal AS with varying degrees of severity and associate myocardial deformation on fetal ultrasound with postmortem histopathological characteristics. During routine fetal echocardiography, speckle tracking recordings of the cardiac four-chamber view were performed to assess myocardial strain as parameter for myocardial deformation. After pregnancy termination, postmortem cardiac specimens were examined using immunohistochemical labeling (IHC) of key markers for myocardial organization, differentiation and fibrosis and compared to normal fetal hearts. Two cases with critical AS presented extremely decreased left ventricular (LV) strain on fetal ultrasound. IHC showed overt endocardial fibro-elastosis, which correlated with pathological fibrosis patterns in the myocardium and extremely disturbed cardiomyocyte organization. The LV in severe AS showed mildly reduced myocardial strain and less severe disorganization of the cardiomyocytes. In conclusion, the degree of reduction in myocardial deformation corresponded with high extent to the amount of pathological fibrosis patterns and cardiomyocyte disorganization. Myocardial deformation on fetal ultrasound seems to hold promise as a potential biomarker for left ventricular structural damage in AS. Fetal aortic stenosis (AS) is a rare congenital heart disease comprising 0.2–0.5 of 1000 live births. It is known for its wide spectrum of clinical severity and difficulty to predict the clinical course in pregnancy and after birth. Even the mildest forms, characterized by only a subtle flow acceleration across the aortic valve at mid-gestation, may develop into a life-threatening critical AS at birth, carrying a high risk of morbidity and mortality ,4,5,6. FMahtab et al. demonstrated that borderline left ventricles (BLV) with similar macroscopic appearances can show great variety in microscopic ventricular organization and differentiation. In particular, a subset of BLV hearts resembled normal hearts, containing a well-organized cardiomyocyte alignment and proper organized intercalated disks with a normal expression pattern of important myocardial differentiation markers such as cardiac troponin-I, N-cadherin, and Connexin43 (Cx43). In contrast, the remaining BLV hearts presented histologic abnormalities similar to HLHS hearts, showing disturbed cardiomyocyte and intercalated disks organization along with an almost completely absent expression of myocardial differentiation markers. Ventricular organization and differentiation could therefore be a predictor of biventricular outcome . In fetaNormal myocardial organization and differentiation allows a coordinated contraction of the myocardium via effective cell–cell coupling, which is necessary for optimal cardiac function. Endocardial damage, such as endocardial fibro-elastosis, also affects myocardial contraction. A non-invasive tool to assess myocardial wall motion in vivo comprises the novel echocardiographic technique speckle tracking. Speckle tracking quantifies the deformation of the myocardial tissue by following the motion of certain myocardial regions, the so-called speckles, over sequential ultrasound frames .We hypothesized that ultrasonographic myocardial deformation corresponds with the level of myocardial organization, differentiation and fibrosis and is therefore an in vivo marker of ventricular damage in fetal AS. Studies assessing the correlation between prenatal imaging and histology are extremely rare. Here, we present four cases of aortic stenosis diagnosed in utero with varying degrees of severity and compare the myocardial deformation based on prenatal ultrasound to postmortem histopathologic characteristics.This study was performed in accordance with the Dutch regulation for the proper use of human tissue for medical research purposes and conducted according to the principles of the Declaration of Helsinki  and Dutch Fetal Tissue Act. The protocol was approved by the local Ethics Committee (NL66978.058.18).An overview flowchart of the methods is depicted in n = 3) gave signed written informed consent to donate cardiac tissue for immunohistochemical analysis. Two women donated the entire heart specimen and one woman gave permission to obtain heart biopsies taken from five segments . The whole cardiac specimens were fixated in formaldehyde followed by storage in ethanol until use and the cardiac biopsies were immediately embedded in paraffin according to standard protocols. Two normal heart specimens (one entire heart specimen and one left ventricle (LV)) of similar gestational age served as immunohistochemical control cases, obtained from legal abortion material.Written informed consent was received from the women to perform speckle tracking recordings during routine fetal echocardiograms. The recordings were analyzed offline, weeks after the initial examination and therefore did not influence clinical decision-making. All women who decided to terminate the pregnancy  using the Canon Aplio i-800 ultrasound machine with abdominal PVI475BX and PVT674 High Frequency convex transducers. For speckle tracking recordings, cine loops of the apical or basal cardiac four chamber-view with an insonation angle of <30° were stored in B-mode setting. We optimized settings by adjusting sector width, depth, gain and zoom box to achieve framerates between 60–120 frames/s. Each clip contained at least 4 cardiac cycles in absence of maternal and fetal movements and was repeated 3 times. A measurement software package  was used for offline analysis; detailed methods of the analysis have been described previously [eviously . In brieImmunohistochemical Analysis. All cardiac specimens were embedded in paraffin according to standardized protocols. Frontal sections of 5 μm from whole hearts and 5 μm sections from the segmental biopsies were mounted on KP-Silane adhesive glass slides (Klinipath). Immunohistochemical and immunofluorescence staining was performed as described previously on the sections comprising the cardiac four chamber view and long-axis view  [ct view) . All useCase 1 was diagnosed with a fetal critical AS at 20 + 3 weeks of gestation that met the international criteria for in utero aortic valvuloplasty  11]. Th. Th11]. Case 2 presented at our fetal cardiology service at 19 + 5 weeks of gestation, revealing a critical AS with postvalvular dilatation . The LV Case 3 presented with a severe fetal AS showing