Immune checkpoint inhibitors (ICIs) are achieving remarkable successes in several cancers, including melanoma, colon, and lung cancers. However, these same successes for ICIs are not yet being realized in estrogen receptor positive (ER+) breast cancer, the most common breast cancer subtype. Compared to triple-negative breast cancer (TNBC), ER+ breast cancers exhibit lower overall response rates to ICI treatments, including anti-programmed cell death (PD)-1, and anti-PD-ligand 1 (PD-L1) . If so, Numerous reports demonstrate that most cells in the body, including tumor cells as well as cells of the TME, express receptors that recognize and respond to viral nucleotide motifs, referred to as pattern recognition receptors (PRRs) . PRRs arin vivo [RIG-I is a cytosolic RNA helicase, recognizing RNA motifs specific to certain viruses. Years of RIG-I research has culminated in the discovery of synthetic non-infectious RIG-I mimetics, comprised of minimal stem-loop RNA (SLR) sequences harboring a 5′-triphosphate motif, capable of potent RIG-I activation in cultured tumor cells and in mice in vivo . Deliverin vivo was enabled by recent advances in nanoparticle (NP)-mediated delivery of RNA interference (RNAi) technologies. We focused on a previously described NP design strategy used with siRNAs, based on its proven in vivo protection of siRNA sequences, longer circulating half-life, increased uptake of siRNA by tumor cells, and enhanced endosomal release of siRNA cargo into the cytoplasm of tumor cells [in vivo [in vivo resulted in increased expression of pro-inflammatory cytokines. Importantly, we found increased CD4+ and CD8+ TILs and heightened ICI sensitivity in tumors treated with NP-SLR, consistent with the larger hypothesis that activation of innate immunity in ER+ breast cancers may prime these tumors for response to ICI.The effective delivery of SLR sequences to the TME or cells . Adaptin[in vivo . SimilarThese findings support clinical translation of RIG-I agonists, which is currently underway (NCT03065023) using the RIG-I agonist from Merck, MK-4621, in advanced and recurrent tumors. Another clinical trial recently opened (NCT03739138) to identify the therapeutic effects of MK-4621 with anti-PD-1 (Pembrolizumab) in patients with advanced and recurrent tumors. These trials are supported by our data demonstrating that RIG-I agonists increase ICI response in breast cancers through at least two mechanisms, tumor intrinsic apoptosis and enhanced immunogenicity of the TME. It is anticipated that, although RIG-I agonists are only in earliest phases of exploration as cancer treatment strategy, the field will move forward at a rapid pace, based on the vast potential for its success in immune-oncology.


Caspase a protease family member, have a vital role in cell death and inflammation process. Caspase-3, an effector caspase controls theregulation of apoptosis and has an anti apoptotic function. The mechanical significance of restoring apoptosis signaling to selectively targetmalignant cells is utilized to develop strong therapeutic strategies by the caspase family of mortality - induction molecules. Caspase-3 hascurrently no clear role in treatment for tumor progression and tumor sensitivity. The present study was aimed to screen caspase forpotential inhibitors using computer aided docking methodologies. For this, zinc natural molecule database molecules were screened usinge-pharmacophore and ADME protocols along with docking studies. Docking analysis selected two molecules, namely ZINC13341044 andZINC13507846 with G-scores -5.27 and -6.19 respectively. These two potential hits are predicted as caspase inhibitors based on the resultsand can be further processed for in vitro validation. The Extracellular matrix (ECM) receptors are imperative controllersof angiogenesis. One of these receptors, integrin α5β1, impacttumor-cell survival, multiplication, and metastasis, since theadversaries of this integrin α5β1 strongly restrain angiogenesis andtumor development The 3D crystalized structure of caspase protein (PDB ID: 5IBC) wasretrieved from the protein data bank Zinc natural molecules database Two methodologies, structure-based drug-design and ligand-baseddrug-design are renowned important in silico screening approachesin drug discovery pipeline. e-Pharmacophore based methodologycombines both structure-based and ligand-based methods to screenthe molecule database The screened molecules were docked into the active site of thecaspase protein with the help of XP docking protocol Glide score =0.065 * vdW +0.130 * Coul + Lipo + Hbond + Metal + BuryP + RotB + SitevdW - van der Waals energy, Coul - Coulomb energy, Liporepresents lipophilic term derived from hydrophobic grid potential,Hbond - hydrogen-bond, Metal - metal-binding term, BuryP - buriedpolar groups, RotB - penalty for freezing rotatable bonds, and Site -polar interactions in the active site.Binding free energies of the final complexes were calculated usingPrime/MM-GBSA Initially, for the generation of pharmacophore hypothesis thecrystal protein was separated into protein and ligand. With thereceptor grid generation application, a grid was generated aroundthe active site and the crystal ligand was docked into that gridboxed active site using Glide XP protocol. The ligand orientationafter docking was cross verified with crystal structure orientation,same orientation was reproduced confirming the docking protocolis valid and further used for docking studies.e-pharmacophore based screening was carried using the re-dockedpose viewer file, a three site hypothesis RRD (R represents ring andD represents donor) was generated . By settThe seven hits attained from the screening studies were subjected tomolecular docking studies. Using Glide XP docking protocol, themolecules binding affinity with the amino acids present in theactive pocket of the protein were studied.Binding Interactions of Caspase-3 with the screened moleculesZINC06036807 molecule made five back-bone hydrogen bonds andone pi-pi interactions with the active pocket amino acids. Aminoacids involved in the hydrogen bond formation are His 121 of chainA and Arg 207, Trp 206, Phe 250 and Ser 251 of chain b with themolecule.one pi-pi interaction was observed between molecule andTrp 206 residue of chain B. Three hydrogen bonds were observedbetween the active pocket of caspase-3 and ZINC13507846 by theend of docking studies. All the hydrogen bonds were maintainedwith the chain B residues, two hydrogen bonds with Ser 205 andthe remaining hydrogen bond was observed with Ser 209 by themolecule. Apart from the hydrogen bond, no other interactionswere observed in this complex. Binding affinity between activepocket amino acids of caspase-3 and ZINC01642250 wasmaintained with two hydrogen bonds and one pi-pi interactions.Chain B residues Ser 205 and Trp 206 made back bone hydrogenbonding with ZINC01642250 and one pi- pi interaction wasbetween Trp 206 and ZINC01642250.A total of five interactions were observed between caspase -3 activepocket and ZINC00056474. Out of five interactions, four are backchain hydrogen bonds and the remaining was a pi-pi interaction.All the interactions were observed with the chain B residues, Arg207, Ser 205 residues made two hydrogen bonds individually andTrp 206 made pi-pi interaction with the ligand molecule.ZINC31167269 molecule produced four back bone hydrogen bondsby the end of docking studies. Three hydrogen bonds were with thechain B residues and one with the chain A residue present in thebinding site of the protein. Chain B residues Arg 207 produced twohydrogen bonds and Ser 205 made one hydrogen bond withZINC31167269. One residue from the chain A, His 121 madehydrogen with the ligand. ZINC13341044 and caspase-3 complexwas maintained through three back bone hydrogen bonds and threepi-pi stacking�s. Two amino acids from chain A, Gly 122 and His121 are involved in the hydrogen bond formation with the ligandmolecule. Chain B residues Arg 207 produced one hydrogen bondand three pi - pi stacking's by the residues Trp 206, Tyr 204 and Phe256 with the ZINC13341044 molecule. Last molecule ZINC13507842made two back bone hydrogen bonds with the Arg 207 and Ser 205of chain B. Prime based MM-GBSA energy calculation was carriedto the seven complexes and their energies were tabled in the From the previous studies, it was evident that Tyr 204, Ser 205, Ser209 and Ser 251 of chain B are very crucial amino acids in the activesite of the caspase 3 for activity Figure 4a-e.The main objective of our present study is to screen naturalmolecules database to select small molecules as inhibitors againstcaspase-3. Analysis selected two potential hits against the targetnamely ZINC13341044 and ZINC13507846 based on the bindingmode with the target active site amino acids, satisfying all theADME important descriptors and with good energy calculations.The caspase-3 inhibition activity for these molecules can bevalidated using in vitro and in vivo methods.Authors declare no conflict of interest.