seriously increased flow velocity (350 cm/s) across the aortic valve at 20 + 1 weeks gestation. The international criteria for in utero aortic valvuloplasty were not met  as the fCase 4 was referred to our fetal cardiology service with an abnormal three vessel view, based on an enlarged aorta, at 21 + 1 weeks GA. We diagnosed a severe AS with an adequate size of the aortic annulus showing increased forward flow across the valve (300 cm/s) and postvalvular dilatation with turbulent flow . As the Speckle tracking values of all cases are depicted in The two cases with severe AS (case 3 and 4) had normal LV appearance by eyeballing, but the left ventricular myocardial wall deformation showed a mild reduction . Right ventricular myocardial deformation was overall normal with a GLS of −17.6% and −21.2%. Again, in all severe AS cases the lateral side of the RVs showed slightly increased SLS values (SLS between −15.3% and 30.0%) and the septal side an overall mild reduction (SLS between −6% and −29.3%).In normal hearts, the cardiomyocytes of both the LV and RV were well-aligned and showed a proper banding pattern. Cx43 and N-cadherin were found as a diffuse pattern in the cytoplasm and were also expressed with more intensity at the lateral borders of the cardiomyocytes, illustrating the presence of gap and adherence junctions. Furthermore, some early intercalated disks were present at the longitudinal ends, mostly solely expressing N-cadherin but co-expression of N-cadherin and Cx43 was also found a–d.In contrast, the LVs of the two cases with critical AS  staining exposed a homogenous pattern of interstitial fibroblasts, without signs of pathological fibrosis a–d. The Strikingly, in contrast to normal hearts, the subepicardium of both LV and RV was significantly thickened in all AS cases, suggesting epicardial activation. The subepicardium contained marked dilated coronary vessels which were surrounded by large TUBB3 positive nerves, while only a few nerve cells were seen in areas without vasculature . Next toFetal AS remains a challenging CHD considering its difficulty to predict left ventricular development over the course of the pregnancy and with that, the postnatal outcome. Myocardial organization, differentiation and fibrosis could be potential biomarkers, relevant for biventricular outcome . The keyMyocardial wall deformation in relation to histopathology. In the current study, fetal AS cases with the most pronounced disturbance of the cardiomyocyte network and endocardial fibrosis patterns showed the utmost impairment of myocardial wall deformation on fetal ultrasound , which changes gradually towards complete localization at the lateral borders and intercalated disks during prenatal and postnatal development . One couPathophysiology of endocardial fibro-elastosis in aortic stenosis. Altered hemodynamics in AS trigger the formation of endocardial fibro-elastosis via myocardial hypoxia [ hypoxia ,42. In t hypoxia . This suThe origin of the accumulated fibroblasts in endocardial fibro-elastosis is still debated, where both endocardium-derived cells (EndMT) or epicardial-derived cells (EMT) have been proposed ,24. EndMRelation to development of HLHS. Remarkably, the degree of cardiomyocyte disorganization in the critical AS cases was identical to the HLHS specimens described by Mahtab et al. [b et al. , which cb et al. . Of noteRight ventricular involvement in a left ventricular disease. The RV has long been neglected in this typical left-sided heart disease. However, there is growing evidence that typical one-sided CHDs are in fact biventricular diseases [diseases . The RV Although the current study material is unique and provides relevant insights in the pathophysiology of fetal AS, the number of studied specimens is too limited for statistical evidence. However, this case series shows that prenatal myocardial function associates to high extent with myocardial histology as the degree of the reduction in myocardial wall deformation on echocardiography corresponded with the histological amount of pathological fibrosis patterns and disorganization of the cardiomyocyte network. Therefore, fetal echocardiography, in particular GLS, seems to hold promise as a potential biomarker for structural myocardial damage. Current promising sophisticated genetic techniques, such as spatiotemporal single-cell RNA sequencing , may pro


The success of ocrelizumab in reducing confirmed disability accumulation in primary progressive multiple sclerosis (PPMS) via CD20-targeted depletion implicates B cells as causal agents in the pathogenesis of PPMS. This review explores the possible mechanisms by which B cells contribute to disease progression in PPMS, specifically exploring cytokine production, antigen presentation, and antibody synthesis. B cells may contribute to disease progression in PPMS through cytokine production, specifically GM-CSF and IL-6, which can drive naïve T-cell differentiation into pro-inflammatory Th1/Th17 cells. B cell production of the cytokine LT-α may induce follicular dendritic cell production of CXCL13 and lead indirectly to T and B cell infiltration into the CNS. In contrast, production of IL-10 by B cells likely induces an anti-inflammatory effect that may play a role in reducing neuroinflammation in PPMS. Therefore, reduced production of IL-10 may contribute to disease worsening. B cells are also capable of potent antigen presentation and may induce pro-inflammatory T-cell differentiation via cognate interactions. B cells may also contribute to disease activity via antibody synthesis, although it's unlikely the benefit of ocrelizumab in PPMS occurs via antibody decrement. Finally, various B cell subsets likely promulgate pro- or anti-inflammatory effects in MS. Multiple Sclerosis (MS) is the most prevalent chronic demyelinating disorder of the central nervous system (CNS) affecting more than 2 million people worldwide and over 700,000 people in the United States . There aB cells have been implicated in the pathology of MS through the presence and diagnostic significance of oligoclonal bands –11, an iThe pathology of PPMS and SPMS are characterized by widespread diffuse inflammation with slowly expanding lesions, abundant cortical demyelination, brain atrophy, and lymphocyte infiltration and microglial activation in normal appearing white matter . In contThere are now over 20 FDA approved disease modifying agents for MS, with one designated by the FDA as an effective option for PPMS. Ocrelizumab is the only FDA approved medication for PPMS, having been approved in early 2017 . OcrelizThe presence of unique oligoclonal bands (OCBs) and increased intrathecal IgG synthesis by antigen experienced B cells has long been recognized as a component of MS . OCBs ar+ B cells and CD83+ B cells , which compartmentalizes the CNS and restricts leukocyte migration into the brain and spinal cord. Historically, the CNS was believed to be an immune-privileged site, but recent evidence has demonstrated a steady trafficking of memory T cells between the periphery and the CNS . It is hFurther studies looking exclusively at PPMS found no proof of ELFs but instead evidence of more widespread disorganized leptomeningeal inflammation . In an aGiven the correlation between both ELFs in SPMS and widespread disorganized leptomeningeal inflammation in PPMS with adjacent cortical pathology, it is possible that leptomeningeal inflammation is an independent driver of disability, particularly in progressive MS , 17. HowThe presence of unique oligoclonal bands in the CSF of MS patients led to the hypothesis that B cells could be contributing directly to MS pathogenesis via autoantibody mediated CNS tissue damage . This idIt should be emphasized that no specific self-antigen has yet been identified that has consistently been verified as an autoantibody target in MS . EvidencClinical data from anti-CD20 treatment of patients with MS argues against a link between treatment benefit and antibody production. B cells down-regulate CD20 expression as they develop into plasma cells and thus mature plasma cells secreting antibodies do not express CD20 . TherefoB cells exert both pro-inflammatory and anti-inflammatory effects depending on distinct cytokine production . B cellsPatients with RRMS and SPMS have a dysregulated cytokine network, specifically demonstrating a decrease in the anti-inflammatory cytokine IL-10 . B cellsLTα is secreted by B and T cells and binding of membrane bound LTα to follicular dendritic cells induces CXCL13 production . CXCL13 Murine EAE is a commonly used animal model that has been used to decipher the immunopathogenic mechanisms of MS and devise novel therapies . EAE is in vitro, partly through IL-6 signaling  and APRIL (A PRoliferation-Inducing Ligand) acting as an antagonist to these ligands and inhibiting receptor activation. These two cytokines are important for B-cell maturation, function, and survival. Atacicept has selective effects on B cells, depleting plasma cells and late stage B cells while sparing B-cell progenitor cells and memory B cells . AtaciceIn a randomized double-blind, placebo-controlled trial of atacicept in patients with RRMS, patients who received atacicept had a higher annualized relapse rate compared to those receiving placebo . For thiex vivo memory B cells play a prominent role in inducing CD4+ self-reactivity, likely through a mechanism of antigen presentation (This hypothesis is supported by further data highlighting these distinct functions of plasma cells and memory B cells. In an EAE mouse model, plasma cells from the gut were found to play an anti-inflammatory role on neuroinflammation in EAE through the secretion of IL-10 . This fientation .Clinical evidence demonstrating that atacicept increases the rate of MS relapses, taken in conjunction with additional findings suggesting an anti-inflammatory role of plasma cells and a pro-inflammatory role of memory B cells, indicates that B cells can have both a pro and anti-inflammatory effect in MS depending on their specific clonal subset, causing either disease mitigation or progression, respectively.The success of ocrelizumab at reducing disability in PPMS, in the context of previous failures of other anti-inflammatory drugs approved for RRMS, in particular the anti-CD20 monoclonal antibody rituximab, raises important questions about the specific mechanisms by which ocrelizumab exerts its therapeutic benefit. One hypothesis put forth regarding the success of ocrelizumab and failure of rituximab derives from phenotypic differences in the types of PPMS patients enrolled in each study. Rituximab and ocrelizumab are both CD20 monoclonal antibodies. CD20 is a cell surface marker expressed on most B cell subsets with the exception of early pro-B cells, late stage plasmablasts and terminally differentiated plasma cells . In clinA recent retrospective study examined the off-label use of rituximab in the treatment of PPMS and found that 41.5% of PPMS patients treated with rituximab had significant disease progression after 3 years . The pat+ B cells, from week 2 to 96 after rituximab treatment (Functional differences in the antibody structure of ocrelizumab compared to rituximab may lead to more favorable safety and tolerability profiles but are unlikely to significantly change the levels of circulating B cells in the periphery and CNS of treated PPMS patients. Rituximab is a chimeric antibody with an Fab domain derived from mouse protein, whereas ocrelizumab is exclusively derived from human protein . Comparereatment . Additioreatment . Anotherreatment . While iOcrelizumab is the first and only FDA approved disease-modifying therapy for patients with PPMS. The characteristics of patients treated in ORATORIO indicate that ocrelizumab likely exerts an anti-inflammatory effect with the most pronounced benefit occurring in younger PPMS patients with a high propensity for disease activity . This idAltogether, the above data indicates that ocrelizumab likely reduces disease progression in PPMS by reducing inflammation. This mechanism of action represents a continuation of the therapeutic paradigm used to treat RRMS in which the primary treatment modality involves drugs that work via reducing inflammation. The benefit of ocrelizumab but the failure of multiple other RRMS anti-inflammatory drugs, in conjunction with the phenotypic differences in PPMS compared to RRMS, has important implications about disease pathogenesis and treatment. Clinical trial data indicates that there is likely a subset of patients with PPMS, typically younger, newly diagnosed patients with gadolinium enhancing lesions on MRI, who have active inflammation contributing to their progressive disability who would benefit from a high potency anti-inflammatory medication. These qualitative