The ER is hub for protein folding. Proteins that harbor a Frizzled cysteine-rich domain (FZ-CRD) possess 10 conserved cysteine motifs held by a unique disulfide bridge pattern which attains a correct fold in the ER. Little is known about implications of disease-causing missense mutations within FZ-CRD families. Mutations in FZ-CRD of Frizzled class receptor 4 (FZD4) and Muscle, skeletal, receptor tyrosine kinase (MuSK) and Receptor tyrosine kinase-like orphan receptor 2 (ROR2) cause Familial Exudative Vitreoretinopathy (FEVR), Congenital Myasthenic Syndrome (CMS), and Robinow Syndrome (RS) respectively. We highlight reported pathogenic inherited missense mutations in FZ-CRD of FZD4, MuSK and ROR2 which misfold, and traffic abnormally in the ER, with ER-associated degradation (ERAD) as a common pathogenic mechanism for disease. Our review shows that all studied FZ-CRD mutants of RS, FEVR and CMS result in misfolded proteins and/or partially misfolded proteins with an ERAD fate, thus we coin them as “disorders of FZ-CRD”. Abnormal trafficking was demonstrated in 17 of 29 mutants studied; 16 mutants were within and/or surrounding the FZ-CRD with two mutants distant from FZ-CRD. These ER-retained mutants were improperly N-glycosylated confirming ER-localization. FZD4 and MuSK mutants were tagged with polyubiquitin chains confirming targeting for proteasomal degradation. Investigating the cellular and molecular mechanisms of these mutations is important since misfolded protein and ER-targeted therapies are in development. The P344R-MuSK kinase mutant showed around 50% of its in-vitro autophosphorylation activity and P344R-MuSK increased two-fold on proteasome inhibition. M105T-FZD4, C204Y-FZD4, and P344R-MuSK mutants are thermosensitive and therefore, might benefit from extending the investigation to a larger number of chemical chaperones and/or proteasome inhibitors. Nonetheless, FZ-CRD ER-lipidation it less characterized in the literature and recent structural data sheds light on the importance of lipidation in protein glycosylation, proper folding, and ER trafficking. Current treatment strategies in-place for the conformational disease landscape is highlighted. From this review, we envision that disorders of FZ-CRD might be receptive to therapies that target FZ-CRD misfolding, regulation of fatty acids, and/or ER therapies; thus paving the way for a newly explored paradigm to treat different diseases with common defects. Frizzled receptors (FZD) are G-protein-coupled receptors (GPCRs), which act as gate-keeping proteins, and are receiving considerable attention in recent years. Observing the domains of FZDs shows an amino-terminal (N′) signal peptide (SP) sequence which localizes FZD polypeptides to the endoplasmic reticulum (ER) membrane Fig. a. SP is Frizzled cysteine-rich domain (CRD), a result of evolutionary membrane fusion, is considered a conserved mobile functional site similar to Frizzled-CRD (FZD-CRD) and is mainly found in Wnt receptors and is shared with other metazoan proteins. The FZD-CRD sequence homology is shared among FZD1–10, and further shares ancestral similarities both in sequence and structure to muscle, skeletal, receptor tyrosine kinase (MuSK); receptor tyrosine kinase-like orphan receptor 2 (ROR2); corin, serine peptidase (CORIN) and the similarity is shown in Fig. CnCnCX8CX6CnCX3CX6,7CnCnC”  in α-helices forming a common Frizzled fold across four α-helices  clustering where postsynaptic differentiation is orchestrated by interactions of the proteoglycan agrin, low density lipoprotein receptor-related protein 4 (LRP4), docking protein 7 (DOK7) and receptor associated protein of the synapse (RAPSN). MuSK FZ-CRD is similar to Frizzled CRDs and interacts with Wnt4, Wnt11, and Wnt9a in vitro  serves as a central hub for efficient protein and lipid synthesis  acts as a folding sensor and reglucosylates Man(7–9)GlcNAc2) to restore the binding site for CXN and CRT to re-guide folding again   is a hereditary condition where retinal blood vessels shows incomplete or no vascularization  sensitivity in vitro assays showed immature proteins. FZD4 protein has two potential N-glycosylation sites in its extracellular domain and upon Endo H treatment, P33S  and SIFT (http://provean.jcvi.org/links.php [in the public domain]) is available in our study , and the amino acid substitutions at the 204 position result in the disruption of a vital cysteine disulphide bond which fails to bind Norrin  pump inhibitors and curcumin among others  found in mammalian cells. Thapsigargin which originates from plants increases cytosolic calcium, and in doing so, results in an enhanced rescue of mutant proteins from the ER , trimethylamine-N-oxide, calcium , is a group of genotypically and phenotypically heterogeneous group of neuromuscular disorders resulting in abnormal signal transmission and AChR clustering at the neuromuscular junction. Mutations in So far, six MuSK missense mutations have been reported in patients with CMS including D38E  rescue of the P344R mutant when treated with 2.5% glycerolProteasome inhibition with MG132 treatment caused the P344R-MuSK protein to increase two-folds compared to WT-MuSK. Further to this, P344R-MuSK showed around 50% of its in vitro autophosphorylation activity on stabilization. The pattern of multi-Ub-P344R-MuSK conjugates were reduced in MG132 treated samples and under chemical chaperone treatment  or autosomal recessive  disorder Robinow . RRS resSFRPs, CPZ, CORIN and COL18A1 also contain FZ-CRDs. Interestingly CORIN has an important role in mammalian cholesterol metabolism where CORIN binds and transports LDL to targeted cells via endocytosis. A reported a S472G missense mutation located within the first FZ-CRD of CORIN Fig. . To our Current targeted strategies have been observed to account for the rescue of several different classes of misfolded and mislocalized proteins, and they are becoming increasingly important as therapeutics  which are attached to cysteine residues  is shown to be an important modification for synaptic transmission and GPCR protein signalling regulation , a DnaJ/Hsp40-family chaperone, shows palmitoylation on cysteine residues and functions to localize the protein on the plasma membrane. Unpalmitoylated CSP shows slow binding capacity and rapidly dissociation from membranes, and ER accumulation  acylation is important in the anterograde secretory pathway and allows for high-affinity interactions between Wnt and FZD8-CRD through Ser residues  are post-translational modifications conserved in Wnts and important for Wnt signalling  .The CRD groove’s amino acid sequence is conserved across all 10 members of the FZDs receptors    member 5 (HSPA5), or specific chaperones such as the CFTR’s Aha chaperone and the Boca chaperone specific for LRP5/6 homologue arrow in Alleviating the pathogenesis disorders of FZ-CRD will require novel emerging therapeutics, such as, structure-guided drug discovery (Winter et al. Insights from this review show a hotspot for the clustering of missense mutations in FZ-CRD resulting in different misfolded proteins, each responsible for eliciting different debilitating diseased states. FZ-CRD can be manipulated with chemical chaperones and therefore is possibly amenable to therapy providing proof-of-principle that conformational disorders can be corrected. Recent work highlights that lipidation of class II proteins is vital for proper folding of proteins and anterograde trafficking. Elucidating the cellular and molecular mechanisms of disease serves as a platform to further understand the proteostasis network, protein-lipid interactions and appreciate the complexity of structural data. Therefore, from this review, it is envisioned that more molecular players in the FZD4, MuSK and ROR2 pathway will be discovered and the folding of FZ-CRD will be sought after to design small molecules for improving expression and stability of the mutant proteins at the cell surface.Our review further opens the door for looking at other conserved modules that cluster mutations across other conformational diseases. It could also possibly lay the foundations for discovering alternative therapeutic measures for FEVR, CMS and RRS patients, the novelty of which is mutation specific which makes it personalized or alternatively targets disorders of FZ-CRD.


TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), alsoknown as APO2L, belongs to the tumor necrosis factor family. By binding to the deathreceptor 4 (DR4) or DR5, TRAIL induces apoptosis of tumor cells without causing sidetoxicity in normal tissues. In recent years TRAIL-based therapy has attracted greatattention for its promise of serving as a cancer drug candidate. However, thetreatment efficacy of TRAIL protein was under expectation in the clinical trialsbecause of the short half-life and the resistance of cancer cells. TRAIL genetransfection can produce a “bystander effect” of tumor cell killing and provide apotential solution to TRAIL-based cancer therapy. In this review we focus on TRAILgene therapy and various design strategies of TRAIL DNA delivery including non-viralvectors and cell-based TRAIL therapy. In order to sensitize the tumor cells toTRAIL-induced apoptosis, combination therapy of TRAIL DNA with other drugs by thecodelivery methods for yielding a synergistic antitumor efficacy is summarized. Theopportunities and challenges of TRAIL-based gene delivery and therapy arediscussed. Nucleic acid-based therapy has been considered one of the most promisingstrategies for the treatment of various diseases . Tumor nc, inducing intrinsic apoptosis viathe mitochondrial pathway in type II cells  (pDMAEMA), poly(β-aminoester)s, and various carbohydrate-based polymers and dendrimers. PEI and itsderivatives are the most commonly used polymeric vectors for gene delivery,with the advantage of the “proton sponge” effect, which facilitates theendosomal escape of gene drugs . Th. Th54]. N-propionic4-acetoxybenzyl ester substituents; the 4-acetoxybenzyl ester group canundergo rapid intracellular esterase-catalyzed hydrolysis, whichsubsequently triggers a reversal of the polymer’s charge from cationic tozwitterionic [Poly(beta-amino ester)s (PBAEs) are a class of cationicsynthetic polymers that can be used as nonviral gene carriers. Thesecompounds are easy to synthesize, bind effectively to DNA, and arehydrolytically degradable under physiological conditions. Tzeng et al.reported the use of PBAE nanoparticles containing pTRAIL for the selectivetransfection of cancer cells