differences in subgroups of PPMS patients have implications for the way we classify patients with PPMS. The recent revisions to the classification of MS to include new qualifiers for active disease and presence of progression represents an effort to further delineate PPMS into more clinically useful groups . ClinicaBroadening our understanding of disease pathogenesis in PPMS and harnessing that knowledge to develop new and effective treatments represents the next frontier in MS research. This goal carries with it unique challenges given the reduced prevalence of PPMS compared to RRMS and SPMS, making clinical trial recruitment more difficult. Additionally, the EAE mouse model, the most widely studied animal model for MS, is of questionable utility in PPMS given the lack of progressive MS pathologic features . DedicatJH drafted the manuscript. JH, RA, NM, and GW provided the content and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DNA double strand breaks (DSBs) are repaired in eukaryotes by one of several cellular mechanisms. The decision-making process controlling DSB repair takes place at the step of DNA end resection, the nucleolytic processing of DNA ends, which generates single-stranded DNA overhangs. Dependent on the length of the overhang, a corresponding DSB repair mechanism is engaged. Interestingly, nucleosomes—the fundamental unit of chromatin—influence the activity of resection nucleases and nucleosome remodelers have emerged as key regulators of DSB repair. Nucleosome remodelers share a common enzymatic mechanism, but for global genome organization specific remodelers have been shown to exert distinct activities. Specifically, different remodelers have been found to slide and evict, position or edit nucleosomes. It is an open question whether the same remodelers exert the same function also in the context of DSBs. Here, we will review recent advances in our understanding of nucleosome remodelers at DSBs: to what extent nucleosome sliding, eviction, positioning and editing can be observed at DSBs and how these activities affect the DSB repair decision. DNA double strand breaks are a highly toxic form of DNA damage, arising from intrinsic and extrinsic sources . EukaryoThe cellular DSB repair pathway decision is made at the step of DNA end resection, the nucleolytic processing of DSB ends . ResectiNucleosomes form obstacles to the resection nucleases . InitialNucleosome remodelers have received attention as regulators of DNA end resection and DSB repair pathway choice. These nucleosome remodelers are enzymes that in ATP-dependent fashion catalyze the breakage of histone-DNA-contacts within the nucleosome and translocate the DNA relative to histone proteins . All eukIn many eukaryotes, DNA end resection is carried out by three resection enzymes, Mre11-C, STR-Dna2 and Exo1, which act specifically at one of the two stages of the resection process initiate and thereby allow to bypass the nucleosome barrier. Currently, such an auxiliary role of Mre11-C in long-range resection lacks experimental support, but recent data suggest that short-range and long-range resection nucleases work in a coordinated fashion .Alternatively, resection enzymes will need assistance by chromatin remodelers to get past nucleosomes and it is therefore important to consider how these enzymes may be able to modify the nucleosome barrier.Eukaryotes express several nucleosome remodelers  and chroNucleosome remodelers are found to be either single protein enzymes or multi-protein complexes. Historically, four major sub-families of remodelers have been proposed , but phyIn vitro and in vivo we can discriminate at least three activities of nucleosome remodelers: 1) sliding/eviction leads to movement of nucleosomes along DNA that can even result in the removal of the entire nucleosome  posicleosome ; 3) editcleosome . Based ocleosome . In the in vivo , which cleaves preferentially non-nucleosomal DNA. Several studies that used MNase to investigate nucleosome localization around a single DSB showed eviction of DSB-proximal nucleosomes, but came to different conclusions as to whether DSB-distal nucleosomes would shift their position , ETL1 (from mouse) and SMARCAD1 (from human) are the prototypical members of a sub-family of nucleosome remodelers that is evolutionary conserved throughout eukaryotes . Historifun30 mutants scored similarly to mutants deficient in the long-range resection nucleases, when they were initially found in screens for resection-dependent repair of DSBs  in thisst cells . Rad9 orst cells , suggestst cells .In budding yeast cells lacking both Fun30 and Rad9, resection and nucleosome eviction are fully functional , suggestAlso human SMARCAD1 antagonizes 53BP1. Depletion of SMARCAD1 stabilizes 53BP1 around DSB sites . HoweverIn all, we think that previous studies collectively indicate that nucleosome remodelers may serve similar roles during DSB repair as during gene transcription with nucleosome eviction, editing and potentially even positioning taking place at DSBs. Knowledge of the specific activities of individual nucleosome remodelers and of their redundancies thereby offers the potential to get to grips with chromatin changes occurring at DSBs. Moreover, we think that studies of DSB resection and repair may be generally inspired by analogies to gene transcription. Both processes appear to be similarly affected by the presence of chromatin, with nucleosomes forming a dynamic barrier and nucleosome remodelers facilitating its bypass.Importantly, while nucleosomes clearly form a barrier to the resection nucleases, nucleosome remodelers equip cells with multiple ways to overcome this barrier. In this review, we have outlined several putative mechanisms of how bypass may occur. These include eviction, sliding and editing of nucleosomes. While we are still only beginning to understand how the nucleosome barrier is overcome, a key future question will be which bypass mechanism is chosen in which cellular scenario. Importantly, the nucleosome barrier and its dynamic nature offers additional possibilities to regulate resection and DSB repair. Moreover, critical factors of the DSB repair decision, such as 53BP1 and BRCA1, are proteins that bind and modify nucleosomes. Therefore, we propose that convergence of resection-regulatory pathways on nucleosomes is a central part of the cellular DSB repair decision.