and the induction of apoptosis in several humancancer cells .Additioterionic . Due to terionic . The samterionic . PQDEA/D3O4-PEI-plasmid complex(FPP) was generated, in which iron oxide NPs were modified by positivelycharged PEI to enable them to carry pACTERT-TRAIL [In addition, some inorganic materials have been modified withpolymers for pTRAIL delivery. For example, a sandwich-type PEI-coated goldnanocomposite coloaded with pTRAIL and nuclear-targeted dexamethasone (Dexa)(termed Au-PEI/pTRAIL/PEI-Dexa) exhibited efficient transfection andsignificantly inhibited the growth of Hep3B tumors . A targeRT-TRAIL . The effDendrimers, which are characterized by their well-defined sizeand low polydispersity index, have been widely used for drug and genedelivery . Jiang eLater, coumarin-anchored low-generation dendrimers (G1 PAMAMdendrimers) were established to improve DNA binding and gene delivery. The couCationic peptides rich in residues such as lysines or argininesare able to condense DNA. Furthermore, conjugation of peptide ligands todelivery systems allows targeting to specific types of cancer cells. A biomiCationic lipids have been widely used for nucleic acid deliveryand for advances in gene delivery through their molecular design. Cationiclipids can spontaneously form specific types of complexes for condensing andencapsulating DNA into particles .Huang et al. reported a novel lipid-3-biguanide-propane (DOBP)) that was elaboratelydesigned by utilizing biguanide as the cationic head group . This no2+ fromthe CaP core that induced DR5 up‐regulation through CaMKII activation  NP to deliver pTRAIL into HCC cells in amouse model of HCC . pTRAILVβ3 receptorsoverexpressed on the neovasculature and most malignant tumor cells. Systemicinjection of the proapoptotic mTRAIL plasmid by RRPHC ternary complexesinhibited the melanoma growth, without noticeable side toxicity. Next, thisgroup reported a similar system of an artificial virus core-shell to targetcancer stem cell-like cells [Gong et al. developed a well-tailored and versatile“core-shell” ternary system (RRPHC) for systemic gene delivery to treataggressive melanoma . Thissyke cells . The intMesenchymal stem cells (MSCs) are a population of fibroblast-like cellsoriginally isolated from bone marrow and other tissues, including adipose tissue,peripheral blood, umbilical cord blood, and Wharton’s jelly, among others, 79. MSC2O4 magneticnanoparticle core and mesoporous silica shell, as well as a surface coating of PEIfor DNA binding. The MC NPs facilitated the intracellular delivery of theheat-inducible plasmid into AD-MSCs via magnetic guidance, and after systemicinjection, the engineered AD-MSCs could home to tumors/metastases. TRAIL expressioncould be specifically activated via the induction of mild magnetic hyperthermia(~41 °C). This system enhanced control over the activation of stem cell-based genetherapies.Lee et al. reported a novel application of magnetic core-shellnanoparticles for the dual purpose of delivering and activating a heat-induciblegene vector that encodes TRAIL in adipose-derived mesenchymal stem cells (AD-MSCs). This grIt was reported that human MSCs were transduced with TRAIL and theIRES-eGFP reporter gene under the control of the tetracycline promoter using alentiviral vector . The tra600-Cyd, prepared by linking low-molecular-weightpolyethyleneimine (PEI) and β-cyclodextrin (β-CD) [Gao et al. transfected pTRAIL into MSCs with a nonviral vector,PEIn (β-CD) . The lunHuman umbilical cord-derived mesenchymal stem cells (HUMSCs) weretransfected by lentiviral vectors coding the sTRAIL with the alpha-fetoprotein (AFP)promoter, and the treatment efficacy of these engineered HUMSCs on orthotopicallyimplanted hepatocarcinomas in mice was examined . HUMSCs Conventionally, MSCs have been genetically modified for cancer therapyby using viral vectors that can elicit oncogenicity, thus limiting their use inclinical trials. Chen et al. used nonviral agents such as a polylysine-modifiedpolyethyleneimine (PEI-PLL) copolymer to generate genetically engineered MSCs withsuicide genes, namely, HSV-TK and TRAIL . The MSCIn addition to MSCs, Hu et al. reported a DC cell-based therapy forcolon cancer cells . TyrosinAlthough TRAIL-induced apoptosis is an attractive therapeutic target,resistance to TRAIL readily develops during treatment. This therapeutic resistancederives from two sources: intrinsic resistance in some highly malignant tumors–99 and aL determine cell survival[TRAIL signaling begins with the binding of TRAIL to the deathreceptors. First, binding to decoy receptors  will not leadto the activation of downstream TRAIL-signaling. Second, mutation or downregulationof the functional DRs  can also result in resistance. Forinstance, transfection of mutated DR4 into SW480 colon cancer cells caused a lowerefficacy of cell killing than transfection of the wild-type counterpart. Low expsurvival, 107. Fusurvival, 109. Hosurvival, 111. ThIn addition, epigenetic changes play a role in TRAIL resistance via theregulation of caspase-8 gene expression, and DNA methylation can restore apoptosisin TRAIL-resistant tumor cells. TRAIL resistance is also related to NF-κB andmitogen-activated protein (MAP) kinases. However, these signaling pathways showedboth proapoptotic and antiapoptotic effects .L, and IAPs contribute to TRAIL resistance, andtherefore, regulation of DRs and inhibition of antiapoptotic proteins resensitizescells to TRAIL treatment [In conclusion, DR dysfunction and overexpression of cFLIP, Bcl-2,Bcl-Xreatment .The resistance of cancer cells to TRAIL has encouraged theinvestigation of combination therapy. In recent years, many multifunctional drugdelivery systems, including liposomes, micelles, and polymeric nanoparticles, havebeen developed for the codelivery of genes and drugs. Here, we summarize thedelivery and therapeutic strategies of combinations of pTRAIL and different types ofdrugs.1.8k (LMW-PEI)was intermolecularly crosslinked via disulfide bonds using sulfhydrylβ-cyclodextrin as a linker  can overcome TRAIL resistance via endoplasmicreticulum stress induction, DR5 upregulation and cFLIP downregulation. Huang eTRAIL exhibits improved efficacy in combination with chemotherapybecause chemotherapeutic agents can sensitize tumors to TRAIL-induced apoptosisvia crosstalk between the intrinsic and extrinsic pathways of cell death. The cheL-lysine(DGL), modified by the T7 peptide and conjugated with DOX via a pH-sensitivehydrazone bond, was used to deliver pORF-hTRAIL to glioma tissue [Jiang et al. investigated the codelivery of the hTRAIL-encodingplasmid open reading frame (pORF-hTRAIL) and DOX using a tumor-targetingcarrier, a peptide HAIYPRH (T7)-conjugated polyethylene glycol-modifiedpolyamidoamine dendrimer (PAMAM-PEG-T7) . In thisa tissue . In addia tissue ,122.Vβ3, whichis overexpressed in the neovasculature and on U87 glioblastoma cells. PTX wasloaded in a CDX–poly(ethylene glycol)–block-poly(lactic acid) micelle. CDX is apeptide derived from the loop II region of the snake neurotoxin candoxin, with ahigh-binding affinity to nicotinic acetylcholine receptors (nAChRs). Dominici etal. investigated the combination of pTRAIL and PTX for MSC therapy [Regarding the combination of PTX and TRAIL, an angiopep-2peptide-modified cationic liposome (ANG-CLP) for the efficient codelivery ofpEGFP-hTRAIL and PTX to glioma was reported. Angiopep-2 can target thelow-density lipoprotein receptor-related protein (LRP) overexpressed on the BBBand on glioma cells . Lu eta therapy . PTX res therapy .These results indicate that TRAIL gene therapy in combination withchemotherapy can be promising for ovarian cancer therapy. Although it is notfully known how chemotherapy sensitizes cells to TRAIL-induced apoptosis, theeffect may be related to a p53-independent pathway, in addition to changes inthe expression of proteins involved in TRAIL signaling, which also play animportant role .As TRAIL induces extracellular apoptosis, its combination withagents mediating the intracellular apoptosis pathway can yield a synergisticeffect. Histone deacetylase inhibitors (HADCi) induce cell cycle arrest andapoptosis in tumor cells, and HADCi can resensitize TRAIL-resistant cancercells. Codelivery of pTRAIL and vorinostat (SAHA) with a reactive oxygen species(ROS)-triggered charge reversal polymer (B-PDEAEA) produced a good antitumoreffect . The incIAPs play an important role in cancer cell resistance to TRAIL, andthe combination of TRAIL with IAP antagonists can help sensitize cells toTRAIL-induced apoptosis. Ge et al. demonstrated that an oncolytic adenoviruscoexpressing TRAIL and Smac, in combination with the cyclin-dependent kinase(CDK) inhibitor SNS-032, synergistically reinforced their individualanti-pancreatic cancer activities, and SNS-032 enhancedZD55-TRAIL-IETD-Smac-induced apoptosis . Shi et Compared with TRAIL monotherapy, combination therapy providesenhanced treatment outcomes. Therefore, combinations including TRAIL-mediatedtherapy are a promising approach for cancer therapy.TRAIL is a promising drug candidate for the treatment of many cancers.The investigation of TRAIL-based therapy in clinical trials focuses on recombinantTRAIL proteins and anti-TRAIL antibodies, but delivery and drug resistance issuesare the main hurdles in the successful translation of these results. TRAIL-basedgene therapy is another potential treatment approach. Importantly, the codeliverysystems loaded with pTRAIL and drugs combined with TRAIL have been activelyexplored. The development of safe and efficient gene vectors is the central issuefor gene therapy. The elegant design of advanced systems provides the tumor-targetedcodelivery of both agents, thus achieving synergistic treatment effects. Inaddition, it is necessary to identify proper biomarkers for maximizing TRAIL-basedtherapy, which can also provide helpful information for the appropriate selection ofdrug combinations. However, clinical trials have made little progress. Effortsshould be directed toward the rational design of appropriate formulations of TRAILto improve its in vivo pharmacokinetic profile and the exploration of biomarkersspecific to TRAIL-based therapy. The future application of TRAIL may benefit from abetter understanding of the anticancer mechanisms of TRAIL as well as itscombination with TRAIL sensitizers. It is expected that as precision medicineprogresses, TRAIL-mediated antitumor functions will be better understood to allowthe development of precise and effective TRAIL-based treatments for patients.


Villin-Cre/Notch-1fl/fl (VN-/-) mice that are selectively deficient in Notch-1 in intestinal epithelial cells. Intestinal epithelial Notch-1 preserved barrier function and integrity, whereas lack of epithelial Notch-1 induced goblet cell hyperplasia, spontaneous serrated lesions, multifocal low- and high-grade dysplasia and colonic mucinous neoplasms in mice. Over time, VN-/- mice displayed high occurrence of colorectal mucinous adenocarcinomas, which correlated with increased levels of mitogenic, angiogenic and pro-tumorigenic gene expression. Finally, we found that the expression of Notch-1 is significantly reduced in human colorectal mucinous adenocarcinoma when compared to colorectal adenocarcinoma. Taken together, our findings reveal a novel and critical protective role for Notch-1 in controlling intestinal tumorigenesis.Increasing evidence links Notch-1 signaling with the maintenance of intestinal architecture and homeostasis. Dysfunction in the common Notch-1 pathway transcription factor recombinant binding protein suppressor of hairless (RBP-J) is associated with loss of epithelial barrier integrity and aberrant conversion of proliferative crypt cells into goblet cells. Furthermore, we have recently discovered that epithelial Notch-1 is indispensable in bridging innate and adaptive immunity in the gut and is required for supporting protective epithelial pro-inflammatory responses. Yet, the epithelial specific function of Notch-1 in intestinal tumorigenesis remains unknown. We generated  The mucosal surface of the intestinal tract is covered with a single layer of epithelium that constitutes a physical and immunological barrier against a variety of foreign antigens derived from food, pathogens and commensal bacteria. Intestinal stem cells located at the crypt bottom sustain continuous turnover of the epithelium by giving rise to transit-amplifying cells that undergo proliferation and differentiation into absorptive (enterocytes) and secretory  cell lineages , 2. KeepThe Notch signaling pathway plays a key role in the embryonic and adult gut epithelium, and is required both for maintenance of intestinal stem cells and for a proper balance of differentiation between secretory and absorptive cell lineages , 8. In tIncreasing evidence underscores the importance of Notch signaling in intestinal tumorigenesis, yet unlike in acute T-cell leukemia, activating mutations in Notch receptors are unusual in colorectal cancer , 16. ConVillin-Cre/Notch-1fl/fl (VN-/-) mice, which have a deletion of Notch-1 under the villin promoter in the intestinal epithelial cells, develop serrated lesions, multifocal epithelial dysplasia and spontaneous colorectal mucinous neoplasms in a background of colonic mucosa with impaired barrier integrity, and enhanced goblet cell metaplasia. The incidence of colorectal mucinous adenocarcinomas in VN-/- mice correlated with increased levels of mitogenic, angiogenic and pro-tumorigenic genes. Concomitantly, Notch-1 expression is significantly reduced in human colorectal mucinous adenocarcinoma compared to non-mucinous colorectal adenocarcinoma. Our findings emphasize the implication of epithelial Notch-1 in i) controlling intestinal tumorigenesis, and, ii) highlighting a molecular signature in a subset of patients with colorectal mucinous adenocarcinoma that may benefit from targeted screening and subsequent therapeutics.In this study, we show that the in vitro and in vivo [Villin-Cre;Notch-1fl/fl; termed VN-/-). These mice were born at the expected Mendelian ratio, and deletion of Notch-1 expression within the intestinal epithelial compartment was confirmed by qPCR (data not shown). To delineate the in vivo implication of Notch-1 in intestinal epithelial integrity, colonic permeability, resistance and tight junction protein expression were assessed in WT and VN-/- mice at 10 weeks of age.We have previously reported that Notch-1 controls intestinal barrier function at homeostasis, both  in vivo –19. More in vivo . In ordeVN-/- mice was significantly decreased and was concomitant with an increase in FITC-dextran flux across the mucosa when compared to WT mice  and trefoil factor-3 (Tff3) mRNA levels were all significantly up-regulated in the VN-/- mice compared to WT mice . No evidence of pericystic or lamina propria inflammation or cyst rupture was seen in association with these mucinous lesions, ruling out a herniation or colitis-associated cystica profunda , c-Myc2, cyclin B1, cyclin E, Angiogenin-4, cyclooxygenase 2 (Cox2), hypoxia-inducible factor 1 alpha (Hif1a), matrix metalloproteinase 10 (Mmp10), Amphiregulin, Epiregulin, and Wnt5a . Expression of Notch-1 mRNA was analyzed in CRC and mucinous CRC tissues in three gene expression datasets using the Oncomine® database. We found a significantly reduced expression of Notch-1 mRNA in mucinous CRC when compared with CRC tissues in the TCGA Colorectal ; Bittner Colon ; and Kaiser Colon  datasets  signaling molecule SMAD3, develop inflammation-associated dysplasia and mucinous colon cancer, but only after chemically induced epithelial injury [Fx-/-) mouse model [Fx-/- mice and these mice further develop mucinous adenocarcinomas. While the VN-/- mice do not develop colitis, the histological features of sessile serrated adenoma like lesions are shared with the Fx-/- model. Both models also have low-grade dysplasia that evolves into high-grade dysplasia over time, due to the loss of Notch-1 [Mouse models of concomitant colonic serrated polyps and mucinous neoplasia have been lacking so far. Transgenic mice with intestinal expression of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the intestine develop colonic serrated polyps, but no dysplasia . Mice exl injury . Additiose model . Interes Notch-1 . Thus, wMin mice, Notch was found to act as a mediator in intestinal tumorigenesis in familial adenomatous polyposis [Most studies on human CRC demonstrate that Notch-1 is elevated in human CRC tissues and correlated with poor differentiation, tumor progression and poor survival , 43–46. olyposis . Conversolyposis , 49. Thiolyposis .One reason for this discrepancy in providing clear and conclusive evidence for the role of Notch-1 in CRC is the lack of studies using patient stratification based on CRC subtypes. This is validated by the description of high, low and no-Notch-1 CRC expressers, in humans . AdditioCRC can be classified into several subtypes with distinct molecular and genomic signatures that will translate into personalized therapies. Our efforts have identified a new molecular signature for the subset of mucinous CRC in humans. Mucinous CRC patients would largely benefit from a more targeted approach of screening, diagnosis and treatments toward the Notch-1 signaling pathway. In summary, our findings reveal a novel role for epithelial Notch-1 in protecting from mucinous colorectal adenocarcinoma and open avenues for the development of personalized medicine and targeted therapeutics to be tailored to specific types of colon cancers.Notch-1fl/fl) and VillinCre mice were purchased from Jackson Laboratories and housed in the Icahn School of Medicine at Mount Sinai specific pathogen-free barrier facility. Notch-1 flox/flox (Notch-1fl/fl) mice are known to have a normal phenotype with no changes in Notch-1 expression [Villin-Cre/Notch-1fl/fl (VN-/-) mouse strain was generated by crossing VillinCre and Notch-1fl/fl mice. All mice were genotyped according to Jackson Laboratories’ protocols at the Transgenix. Mice were used at 10 or 30 weeks of age. Experiments were carried out using age and gender matched groups. The Institutional Animal Care and Use Committee approved all procedures performed in this study.Wild-type , Notch-1 flox/flox  in Ussing chambers  as previously described [Permeability studies were performed on colonic tissues from WT and escribed . Brieflyescribed .Notch-1 and Actin mRNA expression was evaluated using the Solaris qPCR Gene Expression Assay system accompanied by the Solaris probes (Thermo Scientific). For all the other genes, mRNA expression was quantified using the SYBR Advantage qPCR system  . RNA was reverse transcribed using the qScript cDNA system . Paraffin-embedment and hematoxylin and eosin (H&E) stains were performed at the Histology core facility at the Icahn School of Medicine at Mount Sinai. Histologic changes were evaluated and scored by a pathologist blinded to the treatment group. For immunofluorescence, de-waxed paraffin-embedded colonic tissue sections were stained as previously described , 19. BriNotch-1 was obtained from three colorectal (CRC) datasets using the Oncomine database as previously described [https://www.oncomine.com/). Fold change of Notch-1 mRNA expression acquired from each CRC dataset was compared between CRC and mucinous CRC tissues using the following filters: threshold fold change ≥ 1.5X; p value ≥ 1E-04; and gene rank in the top 10%. Expression values of Notch-1 were sorted based on p values and presented in Log2 median-centered intensity ratios for three CRC datasets including: TCGA Colorectal [Gene expression data of escribed  .Statistical significance was determined using two-tailed Mann Whitney test (2 groups comparisons only). A value of


High-grade serous ovarian cancer (HGSC), the most common ovarian carcinoma type, is associated with the highest mortality rate among all gynecological malignancies. As chemoresistance has been demonstrated as the major challenge in improving the prognosis of HGSC patients, we here aimed to identify microRNA (miRNA) biomarkers for predicting platinum resistance and further explore their functions in HGSC.We developed and applied our network vulnerability-based and knowledge-guided bioinformatics model first time for the study of drug-resistance in cancer. Four miRNA biomarkers  were identified with potential in stratifying platinum-sensitive and platinum-resistant HGSC patients and predicting prognostic outcome. Among them, miR-454-3p and miR-183-5p were newly discovered to be closely implicated in platinum resistance in HGSC. Functional analyses highlighted crucial roles of the four miRNA biomarkers in platinum resistance through mediating transcriptional regulation, cell proliferation and apoptosis. Moreover, expression patterns of the miRNA biomarkers were validated in both platinum-sensitive and platinum-resistant ovarian cancer cells.With bioinformatics modeling and analysis, we identified and confirmed four novel putative miRNA biomarkers, miR-454-3p, miR-98-5p, miR-183-5p and miR-22-3p that could serve as indicators of resistance to platinum-based chemotherapy, thereby contributing to the improvement of chemotherapeutic efficiency and optimization of personalized treatments in HGSC. High-grade serous ovarian cancer (HGSC) is the most common histotype accounting for more than 70% deaths from ovarian cancer , the leaMicroRNAs (miRNAs) are approximately 22-nt non-coding RNAs that post-transcriptionally modulate gene expression in a sequence-specific manner via mRNA degradation or translational repression . These mAs platinum resistance is a major obstacle in ovarian cancer treatment, extensive efforts have been made to discover miRNA biomarkers involved in drug resistance. For example, Vecchione et al.  identifiCancer is a complex disease involving dysregulation of multiple signaling pathways. Application of computational approaches to characterize miRNA and/or mRNA signatures from a systems biology perspective has been extensively adopted in various cancers . In our To explore miRNA biomarkers associated with platinum-resistance in ovarian cancer, the mRNA and miRNA expression data and clinical data of 31 platinum-sensitive and 37 platinum-resistant HGSC patients were downloaded from the publicly available International Cancer Genome Consortium (ICGC) based on search term ‘OV-AU’, which has been published by Patch et al.  in NaturTo validate the expression pattern of the identified miRNA biomarkers associated with platinum resistance, miRNA expression datasets of platinum-sensitive ovarian cancer cell lines (A2780_S and OVCAR3_S) and corresponding platinum-resistant ovarian cancer cell lines (A2780_R and OVCAR3_R) were downloaded from gene expression omnibus (GEO) under the accession number of GSE84200.The empirical bayes (eBayes) method in ‘limma’ R package , 19 was To investigate the contributory roles of miRNAs in regulating platinum resistance in ovarian cancer, the platinum-resistance associated