Drosophila melanogaster, that developed on a low-, medium- or high-resource diet, manipulated via yeast content. We found that a low-resource developmental diet reduced the probability of aggressive lunges in adults, as well as threat displays against rivals that developed on a low-resource diet. These effects appeared to be independent of diet-related differences in body mass. Males performed relatively more aggression on a central food patch when facing rivals of a low-resource diet, suggesting that developmental diet affects aggressive interactions through social effects in addition to individual effects. Our finding that resource-poor developmental diets reduce male-male aggression in D. melanogaster is consistent with the idea that resource budgets mediate aggression and in a mass-independent manner. Our study improves understanding of the links between nutrition and aggression.Aggressive behaviours occur throughout the animal kingdom and agonistic contests often govern access to resources. Nutrition experienced during development has the potential to influence aggressive behaviours in adults through effects on growth, energy budgets and an individual’s internal state. In particular, resource-poor developmental nutrition might decrease adult aggression by limiting growth and energy budgets, or alternatively might increase adult aggression by enhancing motivation to compete for resources. However, the direction of this relationship—and effects of developmental nutrition experienced by rivals—remains unknown in most species, limiting understanding of how early-life environments contribute to variation in aggression. We investigated these alternative hypotheses by assessing male-male aggression in adult fruit flies, Significance statementDrosophila melanogaster, we show that a poor developmental diet reduces aspects of adult aggressive behaviour in males. Furthermore, males perform more aggression near food patches when facing rivals of poor nutrition. This suggests that early-life nutrition affects aggressive interactions through social effects in addition to individual effects.Early-life nutrition can influence social behaviours in adults. Aggression is a widespread social behaviour with important consequences for fitness. Using the fruit fly, The online version contains supplementary material available at 10.1007/s00265-021-03050-z. Aggression is widespread amongst animals  that develop on carbohydrate-rich diets  to vials containing food media  and incubated them until eclosion. For ease of diet manipulation, we modified a simple sugar-yeast-agar medium (yeast:sugar ratio of 2:1) to create a ‘low’ , ‘medium’  and ‘high’ resource media  containing a food patch of molasses-based media combined with yeast paste (5-mm diameter), a standard protocol for assessing aggression in  Dierick . In each Dierick . To avoiA single observer blind to treatment scored the videos using JWatcher , and weighed them again after they were dried for 48 h at 60 °C (dry mass).D. melanogaster . We expressed behavioural data as total durations (in seconds) or number of bouts of each behaviour. To test the influence of focal male diet, rival male diet and their interaction on focal male aggression, we first analysed lunging behaviour, the principal form of aggression displayed by male Wing threat behaviour is a non-contact threat component of male aggressive behaviour. We analysed the influence of developmental diet on the number of focal male wing threats using a negative binomial distribution.We then analysed the influence of developmental diet on total aggression duration, summed over lunging, fencing, tussling and wing threat, using a general linear model. Because our findings for total aggression differed from those for lunging, we secondarily explored total aggression further by conducting general linear models for the duration of fencing, and likelihood and duration of chasing independently. Chasing data were also zero-inflated , so we analysed the influence of diet on the likelihood of chasing in a binomial general linear model, and then on chase durations in the subset of flies that displayed chasing using a Gaussian general linear model. Because there were only two incidents of tussling, we did not analyse tussling separately. Because diet might affect locomotion, which might impact aggressive encounters, we assessed the influence of focal and rival diet on locomotion in a linear model.To assess the interdependence of behaviour of the two individuals in a pair, we used a chi-square test of independence to assess whether the likelihood of a focal male lunging was related to the likelihood of his rival lunging. We then assessed the influence of rival lunge number on focal lunge number in the subset of males that lunged, and the influence of rival total aggression duration on focal total aggression duration amongst all males in linear models. To test whether this relationship was influenced by developmental diet, we performed chi-square tests of independence for each focal developmental diet treatment separately, and we ran an additional model including focal and rival diet and their interaction with rival aggression on focal total aggression.D. melanogaster, food patches provide access to mates and males aggressively defend these sites , and sequential sums of squares analysis revealed no further effect of focal body mass after focal male developmental diet was accounted for . Within the low-resource treatment, focal males of a lower dry mass were less likely to lunge , although there was no within-treatment effect of wet mass . Within medium and high-resource treatments, focal mass had no influence on lunge probability .Our data do not support the hypothesis that high-resource males lunge more often solely as a consequence of their larger body mass, as focal male lunging probability was not related to focal male mass  and no interaction between focal and rival mass . Furthermore, sequential sums of squares analysis revealed no interaction between focal and rival body mass after focal and rival developmental diet were accounted for .Males of a high-resource developmental diet showed increased wing threat compared with males that developed on a lower-resource diet, but only when paired with rivals of a low-resource diet Table ; Fig. 2cThreat displays can represent strategies to settle contests without costly escalation. However, we found no evidence that wing threat reduced escalated fighting, as there was a positive correlation between lunge number and wing threat number . However, focal males performed relatively more of their aggression on the food patch (as opposed to off the food patch) when competing against rivals of a low-resource developmental diet . Although focal males displayed relatively more of their aggression on the food patch as rival mass decreased .We investigated how aggression related to access to the food patch because food patches represent valuable breeding sites for male 21 = 18.5, p < 0.001). We found some evidence that diet influences this relationship, because although focal and rival male lunging was related in the low- and medium-resource focal developmental diet treatments , we found no relationship the high-resource focal developmental diet treatment . Amongst those focal males that lunged, rival lunge number did not influence focal lunge number . Furthermore, the total duration of focal male aggression was not related to the total duration of rival aggression , and this was not influenced by differences in developmental diet .Because rival behaviour might influence focal male behaviour, we examined the relationship between the two. Chi-square analysis revealed that the probabilities of a focal and rival male performing at least one lunge are not independent  and wing threats . The influence of developmental diet on aggression appears above and beyond any influence of body mass. The developmental diet of rival males influenced where males performed aggression: focal males concentrated their aggression to the food patch when competing with low-resource rivals, suggesting that males may be better able to access food resources against nutritionally poorer rivals. Our findings suggest that adult D. melanogaster alter aggressive behaviour in light of their own developmental diet, through factors distinct from body mass, and that the nutritional experience of social partners also impacts contest characteristics.We found that developmental diet influenced adult male aggression in D. melanogaster Below is the link to the electronic supplementary material.