miRNA-mRNA network in HGSC (PRMNH) was constructed based on the following two steps. Firstly, a human miRNA-mRNA regulatory network was generated using both experimentally validated and computationally predicted miRNA–mRNA regulatory interactions from public databases as used in the miRNA-BD tool . SecondlP < 0.01, Wilcoxon signed-rank test) in PRMNH were identified using MicroRNA-BD tool. As a miRNA that regulates platinum resistance-associated target genes is more likely to play key roles in resistance to chemotherapy, the selected miRNAs were further filtered based on the existence of platinum resistance-associated target genes in PRMNH. The functions of miRNA target genes were further searched in the PubMed database. Known platinum resistance-associated miRNAs in ovarian cancer were obtained from the review by Zhang et al. [As reported by Lin et al. , miRNAs g et al.  and papeg et al.  and Arrig et al. .To evaluate the sensitivity and specificity of identified miRNA biomarkers for distinguishing between platinum-sensitive and platinum-resistant patients, ROC curve analyses were conducted using the ‘ROCR’ package in R .P < 0.05).To establish the prognostic value of miRNA biomarkers, patients were divided into high-expression and low-expression groups using the upper quartile of miRNA expression level as the threshold. Then, Kaplan–Meier survival analyses were conducted to evaluate the differences in progression free survival (PFS) and overall survival (OS) times between high-expression and low-expression groups by the ‘survival’ package in R, and the log-rank test was employed to assess the statistical significance of the survival curves  and all miRNAs in the human miRNA-mRNA network   and miR-98-5p  had a significantly shorter PFS time, while those patients with high expression level of miR-183-3p  and miR-22-3p  had a significantly shorter OS time  of miRNA biomarkers between platinum-resistant and platinum-sensitive HGSC tissue (termed as HGSC samples (R/S)) was compared with that between platinum-resistant and platinum-sensitive ovarian cancer cell line A2780 (termed as A2780 (R/S)) or OVCAR3 (termed as OVCAR3 (R/S)). As shown in Fig. To further validate association of the identified miRNA biomarkers with platinum resistance, their expression pattern between platinum-sensitive and platinum-resistant ovarian cancer cell lines were analyzed using miRNA expression datasets from GSE84200. Then, the logIn this study, to gain insights into the mechanisms underlying chemoresistance in HGSC, an integrated method was proposed for identifying putative platinum resistance-associated miRNA biomarkers through constructing PRMNH that consists of regulatory miRNA-mRNA pairs with differential expression patterns between platinum-resistant and platinum-sensitive HGSC patients. Given the critical importance of vulnerable nodes in the biological network structure, this approach was improved by introducing the criteria of number of platinum resistance-associated genes (NPRG) among miRNA targets based on the theory of single-line regulation, which has been successfully applied to identify miRNA biomarkers for detection of pediatric acute myeloid leukemia and prostate cancer metastasis , 20. ThuBased on the computational approach, four miRNAs  were identified as putative biomarkers for predicting platinum resistance in HGSC. The expression patterns of these miRNAs were further validated in ovarian cancer cell lines, A2780 or OVCAR3 Fig. . ConsistIt’s well known that prognosis assessment is of great significance for making an appropriate therapeutic strategy. Due to tumor heterogeneity , we obseEfforts have been made in the present study to explore the functional mechanism of the identified miRNA biomarkers by GO and IPA pathway enrichment analysis of their target genes. Notably, target genes of the four miRNA biomarkers were closely implicated in cancer progression-related processes, such as transcriptional regulation, morphogenesis, and cell migration and proliferation Fig. a. PlatinIn summary, by employing a network-based computational method, we discovered four miRNA biomarkers, miR-454-3p, miR-98-5p, miR-183-5p and miR-22-3p that could potentially serve as indicators of resistance to platinum-based chemotherapy thereby contributing to reduce treatment costs and improve patients’ prognosis. The functions of the identified miRNA biomarkers were further explored via GO and IPA pathway enrichment analyses, providing insights into the mechanisms by which these miRNAs contribute to platinum resistance in HGSC.Additional file 1. Differentially expressed miRNAs between platinum-resistant and platinum-sensitive HGSC patients.Additional file 2. Differentially expressed mRNAs between platinum-resistant and platinum-sensitive HGSC patients.Additional file 3. The PRMNH composed by differentially expressed miRNAs and mRNAs.Additional file 4. Known miRNAs associated with platinum resistance in ovarian cancer.Additional file 5. The feature of target genes of the identified miRNA biomarkers in PRMNH.Additional file 6. The Wnt signaling pathway enriched by targets of the identified miRNA biomarkers in IPA. Objects with purple circles or triangles were acting locus by mapped genes.Additional file 7. The axon guidance signaling pathway enriched by targets of the identified miRNA biomarkers in IPA. Objects with purple circles or triangles were acting locus by mapped genes.Additional file 8. The ATM signaling pathway enriched by targets of the identified miRNA biomarkers in IPA. Objects with purple circles or triangles were acting locus by mapped genes.Additional file 9. The SAPK/JNK signaling pathway enriched by targets of the identified miRNA biomarkers in IPA. Objects with purple circles or triangles were acting locus by mapped genes.


Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming.  •Genome-wide identification of miRNAs regulated in human endocrine cell development•Endocrine-enriched miRNAs promote cell cycle exit and endocrine cell formation•Network modeling predicts miRNA-regulated TFs and downstream cell cycle regulators Molecular Mechanism of Gene Regulation; Molecular Network; Endocrinology; Stem Cells Research The potential to generate pancreatic beta cells from human pluripotent stem cells (hPSCs) or via cell reprogramming from other cell sources holds promise for modeling causes of diabetes and cell replacement therapies . KnowledDicer1, an enzyme that is universally required for the functional maturation of miRNAs, results in reduced endocrine cell numbers (Dicer1 disruption in beta cells impairs insulin biogenesis (MicroRNAs (miRNAs) are a group of small non-coding RNAs (∼22 nucleotides) with known roles in the regulation of gene expression in development, mature cell function, and disease . Studies numbers , whereasogenesis . At the ogenesis  and miR-ogenesis  have beeGenerally, miRNAs are thought to repress target mRNAs and act by destabilizing mRNAs through base pairing between the miRNA seed sequence (nucleotides at position 2–8) and a complementary sequence in the target mRNA . HoweverIn this study we used genome-wide small RNA sequencing to identify candidate miRNAs with possible roles in human endocrine cell differentiation. By comparing miRNA profiles of hPSC-derived pancreatic progenitors and human cadaveric beta and alpha cells genome-wide, we identified miRNAs that are induced during endocrine cell differentiation. Through gain-of-function experiments during hPSC differentiation, we show that islet cell-enriched miRNAs act to promote cell cycle exit and hence islet cell differentiation. Integrating RNA-seq, CLIP-seq, and chromatin state data, we applied a network modeling approach to identify candidate miRNA-regulated TFs that explain the impact of islet cell-enriched miRNAs on cell cycle regulation during endocrine cell differentiation. Our findings provide a systems-level view of how miRNAs regulate human endocrine cell differentiation, which has implications for programming islet endocrine cells from hPSCs or other cell sources.To identify miRNAs that are regulated during pancreatic beta cell differentiation, we conducted genome-wide small RNA sequencing in pancreatic progenitor cells derived from CyT49 human embryonic stem cells (hESCs)  and primTo identify mRNAs regulated by these miRNAs, we selected several miRNAs for over-expression in hESC-derived PE cells. We included the top three beta cell-enriched miRNAs , as well as Let-7g and miR-200a, as they are highly induced during endocrine cell differentiation. miR-7 was not included because of its inhibitory role in endocrine cell differentiation in mice .in vitro  were repressed by all five miRNAs, indicating distinct regulatory roles for each one of the miRNAs. Together, these results suggest distinct but converging miRNA targets in regulating cell division in pancreatic progenitors.To identify miRNA-regulated transcripts with likely roles in endocrine cell differentiation, we analyzed sets of genes that were down-regulated by forced expression of each miRNA  and also down-regulated in islets as compared with PE cells  lentiviral construct that drives the expression of Let-7g, Let-7a, miR-200a, and miR-375 under the control of a single promoter. miR-127 was excluded because overall it repressed fewer genes than the other miRNAs F. We expTo decipher mechanisms by which Let-7g, Let-7a, miR-200a, and miR-375 regulate cell cycle genes, we sought to distinguish direct and indirect targets of the four miRNAs A. We defCCND3, CDC45, MCM7, and CKS1B . In addition, TFs predicted to be directly regulated by the miRNAs included TFs involved in the regulation of endocrine cell development and maturation, such as NKX6.1 , whereas a decrease was associated with lower mRNA levels (p = 1.9 × 10−36). Finally, to construct the network, we linked open chromatin regions with dynamic histone marks to miRNA-regulated TFs by identifying those regions with a matching TF-binding motif. Validating our miRNA-gene regulatory network, GO analysis showed that the 1,307 genes comprising the network were enriched for cell cycle regulators  stage, when insulin present A. Sectio by forced miRNA expression   A and S7C SLC2A1) A, suggesHere, we identified 14 miRNAs , that are induced during human beta cell differentiation. We further studied five miRNAs  with a high fold-change during endocrine cell differentiation and experimentally show that these miRNAs induce cell cycle exit in pancreatic progenitor cells. By constructing an integrated miRNA-gene regulatory network of endocrine cell differentiation, we show that these miRNAs likely contribute to endocrine cell differentiation by directly regulating different sets of cell cycle-associated TFs.To analyze how islet cell-enriched miRNAs cooperate to drive endocrine cell differentiation, we developed a computational method to model the relationship of miRNAs, TFs, and miRNA-regulated genes. Our computational model builds on a previously published approach for constructing miRNA regulatory networks  and intemiR-375-deficient mice, which exhibit decreased beta cell proliferation   , which cNEUROG3) . We obse(NEUROD1 . Based of Cdkn1a , miRNA-min vitro system where growth factor conditions have been optimized for efficient beta cell differentiation. Therefore, the miRNAs might not be limiting in the context of these optimized conditions. Studies in model organisms underscore the idea that miRNAs confer robustness to developmental processes and become limiting only under conditions of stress. For example, loss of miR-7 has little effect on Drosophila sensory organ development under normal conditions, but when environmental stresses are added to the developing organism, miR-7 becomes necessary (Gain- and loss-of-function studies in model organisms have shown that the repressive effects of miRNAs on their targets is mostly modest, which has led to the view that miRNAs act to fine-tune gene expression. Consistent with this view, we observed relatively small effects of miRNA over-expression on gene expression, cell proliferation, and endocrine cell differentiation. However, these results do not mean that the miRNAs are not important for endocrine cell differentiation. We over-expressed islet cell-enriched miRNAs in an ecessary . Similarecessary . Furtherecessary . TherefoOne limitation of our approach for identifying endocrine cell differentiation-relevant miRNAs is the focus on miRNAs that repress mRNAs. There is evidence that miRNAs can activate gene expression or directly reduce protein levels . It is pAll methods can be found in the accompanying