Epithelial downgrowth is a rare complication after Descemet stripping automated endothelial keratoplasty (DSAEK), which usually leads to poor visual outcome despite multiple available options of treatment.A 50-year-old man underwent DSAEK procedure due to pseudophakic bullous keratopathy. Three months later, the patient presented with gradual visual loss; slit-lamp examination revealed detachment and folding of the DSAEK lenticule, which was confirmed by anterior segment optical coherence tomography. On confocal scanning, epithelial cell sheets were detected in the interface leading to the wrinkling of the donor tissue and donor detachment. Surgical debridement and transient fixating with straight 10–0 prolene needles were performed followed by air injection into the anterior chamber. The cornea turned clear in the one-year follow-up with uncorrected-visual acuity of 20/30 and best-corrected visual acuity of 20/25.Early diagnosis and treatment of epithelial downgrowth may be associated with a good prognosis and prevent from more aggressive treatments such as repeat of grafting. In this case, mechanical debridement and transient fixation of lenticule by 10–0 prolene needles was performed to manage post-DSAEK epithelial downgrowth and lenticule detachment, which was successful without requiring of additional re-grafting. It seems this is a feasible technique with acceptable long-term outcomes.The online version contains supplementary material available at 10.1186/s12886-022-02264-7. Penetrating injuries and intraocular surgeries may be complicated with invasion of the epithelium into the anterior chamber, which is called epithelial downgrowth (ED) or ingrowth . MechaniTo the best of the author’s knowledge and database search, few cases of ED after DSAEK have been reported in the last decades. We report a case of lenticule detachment and epithelial downgrowth after Descemet Stripping Automated Endothelial Keratoplasty, which managed through surgical debridement and transient fixating of lenticule by straight 10–0 prolene needles not requiring a re-grafting.A 50-year-old man presented with pseudophakic bullous keratopathy after cataract surgery. His visual acuity was counting fingers at 1 m; the posterior capsule was intact with well-positioned intraocular lens (IOL) in the bag. The patient was scheduled for DSAEK surgery. In the DSAEK procedure, recipient corneal epithelium was removed and four venting incisions were made in the midperiphery of the cornea before Descemet stripping. The donor cornea was inserted using a Busin glide; after DSAEK lenticlue insertion, air injection was done up to the border of DSAEK lenticule. The surgical procedure resulted in a clear cornea. However, the patient presented with gradual visual loss three months later and the DSAEK lenticule was detached, non-mobile and folded confirmed by anterior segment optical coherence tomography (ASOCT)  , glaucoma filtering surgery, Descemet membrane endothelial keratoplasty (DMEK), and DSAEK may be complicated with ED . It seemPrevious studies have been shown that different risk factors such as several intraocular operations, poor wound closure, iris or vitreous incarceration, long-standing inflammation, corneal vascularization, and low intraocular pressure (IOP) may prone the eye to ED . MoreoveSubtypes of ED include epithelial sheets, pearls, and cysts. Sheet type is the most common type of ED as occurred in our patient. Also, this type is the most destructive type, which may cause vision loss through glaucoma and corneal endothelial failure . One stuThese patients usually turn symptomatic with decreased vision or pain. Non-specific features such as a hazy cornea, inflammation, and high IOP may make the diagnosis difficult. So, clinical suspicious can be helpful in these situations, especially in cases with history of previous ocular surgery. ED must be differentiated from iridocorneal endothelial (ICE) syndrome and metastatic carcinoma .The gold standard method of the diagnosis is a histological assessment, which can also differentiate between the corneal or conjunctival origin of ED through the presence of goblet cells in cases with conjunctival source. Also, argon laser photocoagulation has a diagnostic value, which shows whitening of burn spots on invaded iris surface whereas healthy areas appear dark. Other noninvasive diagnostic modalities include ASOCT, confocal scanning, and specular microscopy .Although there are some reports of spontaneous resolution of ED, various treatment options including surgical removal, cryotherapy, wide excision with adjunctive argon laser ablation, re-grafting through PK, DMEK, or DSAEK, and injection of antimetabolites like 5-FU into the anterior chamber are available. However, the failure rate of treatment is remarkable. It could be mentioned use of 5-FU was not successful in some cases . Informa2 was changed to 2109 cells/mm2.The unique aspect of our report is management of epithelial downgrowth without requirement to re-grafting. Unlike previous reports of epithelial downgrowth after DSAEK which led to re-grafting (re-DSAEK or PK) after a short duration despite primary management with debridement or using 5-FU, our patient is currently stable through our novel technique without re-grafting . It should be mentioned that the presence of epithelial sheets and diagnosis of ED was made using confocal scanning since unlike the typical cases of epithelial downgrowth, epithelial sheets were so fine in our complicated case leading to poor visibility through surgical microscope. It’s of note access and cleaning of epithelial sheets was done from venting incisions and interface scraping (but not excision) was tried to decrease the epithelial downgroth as less as possible. The authors have no claim regarding complete removal of epithelial sheets since this is not rationally possible and we think the reason of refractory nature of lenticule to repositioning is presence of remnant epithelial sheets. In this condition use of 10–0 prolene needles beside air injection was helpful in stretching of lenticule resulting in attachment. The concern of further endothelial damage by penetration of lenticule is logically right but it should be mentioned the needles have very tiny cross section and we believe endothelial damage following possible penetration was not significant. Our witness for this claim is comparison between preoperative and postoperative endothelial cell count (at 1st year of follow-up) in which 2327 cells/mmAt the time being, no study is available on direct comparison between outcomes of graft exchanging and debridement. Although this condition is so rare, future studies may be helpful to address the preferred option. Nevertheless, we deeply believe that giving a chance to the patients for prevention from re-grafting and its subsequent issues is a logical approach.In conclusion, early diagnosis and treatment of epithelial downgrowth may be associated with a good prognosis and prevent from more aggressive treatments such as repeat of grafting. In this case, mechanical debridement and transient fixation of lenticule by 10–0 prolene needles was performed to manage post-DSAEK epithelial downgrowth and lenticule detachment, which was successful without requiring of additional re-grafting. It seems this is a feasible technique with acceptable long-term outcomes.Additional file 1: Video 1. Shows the used surgical technique.