For many years, conventional oncologic treatments such as surgery, chemotherapy, and radiotherapy (RT) have dominated the field of non-small-cell lung cancer (NSCLC). The recent introduction of immunotherapy (IT) in clinical practice, especially strategies targeting negative regulators of the immune system, so-called immune checkpoint inhibitors, has led to a paradigm shift in lung cancer as in many other solid tumors. Although antibodies against programmed death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) are currently on the forefront of the immuno-oncology field, the first efforts to eradicate cancer by exploiting the host's immune system date back to several decades ago. Even then, researchers aimed to explore the addition of RT to IT strategies in NSCLC patients, attributing its potential benefit to local control of target lesions through direct and indirect DNA damage in cancer cells. However, recent pre-clinical and clinical data have shown RT may also modify antitumor immune responses through induction of immunogenic cell death and reprogramming of the tumor microenvironment. This has led many to reexamine RT as a partner therapy to immuno-oncology treatments and investigate their potential synergy in an exponentially growing number of clinical trials. Herein, the authors review the rationale of combining IT and RT across all NSCLC disease stages and summarize both historical and current clinical evidence surrounding these combination strategies. Furthermore, an overview is provided of active clinical trials exploring the IT-RT concept in different settings of NSCLC. Radiotherapy (RT) has earned its place as one of three main pillars in non-small cell lung cancer (NSCLC) treatment, alongside surgery and systemic agents. Traditionally considered as a means of achieving local tumor control through induction of irreversible deoxyribonucleic acid (DNA) damage in irradiated tumor cells, RT is used in routine clinical practice across all NSCLC disease stages, whether with curative or palliative intent . HoweverClinicaltrials.gov was queried for currently active trials in the area of NSCLC combining both treatment modalities.For this review, we conducted a search of PubMed, Embase and Web of Science for original research, review articles and meta-analyses relevant to the combination of RT and IT in NSCLC from inception until March 2019, yielding a total reference count of 708. After removal of duplicates, 394 abstracts were screened by one reviewer, of which 62 qualified for full text screening. In case of multiple publications reporting on the same study population, manuscripts with the longest follow-up were selected for inclusion. The cited and citing references of the included studies were checked for additional relevant publications. Finally, a total of 42 published original research papers and abstracts were included and their results will be discussed below. Furthermore, using the search terms “radiotherapy,” “immunotherapy,” “immune,” “vaccine,” and “checkpoint,” the international clinical study database It has been over a decade since new insights into the complex interplay between cancer and the host's immune system, known as the cancer immunoediting hypothesis , have reThe principal mechanism of action of ionizing radiation is the induction of irreparable DNA damage in tumor cells—either directly or indirectly through free radicals. Under the right circumstances, radiation-damaged tumor cells may in turn undergo a phenomenon called “immunogenic cell death,” whereby an increased expression of calreticulin facilitates their phagocytosis by dendritic cells (DCs) and promotes the secretion of pro-inflammatory cytokines  5). In . In 5). in-situ vaccination, as demonstrated by the detection of TAA-specific CD8+ T-cells in the circulation of colorectal  per fraction] may elicit profound immunosuppressive responses in tumors. Such effects include recruitment of notoriously pro-tumorigenic myeloid-derived suppressor cells (MDSCs) and M2 tumor-associated macrophages (TAM), as well as a preferential increase of the regulatory T-cell (Treg) population, either independently, due to their intrinsic radioresistance or as a consequence of RT-induced upregulation of transforming growth factor beta , 17–20. lorectal  and proslorectal  receivinlorectal . For exalorectal . Other rlorectal , 24.Interestingly, the enhanced immunogenicity of increasing radiation doses does not seem to extend beyond a certain dose range . This coin vitro data demonstrate an increase in the MHC-I-associated peptide pool after approximately 8 h and up to 11 days or more following high doses of radiation , for instance, rely on the generation of a potent cellular immune response against a specific TAA. A prime example of this is tecemotide, a peptide vaccine containing a liposomal formulation of tumor-associated mucin 1 (MUC1), which is designed to elicit a MUC1-specific T-cell proliferative response. As the initial depletion of effector cell types from the tumor microenvironment caused by repeated moderate doses of RT could abrogate any previously induced immune infiltrate, ASI such as tecemotide may prove most useful when administered after RT, serving as a booster for the immune cells generated by Similarly, RT may be used to prime tumors in order to render them more susceptible to adoptive IT, such as lymphokine-activated killer cell (LAK), DC, cytokine-induced killer cell (CIK), tumor-infiltrating lymphocyte (TIL) and chimeric antigen receptor (CAR) T-cell therapy. Important work by Klug et al. demonstrated in murine as well as xenotransplanted and human primary tumors that even single low doses of RT ( ≤ 2 Gy) may be capable of reprogramming the tumor microenvironment—in particular polarizing TAMs to an M1 phenotype—causing inducible nitric oxide synthase-mediated vascular normalization, and thus facilitating tumor homing of transferred T-cells . In addiin-situ vaccination effect of RT. Additional benefits of ICB in the context of RT include: depletion of intratumoral Treg cells (anti-CTLA-4), counteracting RT-induced T-cell exhaustion through upregulation of PD-L1 (anti-PD-(L)1) and a reduction of MDSC populations in the tumor microenvironment (anti-PD-(L)1)  have garnered the most interest in recent years. Simply put, the rationale of a potential synergy between both treatments is that the inhibition of immune checkpoints, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1), liberates T-cells from immunosuppression, thereby increasing the PD-(L)1) . RT can PD-(L)1) . AltogetPD-(L)1) . NeverthPD-(L)1) .Less is known about the underlying mechanisms of interaction between RT and non-specific immunomodulatory drugs, such as levamisole, or so-called active immunotherapy , making it more difficult to theorize their optimal sequencing.As illustrated above, lymphocytes play a crucial role in antitumor immunity. Unfortunately, one of the major drawbacks of conventional oncological treatments is their tendency to cause systemic lymphopenia, which is increasingly being recognized to profoundly influence outcome. Indeed, several reports have shown radiation-induced lymphopenia (RIL) to be an independent predictor for poor survival in solid tumors , 34. MorIn addition to the direct hit of lymphocytes by RT, pre-clinical data suggest irradiation of the draining lymph nodes may also attenuate adaptive immune responses through altered intratumoral chemokine expression and CD8+ T-cell trafficking as compared to RT to the primary tumor alone . This inAdding to the list of OARs to consider, Price et al. demonstrated irradiating major areas of skin may also have detrimental effects on antitumor immune responses. Their experiments showed RT mobilizes Langerhans cells, a DC subset present in the epidermis, which may subsequently migrate to the draining lymph node where they cause an accumulation of Treg cells .On the other end of the benefit-risk balance of IT-RT strategies stands the possibility of increased toxicity of combined treatment. RT-induced immune-cell infiltration and subsequent inflammation-associated normal tissue responses underlying the occurrence of radiation-induced lung injury  warrant further inquiry into a potential interaction with immunotherapy . Most noApproximately one in four new NSCLC cases are diagnosed with locally-advanced (LA-NSCLC) disease . In thisThe earliest clinical data on IT-RT combinations in NSCLC date back to the 1970's and 80's, when immuno-oncology was still in its infancy. At the time, experience in leveraging the immune system in order to eradicate cancer cells was mostly limited to the use of non-specific immunostimulants.One in particular, BCG, had garnered substantial interest due to its encouraging results in hematologic malignancies, which soon inspired the development of several pre-clinical experiments and clinical studies in various other cancer types . SpecifiThe same allegedly applied to levamisole, a drug used as an anthelmintic before its immunotropic properties were discovered in animal cancer models. Several trials investigated levamisole in conjunction with (chemo)radiation, among which a phase III study initiated by the Radiation Therapy Oncology Group (RTOG), unfortunately showing no difference in OS and PFS compared to placebo, in both resectable and unresectable NSCLC –67.Meanwhile in Japan, research efforts were aimed at combining RT with other non-specific immunopotentiators, namely Picibanil (or OK-432) and polysaccharide K (PSK or Krestin). In their non-randomized trial, Ogawa et al. demonstrated an increased survival associated with the use of OK-432 and/or PSK in addition to chemo-radiotherapy, but did not report significance .in-situ vaccination, it seems increasing systemic exposure during RT may overstimulate these pathways, thereby inducing radiation damage where it is least needed. Whether this effect could be mitigated if RT doses were delivered more precisely and conformally is an interesting hypothesis but remains unproven.A decade later, the first clinical data on experimental RT regimens integrating interferons as radiosensitizers in LA-NSCLC started to emerge. Two smaller studies showed that combining natural alpha-interferon or human recombinant IFNɤ with hyperfractionated RT  resulted in a marked increase in severe treatment-related toxicity  , 70. WhiA final member of the non-specific immunomodulatory drug family, referred to as bovine dialyzable leukocyte extract, was evaluated concomitantly with chemoradiation in a phase I study, demonstrating its safety and ability to increase certain T-cell subpopulations .p = 0.016), as opposed to those who had received sequential chemo-radiotherapy . This was confirmed in the updated results published a few years later by Mitchell et al., adding exploratory biomarker analyses which demonstrated a potential positive predictive value of high blood levels of soluble MUC1 (sMUC1) for tecemotide therapy . As maintenance treatment after chemoradiation for stage III disease, the anti-MUC1 vaccine first demonstrated an acceptable safety and tolerability profile . Subsequ therapy . Given tMore recently, Patel et al. hypothesized that vascular endothelial growth factor-inhibitor bevacizumab may provide additional immunomodulatory benefits when combined with tecemotide following definitive chemoradiation for LA-NSCLC . Their pA second peptide-based vaccine studied in LA-NSCLC, called GV1001, is derived from the functional domain of human telomerase reverse transcriptase. Overexpression of this enzyme in cancer cells helps maintain the integrity of telomere sequences, thereby allowing them to avoid senescence. In their phase II trial of maintenance vaccination following radiotherapy and docetaxel for stage III NSCLC, Brunsvig et al. demonstrated a trend toward improved PFS in patients exhibiting a GV1001-specific T-cell response . The benn = 12) integrated RT into protocol treatment, adding the MAGE-A3 immunotherapeutic after sequential chemoradiation in unresectable stage III NSCLC. In addition to demonstrating an acceptable toxicity profile, a higher prevalence and magnitude of MAGE-A3-specific T-cell responses were observed following chemoradiation in comparison to other study arms not incorporating RT, suggesting irradiated tumor tissue may present an ideal substrate for this particular vaccine.The cancer/testis antigen, MAGE-A3, has also been proposed as a target for ASI in NSCLC and was investigated in one phase I trial . One of p < 0.001)  to conventional NSCLC treatments significantly improved 2-year OS  .p = 0.0025) across all prespecified subgroups -1 receptor interaction. First, consolidation pembrolizumab (anti-PD-1) after chemoradiation in patients with unresectable stage III NSCLC demonstrated increased 2-year PFS (44.6%) and time to metastatic disease or death  compared to historical controls , but theubgroups . This sun = 21) established that the addition of nivolumab (anti-PD-1) concurrently to chemoradiation followed by nivolumab maintenance, did not lead to the occurrence of grade ≥3 pneumonitis by 3 months post-RT (Table 3) are eagerly awaited.The proof of durvalumab's manageable safety profile as a sequential treatment after chemoradiation, in line with previous studies of ICB monotherapy, encouraged researchers to attempt combining all three treatment modalities concurrently. In the DETERRED trial, 40 LA-NSCLC patients were randomized 3:1 to receive standard chemo-radiotherapy with or without concomitant atezolizumab (anti-PD-L1) followed by atezolizumab maintenance treatment . No incr post-RT . Regrett post-RT . Furtherin-situ vaccination and the induction of abscopal responses, while debulking sites of gross disease in polymetastatic patients. IT, in turn, may reduce tumor loads or slow down the process of metastatic spread, creating a window of opportunity for local ablative treatments, such as SBRT.About half of all lung cancer patients are diagnosed with advanced disease . Though As in LA-NSCLC, research efforts into IT-RT combinations for the treatment of M-NSCLC date back to a time when ICB had not yet been developed. Studies with limited patient numbers have explored the potential benefit of different IT-RT combinations.Among the first experiments with RT and immunocytokine therapy in lung cancer demonstrated an acceptable safety profile and potential immunogenicity of Selectikine or NHS-IL2 , administered after local irradiation of a single pulmonary lesion in 13 M-NSCLC patients . Interesn = 18), four of whom demonstrated a partial (2/4) or complete (2/4) response in non-irradiated lesions. In addition, patients experiencing an abscopal response  showed significantly better OS (HR 2.06).Recognizing the importance of DCs in RT-induced immune activation, Golden et al. attempted to stimulate DC maturation by combining radiation with granulocyte-macrophage colony-stimulating factor (GM-CSF), ultimately aiming to elicit abscopal responses . Almost Similar to GM-CSF, the feline McDonough sarcoma-like tyrosine kinase 3 ligand CDX-301 has the ability to induce differentiation of bone marrow precursor cells into distinct subsets of DCs. Preliminary results from an ongoing trial investigating SBRT to an intrathoracic disease site and concurrent CDX-301 in M-NSCLC are promising, with more than half of patients (5/9) achieving partial remission . Of noteThough disappointing phase III results, such as those of the START trial in LA-NSCLC , 73 and The largest body of available IT-RT evidence in M-NSCLC describes the marriage of radiation and ICB. While Formenti et al. performed a pioneering study on abscopal effects with ipilimumab , researcEarly results of two phase I trials, comparing different—radical or palliative—RT dose-fractionation schedules given concurrently with escalating doses of pembrolizumab, demonstrated acceptable toxicity of combined treatment and encouraging disease control rates (DCR) of up to 68% , 90. MorEarly safety data on the concurrent use of other anti-PD-1 drugs  with RT appear to be reassuring , althougp = 0.04). Moreover, subgroup analyses showed patients benefiting most from SBRT were those with PD-L1 negative tumors at baseline. This finding is particularly intriguing, since this is a population for which single-agent PD-(L)1 inhibition is known to be of limited benefit. Perhaps, pembrolizumab following SBRT may represent a less toxic alternative to chemoimmunotherapy when aiming to enhance response rates in M-NSCLC patients with a low PD-L1 tumor proportion score.Sequential approaches were also evaluated, initially for nivolumab , but morDespite promising results, some authors have advocated abandoning the single-site abscopal approach and instead propose to irradiate as much of the tumor burden as can be safely achieved . They hyEarly-stage NSCLC (ES-NSCLC) represents about 15–20% of all new lung cancer diagnoses . AccordiAs of yet, the lion's share of evidence demonstrating clinical benefit with combined immunotherapy-radiotherapy strategies for NSCLC is situated in locally-advanced disease. Even so, a growing body of pre-clinical and clinical data has allowed further insights into their synergy, providing a strong rationale for extending this potential to other disease entities, such as metastatic NSCLC. In addition, the excellent tolerability profile of many novel immunotherapeutic drugs, both as monotherapy and in conjunction with radiotherapy, as well as technological and technical radiotherapy advances have created a window of opportunity for the development of combined strategies in earlier disease stages. Nevertheless, the success of future trials will require well-reasoned hypotheses for radiotherapy timing, dose and fractionation, in addition to selection of the appropriate partner immunotherapy.MS performed the literature review and search of ongoing clinical trials, on which both authors structured and synthesized the evidence for the manuscript. YL critically revised all the drafts and approved the final version for submission.YL reports expert positions for AstraZeneca and RaySearch, outside the current work. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While allergen exposure in sensitized subjects mainly boosts IgE production and T cell activation, successful allergen-specific immunotherapy (AIT) induces the production of allergen-specific IgG antibodies and reduces T cell activity. Under both circumstances, the resulting allergen-antibody complexes play a major role in modulating secondary allergen-specific immune responses: Allergen-IgE complexes induce mast cell and basophil activation and perpetuate allergen-specific T cell responses via presentation of allergen by allergen presenting cells to T cells, a process called IgE-facilitated antigen presentation (FAP). In addition, they may induce activation of IgE memory B cells. Allergen-induced production of specific IgGs usually exerts ameliorating effects but under certain circumstances may also contribute to exacerbation. Allergen-specific IgG antibodies induced by AIT which compete with IgE for allergen binding  inhibit formation of IgE-allergen complexes and reduce activation of effector cells, B cells and indirectly T cells as FAP is prevented. Experimental data provide evidence that by binding of allergen-specific IgG to epitopes different from those recognized by IgE, allergen-specific IgG may enhance IgE-mediated activation of mast cells, basophils and allergen-specific IgE+ B cells. In this review we provide an overview about the role of allergen-specific antibodies in regulating secondary allergen-specific immune responses.Immunoglobulin E (IgE)-associated allergy is the most common immunologically-mediated hypersensensitivity disease. It is based on the production of IgE antibodies and T cell responses against  Besides environmental factors also genetic factors are very important for the development of IgE responses. They include genetic factors promoting Th2 immune responses and IgE production in general as well as genetic restriction of allergen-specific immune responses –3. Allerpatients . The effpatients  may alsopatients  and assopatients , 10 lead4 and IgG1 subclass, is observed both in serum  is the only disease-modifying treatment in allergy with long lasting clinical effects and modulation of the allergic immune response , 12. Thein serum –19 as wein serum , 21. AITin serum . Though otherapy . Importanditions , 25. Idenditions . By blocnditions , 27.Already in 1903, long before allergy was recognized as an immunologically-mediated hypersensitivity disease, Dunbar demonstrated that allergic reactions in patients could be ameliorated when the disease-causing allergens were neutralized with an allergen-specific antiserum  Figure  and beca+ B cells and long-lived IgE plasma cells in human subjects are still debated  that has previously been internalized by fluid phase endocytosis Figure . Howeverpatients , 57–60. patients , 62. In patients  and havepatients . Surfacepatients . Though patients . The latpatients , CD23-mepatients , 57, 65.o by 50% . In addio by 50% . Furthero by 50%  as discu+ memory B cell subsets was suggested  upon binding. This potential mechanism of further oligomerizing IgE-allergen-complexes by allergen-specific IgG may be called “super-crosslinking” and obviously will depend on and vary with the concentrations, affinities and specificities of allergen-specific IgG . Se. Se+ memG Figure –77. For G Figure –80. Bet G Figure  and wereG Figure . Alternauggested . Using PThe presence of an allergen-specific “inhibiting or immune type of substance” in post immunotherapy sera was first reported by Cooke and colleagues in 1935 Figure . They paAllergen binding to IgE bound to FcεRI on the surface of mast cells and basophils induces cross-linking of the receptors and hence activation of these cells . The int1 and IgG4 subclass. Interestingly, this vaccine did also not induce an initial rise of IgE levels as observed during allergen extract-based SCIT and SLIT.Despite an initial transient rise in IgE levels upon start of subcutaneous immunotherapy (SCIT) with extracts, the allergen-specific IgE increase observed after seasonal natural pollen exposure is blunted during SCIT , 93–95. As an alternative to subcutaneous application, allergen extract can also be applied in a sublingual form (SLIT). However, direct comparison of SLIT and SCIT vs. placebo for a period of 3 years did not reveal any effect of SLIT on seasonal changes in allergen-specific IgE levels in contrast to SCIT .+ BCR and thus reducing the activation of memory IgE+ B cells ., 4.+ BCRSuccessful AIT is also associated with a reduction in the T cell-mediated late phase responses in target organs of allergy such as the skin, lung and nose , 102–1044 antibodies. IgG1 may have the same effects but this has not been studied in depth  which leads to the establishment of an allergic immune response. However, there is compelling evidence that once allergic sensitization has occurred in an allergic individual, the secondary allergen-specific IgE and T cell response is regulated by allergen-specific IgE and IgG antibodies. It has been demonstrated that allergen-specific IgG antibodies which are for example induced during AIT and are directed against the IgE binding sites of allergens can prevent allergen-induced immediate and delayed allergic inflammation as well as specific IgE production. This can be achieved by inhibition of allergen-induced cross-linking of IgE bound to high affinity receptors for IgE on mast cells and basophils, by inhibition of IgE-facilitated allergen presentation to T cells and by preventing boosts of allergen-induced IgE production by IgE memory B cells by blocking IgG antibodies. Much less is known about the potential role of allergen-specific IgG in enhancing allergen-specific immune responses but it is possible that under certain conditions allergen-specific IgG may super-crosslink allergen-IgE immune complexes on B cells, mast cells, basophils and eventually APCs and thus contribute to boosting of secondary allergen-specific IgE production in the absence of T cell help, to the enhancement or blocking of allergen-induced basophil and mast cell activation and may also have as yet unknown effects on IgE-facilitated allergen presentation and T cell activation. The role of the latter mechanisms and their biological effects need further investigations.JE-D, SV-M, BL, and RV wrote the manuscript. JE-D, RV and SV-M designed the Figures and Table. AK, YZ, MK, and VN-L critically read and revised the manuscript.RV has received research grants from Biomay AG and Viravaxx, Vienna, Austria and serves as a consultant for these companies. The remaining 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.