The final biochemical and mechanical performance of an implant or scaffold are defined by its structure, as well as the raw materials and processing conditions used during its fabrication. Electrospinning and Additive Manufacturing (AM) are two contrasting processing technologies that have gained popularity amongst the fields of medical research i.e., tissue engineering, implant design, drug delivery. Electrospinning technology is favored for its ability to produce micro- to nanometer fibers from polymer solutions and melts, of which, the dimensions, alignment, porosity, and chemical composition are easily manipulatable to the desired application. AM, on the other hand, offers unrivalled levels of geometrical freedom, allowing highly complex components  to be built inexpensively within 24 hours. Hence, adopting both technologies together appears to be a progressive step in pursuit of scaffolds that better match the natural architecture of human tissues. Here, we present recent insights into the advances on hybrid scaffolds produced by combining electrospinning (melt electrospinning excluded) and AM, specifically multi-layered architectures consisting of alternating fibers and AM elements, and bioinks reinforced with fibers prior to AM. We discuss how cellular behavior  is influenced by the co-existence of these micro- and nano-features. Electrospinning is a nanofabrication technique based on the use of high electric voltages to produce polymeric fibers with micro-to nanometer diameters and distinct morphological features . A typicThe biomedical sector particularly benefits from electrospinning for the development of advanced systems that are relevant to tissue engineering and drug delivery . One maiStrategies to further enhance the relevance of electrospun materials for tissue engineering applications have recently led to novel three-dimensional (3D) scaffolds with multi-scale hierarchical architectures . These hThis review focuses on recent advances in hybrid scaffold fabrication through the combination of electrospinning (melt electrospinning excluded) and additive manufacturing, and discusses how cellular behavior, such as attachment, migration, and differentiation, is influenced by the co-existence of micro- and nano-features.In the literature, two main fabrication approaches that combine electrospinning and additive manufacturing have been reported so far to produce scaffolds for tissue engineering . These ain-vivo tests. Animal tests showed that the presence of keratin promoted would closure, epithelialization, proliferation of both keratinocytes and fibroblasts, and collagen deposition at the wound site. In this work, fiber’s diameter was effectively used to control cellular growth, and promote the formation of distinctive layers of keratinocytes and fibroblasts by stopping fibroblasts to infiltrate the nanofibrous layer.Depositing electospun mats upon 3D printed substrates is one strategy used to develop multi-scale composite scaffolds . One semIn another study, a rotating drum electrospinning system was adopted to control the density (fibers per unit area) and the degree of alignment of electrospun fibers . Furtherl-lactide)/octa-armed polyhedral oligomeric silsesquioxanes (PLLA/POSS-(PLLA)8) to mimic the annulus fibrosus (AF) structure; a gellan gum/poly (ethylene glycol) diacrylate (GG/PEGDA) double network hydrogel that encapsulated living bone marrow mesenchymal stem cells (BMSCs) to reproduce the nucleus pulposus (NP) structure. 3D printing offered great control over size and shape of the scaffolds that were tested in an animal model of IVD replacement at rat tail vertebra. The tests revealed that, after 6 months of implantation, the artificial scaffolds were effective as reimplanted autologous discs in terms of maintained disc height and production of new ECM (proteoglycans and collagen). This work demonstrates the potential of the hybrid technology (3D printing and electrospinning) for the manufacturing of personalized medical devices for IVD regeneration.Recently, complex scaffolds have been manufactured to treat intervertebral disc (IVD) degeneration . They weElectrospun layers can be used not only as biomimetic interface to direct cellular attachment and migration, but also as mechanical reinforcement elements, particularly when hydrogels are 3D printed (instead of thermoplastic polymers). For example, PCL electrospun mats were combined with alginate hydrogel (3D bioprinting) to create multi-layered hybrid constructs .Alginate-PCL scaffolds recorded a 4-fold increase in compressive tangential modulus ( ∼ 89 kPa with nanofibers and ∼ 23 kPa without nanofibers) at 30% strain. Furthermore, alginate-PCL scaffolds witnessed elastic recovery (without failure) up to a strain limit of 45% vs. 30% for specimens constructs without fibers. These results were credited to the elastic nature of PCL, which improved the resilience of the scaffolds. In addition, the nanofibrous layers increased the proliferation rate of NIH 3T3 fibroblasts (1.8-fold increase compared to constructs without fibers) by offering favorable perfusion conditions i.e., open porosity throughout their scaffold structure.Electrospun nanofiber interleaves have been proposed to improve the mechanical properties of PLA 3D printed structures . The nanin vitro cytotoxicity tests showed that murine fibroblasts L929 attached and proliferated onto the gelatine layer, indicating that the composite scaffolds were biocompatible.Through the combination of electrospinning techniques and flexible material selection, naturally derived polymer scaffolds that can release bioactive compounds to promote soft tissue engineering, such as cartilage regeneration, have been explored. For example, fused filament fabrication (FFF) technologies were used to manufacture poly l-lactic acid (PLLA) scaffolds, prior to the deposition of electospun gelatine and osteogenon  nanofibers onto a single scaffold surface . AlthougLiterature suggests that introducing electrospun nano-fibers into multi-layered scaffolds (produced by combination of electrospinning and 3D printing) has the ability to manipulate both the porosity and mechanical performance of the 3D printed constructs, as well as modulate cellular behavior (elongation and differentiation) through their intrinsic topological features. Structures made of various biocompatible and biodegradable polymers  can be easily embedded within the same scaffold to obtain the desired functionality. Moreover, cell infiltration and proliferation within the scaffold can be promoted by altering density and thickness of electrospun fibrous membranes. So far, biodegradable aliphatic polyesters  have been primarily used for the 3D printing process, because they have a relatively low melting point, are generally inexpensive and widely available, and do not need post-processing to be crosslinked . These pin vitro with chondrocytes and in vivo on mice. After 6 weeks of cell culture, chondrocytes produced ECM to cover the 3D scaffolds and cartilage-like tissue was formed. Similarly, 8 weeks after animal implantation, cartilage tissue with the structure and composition to the native tissue was generated . These results were attributed to the porosity of the scaffolds, that allowed uniform cells infiltration and nutrients diffusion.The incorporation of electrospun nanofibers into hydrogels is discussed in the literature to create injectable composite systems that can stimulate, for example, the growth of nerve cells . HoweverIn follow-up studies, the bioactivity of such composite bio-inks and their regenerative potential were further enhanced by adding cartilage decellularized matrix (CDM) . CDM parMulti-Layered Architectures Section, because it requires additional dehydration, freeze-drying and cross-linking procedures. Nevertheless, it offers advantages in terms of obtaining scaffolds with enhanced porosity, controlled mechanical properties, and improved cellular viability. This is in line with previous research on the 3D bioprinting of hydrogels reinforced with polymeric nanofibers but not electrospun ones, including inks containing PLA nanofibers (produced by extrusion) (The literature here discussed shows that the inclusion of electrospun nanofibers in bio-inks for additive manufacturing is an approach far more complex and time-consuming than the multi-layering methods described in trusion) , and siltrusion) .The need to create functional and structurally complex scaffolds for tissue engineering has motivated the research of novel manufacturing methods that operate across different length scales. One strategy that has shown potential is the combination of electrospinning and 3D printing to enable, as discussed in this review, the production of scaffolds with multiscale porosity and hierarchical architecture. The presence of electrospun nanofibers and 3D printed micro-features within the same scaffold provides morphological and biomechanical cues that can regulate cellular behavior and promote tissue regeneration. Although a promising approach, combining these two techniques is currently time consuming and limited to research laboratories, because two separate set-ups, namely one for electrospinning and one for 3D printing, are typically required. In addition, post-processing procedures, such as freeze drying and/or crosslinking, are used to stabilise the complex structures created. Future works could focus on developing high-throughput automated systems, which integrate electrospinning and 3D printing , to achiIn a recent work, a 3D printer was specifically modified to include electrospinning units (within its construct), for vascular tissue engineering applications . The hybThe inclusion of multiple-electrospinning units within the confines of the hybrid technology could enable the fabrication and precise placement of nanofibers, constructed from different materials. This could promote localized cell-specific differentiation from Mesenchymal Stem Cells (MSCs) within the scaffold, leading to the generation of highly tunable interconnected multi-cellular/tissue systems. Such scaffolds could be used to develop sophisticated tissue models, better resembling natural tissue architectures, for future drug delivery and tissue replacement therapies. Alongside such prospects, protocols surrounding best practice e.g., sterilization methods for multi-cellular scaffolds would need to be investigated prior to implantation.


Neuropathic arthropathy is the painless destruction of weight-bearing bones and joints that is caused by a neurosensory deficit. Minimally displaced neuropathic fractures may be managed non-operatively. However, larger displacements often require surgical fixation. Nonunion is a rare entity in calcaneal fractures, and its occurrence in a neuropathic setting is an even rarer scenario. An unusual clinical scenario means there is a paucity of literature to guide the optimum treatment. Here we describe a patient with neuropathic arthropathy due to meningomyelocele in childhood, presenting with neuropathic nonunion of the calcaneum, managed with a minimally invasive surgical approach using an arthroscopic burr and fixation with percutaneous screws. Despite the high risk of complications in the operative treatment of neuropathic fractures, good functional results and successful limb salvage may be achieved with minimally invasive techniques. Neuropathic arthropathy is the painless destruction of weight-bearing bones and joints caused by a neurosensory deficit . The neuNeuropathic arthropathy of the ankle and foot is commonly associated with spontaneous calcaneal fractures . There iA 22-year-old male patient presented to us with swelling in his right heel. He was a known case of lumbosacral meningomyelocele with a low-lying tethered cord and had a partial sensory loss in the L4-L5 dermatomal distribution.There was broadening of the heel with non-tender bony hard swelling all around the calcaneum. Radiographs showed an extra-articular undisplaced calcaneal fracture which was treated with a non-weight-bearing below-knee plaster cast for three months, followed by an ankle-foot orthosis (AFO).Follow-up radiographs Figure  and compThe patient was operated in spinal anesthesia under tourniquet control. Nonunion site was approached through a stab incision made over the medial side of the heel region Figure . An arthThe postoperative period was uneventful. Ankle range of motion was started immediately post-surgery. Partial weight-bearing was allowed at three months post-surgery and full weight-bearing at five months after clinical and radiological union of the fracture Figure . At the Both physiological and mechanical factors contribute to the Charcot calcaneus. There is autonomic neuropathy, which causes increased blood flow and hence bone resorption in the affected area . Due to Three main patterns of neuropathic calcaneal fractures have been described in previous reports: posterior avulsion, joint depression, and anterior process fracture . A posteThe fracture pattern in the present case Figures , 2 is unTreatment goals for neuropathic fractures include maintenance of plantigrade foot alignment, prevention of ulceration, and achieving a braceable foot. Nonoperative treatment is recommended in most cases, which consists of initial control of the acute Charcot process by limb elevation, non-weightbearing, compression bandaging, and total contact casting. These are continued until the acute (Eichenholtz stage I) inflammation like phase has resolved . Long-teMinimally displaced tuberosity avulsion fractures are treated with cast immobilization in equinus. Surgical fixation with cannulated screws has been recommended for larger displacements . SurgicaNeuropathic nonunion is a rare entity in calcaneal fractures, which means there is a paucity of literature to guide the optimum treatment. Suitable options include open reduction and internal fixation with screw/plate fixation with or without bone grafting, corrective osteotomy with bone grafting, and subtalar arthrodesis . HoweverThrough this case report, we want to highlight our management technique and add to the literature on the management of nonunion of neuropathic calcaneal fracture.In conclusion, nonunion of a neuropathic calcaneal fracture is a rare clinical scenario with little guidance available for optimum treatment. Despite the high risk of complications, good functional results and successful limb salvage may be achieved with minimally invasive techniques. Freshening the fracture margins with arthroscopic burr under fluoroscopic guidance followed by percutaneous lag screw fixation seems to be an acceptable treatment option to achieve a well aligned shoeable/braceable foot.