Bacteriodetes hosts. CrAssphage, the first crAss-like phage to be discovered, contains a protein encoding a Bacteroides-associated carbohydrate-binding often N-terminal (BACON) domain tandem repeat. Because protein domain tandem repeats are often hotspots of evolution, BACON domains may provide insight into the evolution of crAss-like phages. Here, we studied the biodiversity and evolution of BACON domains in bacteriophages by analysing over 2 million viral contigs. We found a high biodiversity of BACON in seven gut phage lineages, including five known crAss-like phage lineages and two novel gut phage lineages that are distantly related to crAss-like phages. In three BACON-containing phage lineages, we found that BACON domain tandem repeats were associated with phage tail proteins, suggestive of a possible role of these repeats in host binding. In contrast, individual BACON domains that did not occur in tandem were not found in the proximity of tail proteins. In two lineages, tail-associated BACON domain tandem repeats evolved largely through horizontal transfer of separate domains. In the third lineage that includes the prototypical crAssphage, the tandem repeats arose from several sequential domain duplications, resulting in a characteristic tandem array that is distinct from bacterial BACON domains. We conclude that phage tail-associated BACON domain tandem repeats have evolved in at least two independent cases in gut bacteriophages, including in the widespread gut phage crAssphage.The human gut contains an expanse of largely unstudied bacteriophages. Among the most common are crAss-like phages, which were predicted to infect  Bacteriophage populations are essential for stability and proper functioning of the human gut microbiome . Gut phaBacteroides-associated carbohydrate-binding often N-terminal (BACON) domains [Bacteroidetes bacteria, but despite their name, a direct role in carbohydrate-binding for BACON domains is yet to be proven. The only currently published BACON domain X-ray crystallography structure in human gut Bacteroidetes indicated that they form links between carbohydrate-binding domains and are anchored onto the surface of Bacteroides cells and do not perform a carbohydrate-binding function themselves [One characteristic crAssphage protein (gp64 in NC_024711.1) encodes a tandem repeat of eight  domains . BACON demselves . This X-emselves . This maemselves . Phage iemselves  and carbemselves , and areemselves ,17, p2 [emselves , HK620 [emselves , p22 [20emselves  and Sf6 emselves . LikewisProteins with domain tandem repeats are found throughout the tree of life . BecauseHere we studied the evolution of BACON domain tandem repeats in crAss-like phages. First, we constructed a profile hidden Markov model (HMM) of the crAssphage-like BACON domain (crAss-BACON), which we identified in half of the known candidate crAss-like phage lineages and in two novel gut phage lineages. Tandem repeats of the crAss-BACON were exclusive to two crAss-like phage candidate genera and one of the novel lineages, and were always genomically flanked by tail proteins. In phage lineages with single (non-repeated) crAss-BACONs, they were associated with the replicative and head regions of the phage genome. By studying crAss-BACON evolution, we showed that crAss-BACON tandem repeats are divergent from bacterial domains, whereas individually occurring crAss-BACON domains are more closely related to bacterial sequences, indicating frequent horizontal transfer between viral and cellular organisms. Finally, we showed that the crAss-BACON tandem repeat in the most widespread crAss-like phage lineage has evolved through multiple single-domain duplications.Bacteroidetes bacteria, was downloaded from the Pfam database v32.0 on 1 April 2019 [We identified BACON domains across a broad range of viruses using two datasets that included both genomic and metagenomic sequences. The first dataset consisted of 661 crAss-like phage sequences from five recent publications ,10,11,29ril 2019 . The 304All bioinformatics tools mentioned below were run with default settings and cut-offs, except where stated explicitly.Before searching for BACON domains, we predicted all ORFs in both datasets of viral sequences using Prodigal v2.6.1  with trap-values was constructed with the R function dist and used for clustering with the Ward.D2 algorithm as incorporated in R. The optimal number of clusters was determined using the NbClust v3.0 R package [To obtain a tentative taxonomical classification of the crAss-BACON-containing contigs, we clustered them based on shared protein content ,38,39. T package , whereas package .To analyse the conservation of the cluster 1 crAss-BACON tandem repeat, all cluster 1 crAss-BACON ORFs with eight domains were selected and aligned with Clustal Omega v1.2.1 . InformaMost crAss-BACON-containing contig clusters contained members of previously defined candidate crAss-like phage subfamilies and genera, except clusters A and B which consisted of highly dissimilar viral sequences (see Results and Discussion). To provide an initial sequence-based characterization of these novel viruses, the largest contigs from each cluster were extracted and annotated using the PROKKA v1.11 software tool , with thTo further study the relation of the newly described contig clusters to known crAss-like phages, we performed a phylogenetic analysis of crAss-like phage head proteins. Five crAss-like phage head proteins that were previously used to determine crAss-like phage taxonomy  were extHosts were predicted for all crAss-BACON contigs with a length over 75,000 bp using the host prediction algorithm WiSH v1.0 . This prE-value ≤10−5) to proteins with a “hypothetical protein” function description. Because this provided no additional insight into the role of these 121 crAss-BACON ORFs, they were discarded from the analysis. All other neighbourhoods were plotted using the ggplot2 v3.1.0 R package.To study the genomic neighbourhood of crAss-BACON ORFs, these plus five ORFs up- and downstream were collected. All ORFs were queried against the NCBI non-redundant protein sequences collection  with BLATo study crAss-BACON evolution, we constructed an approximate maximum likelihood tree. CrAss-BACONs were first aligned with Clustal Omega v1.2.1 . While cTo analyse whether the cluster 1 crAss-BACON array had evolved through simultaneous duplication of multiple domains, the eight domains from crAssphage were subjected to a BLASTp all versus all using BLAST+ v2.9.0 , and bitBacteroides-derived BACON domain that was available in Pfam (PF13004) [We started our study of BACON domain diversity and evolution by constructing a profile HMM distinctive to crAssphage-like BACON domains (crAss-BACONs). The PF13004)  containePF13004) ,27 out oPF13004) . These aAlpha-, Beta-, Gamma- and Deltacrassvirinae, but not in all ten candidate genera. Candidate genera I, II, III, V, VI and VII contained crAss-BACONs, whereas IV, VIII, IX and X did not. Alphacrassvirinae is the most widespread subfamily that contains candidate genus I and the prototypical crAssphage. This subfamily contains two candidate genera with crAss-BACONs (I and III) and two without it (IV and IX). BACON domains are particularly prevalent in candidate genus I, where 55 of the 63 genomes that were previously categorised in this genus contain crAss-BACONs. In candidate genus III, 7 of the 22 genomes contain crAss-BACONs. Likewise, Deltacrassvirinae contains one candidate genus with crAss-BACONs  and two without it (VIII and X). We found crAss-BACONs in Betacrassvirinae , which contains the first isolated crAss-like phage [Gammacrassvirinae . Thus, crAss-BACONs were absent from some lineages, even though the search space contained genome sequences from all established candidate crAss-like phage genera that a previous publication showed were (near-)complete [The dataset in which we searched for BACON domains included contigs from an earlier study that presented a preliminary crAss-like phage taxonomy . That stcomplete . We concn = 2), chicken ceca (n = 2), sheep rumen (n = 1) and wombat intestines (n = 1), as well as three contigs originating from human oral metagenomes [As BACON domains were found in taxonomically diverse crAss-like phages, we next determined how widespread they were in other phages. For this, we queried 2,147,193 viral sequences from seven metaviromics studies for the crAss-BACON profile HMM b and ideagenomes   and Klebsiella pneumoniae (14 ORFs). Multiple cluster B ORFs hit phage structural proteins located in bacterial genomes, including three in A. baumannii, one in K. pneumoniae, one in Desulfovibrio alaskensis and one in Rhodobacteriacea. Like the cluster A contig, the cluster B contig may thus have a temperate lifestyle. The high number of ORFs with hits in Proteobacteria, coupled with only three ORF hits to Bacteroidetes species, could indicate that this cluster B phage infects Proteobacteria, a different phylum than the crAss-like phages [Bacteroidetes as previously thought [Bacteroidetes hosts for cluster B contigs, consistent with crAss-like phages [To further analyse the phage lineages in clusters A and B, we functionally annotated the protein-encoding genes on the longest sequences from clusters A and B. These contigs were 154,123 bp (cluster A) and 87,116 bp (cluster B) long and contained 180 and 110 ORFs respectively . These phages infect four different phyla and are unrelated to crAss-like phages. The above results show that cluster A and B genomes likely represent newly described phage lineages.Few ORFs in either the cluster A or B contigs are homologous to phage proteins in the RefSeq database see . Of the After establishing that crAss-BACONs are widespread in crAss-like phages and beyond, we next looked at differences in crAss-BACON architecture among the phage clusters. Domain tandem repeats in cellular organisms tend to rapidly evolve following domain duplications, after which they remain stable and conserved within species . The tanOther crAss-BACON architectures were found outside of cluster 1 where all crAss-BACON-containing ORFs had fewer than eight domains. Most crAss-BACON ORFs in clusters 2, 5 and 6 contain a single domain a. MultipE-value of 6 × 10−8) and a putative T9SS sorting domain-containing protein , were solely based on an alignment to BACON domains. To circumvent this lack of detectable sequence similarity of crAss-BACON ORFs to proteins with predicted function, we instead analysed their genomic neighbourhoods , the genomic regions that contained crAss-BACON ORFs had high sequence similarity, whereas the rest of the phage genomes did not . This suTo investigate BACON domain evolution in phages and bacteria, we created a phylogeny of 1509 BACON domains a. This iThe crAss-BACONs in the cluster 7 and B tandem arrays showed a greater spread in the tree c,d. LikeAlphacrassvirinae candidate subfamily that contains the longest and most conserved crAss-BACON tandem array  belongsAlphacrassvirinae). In this lineage, we showed how a stable tandem repeat of eight crAss-BACONs has resulted from multiple single domain duplication events. The presented results show that focus on uncharacterised proteins can provide insight into the enormous biodiversity and evolutionary dynamics of the viral world.We investigated the evolution of BACON domain tandem repeats in crAssphage and other gut bacteriophages. We demonstrated that crAss-like phage BACON domains (crAss-BACONs) are widespread in the crAss-like phage family, and we identified crAss-BACONs in two novel gut phage lineages. We further found that crAss-BACON tandem repeats are associated with tail fibres in three gut phage lineages. In two lineages (clusters 7 and B), these repeats are the result of horizontal transfer and tandem duplication. The third lineage with tail-associated crAss-BACON tandem repeats includes the prototypical crAssphage (cluster 1, candidate subfamily