Pseudomonas aeruginosa efflux pumps involved in the development of antibiotic resistance. Several studies developed with laboratory strains or using a few clinical isolates have reported that the regulation system of MexEF-OprN is involved in the final levels of MexAB-OprM expression. Therefore, this study was aimed to determine the interplay between MexAB-OprM and MexEF-OprN in 90 out of 190 P. aeruginosa clinical isolates with an efflux pump overexpression phenotype. Regarding oprD, 33% (30/90) of isolates displayed relevant modifications (RM) defined as frameshift or premature stop, both related to carbapenem resistance. On the other hand, 33% of the isolates displayed RM in nalC, nalD or mexR, which were significantly associated with multidrug resistance (MDR), non-susceptibility to carbapenems, OprD alterations and strong biofilm production. Meanwhile, the RM in MexS were associated with presence of pigment (p = 0.004). Otherwise, when all the regulators were analysed together, the association between RM in MexAB-OprM regulators and MDR was only significant (p = 0.039) when mexS was the wild type. These data show the modulatory effect of MexEF-OprN on MexAB-OprM in a clinical population of P. aeruginosa. Further studies may contribute to design of novel molecules acting on this interplay to fight against antimicrobial resistance.MexAB-OprM and MexEF-OprN are  Pseudomonas aeruginosa is an opportunistic human pathogen characterised by intrinsic resistance to a variety of antimicrobial agents. This property results from the interplay between drug efflux systems and the low outer membrane permeability of this microorganism4. P. aeruginosa possesses at least 12 structural genes for multidrug efflux pumps belonging to the resistance – nodulation – cell division (RND) family of transporters2. Of these, MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM efflux pumps have shown to be of clinical relevance5. The MexAB-OprM efflux system contributes to the intrinsic resistance of this organism to quinolones, tetracycline, chloramphenicol, novobiocin, macrolides and β-lactams, and its overexpression confers cross-resistance or reduced susceptibility to several antibiotics6. In addition, it has been reported that MexAB-OprM exports quorum-sensing mediators such as acylhomoserine lactones including N-butyryl-L-homoserine lactone (C4-HSL), which induce the production of virulence factors, including proteases, rhamnolipids, exotoxin A, exoenzyme S, and pyocyanin7. On the other hand, the MexEF-OprN system is not expressed during growth, and under laboratory conditions it is expressed in nfxC multidrug-resistant mutants2.9, whereas MexEF-OprN expression is enhanced by a positive regulator, MexT, and impaired by MexS expression7. In addition, MexT down-regulates oprD, the gene encoding the porin OprD which is used by imipenem for cell entry11.The presence of mutations in MexR, NalC and NalD repressors of MexAB-OprM up-regulate its expressionP. aeruginosa clinical isolates, suggesting alternative regulation pathways13, the regulation system of MexEF-OprN is also involved in the final levels of MexAB-OprM expression1. Thus, it has been proposed that MexT down-regulates MexAB-OprM14. Nonetheless, further studies by Richardot et al.15 have shown that equivalent levels of mexT expression may or not drive to down-regulation of the mexB gene. This mexB down-regulation was associated with overexpression of mexE related to the lack of functionality of mexS, suggesting a more complex interrelation between the two efflux pumps15. In this sense, the NfxC type mutants (overexpressing MexEF-OprN) exhibit low-level production of MexAB-OprM and virulence factors including a lower ability of biofilm formation16.Although, the concomitant overexpression of both efflux systems have previously been described in several 17. Nonetheless, this finding may also cause differences in virulence, antimicrobial resistance or specific properties1.This inverse relationship between the expression of MexAB-OprM and the expression of MexEF-OprN could reflect an overlapping of antimicrobial substrates or similar cell-associated extruded products by any of these systems16, however most of them have mainly been developed in laboratory strains or a few clinical isolates. The purpose of this study was to determine the interplay between MexAB-OprM and MexEF-OprN in antimicrobial resistance, the oprD gene, biofilm formation, swarming motility and pigment in a wide variety of clinical isolates of Pseudomonas aeruginosa from two Peruvian hospitals.Overall, several studies have proposed the interaction between the two efflux systemsOverall, 58% (110/190) of the isolates were non-susceptible to levofloxacin (LVX), with no differences between the two hospitals ; 55% (62/112) in HNCH and 62% (48/78) in HAL. All isolates grew in the presence of phenylalanine-arginyl ß-naphthylamide (PAβN). The EPO phenotype was observed in 47% (90/190) of the isolates, with similar values in HNCH and HAL .p = 0.0006). In addition, the EPO phenotype was associated with LVX and carbapenem susceptible isolates   may be easily detected using a fluoroquinolone such as LVX together with an efflux pump inhibitor (EPI)1. Nonetheless, the present results agree with a limited effect of efflux pumps, by itself and in the absence of other mechanisms, on the change of the clinical strain classification from Susceptible to Intermediate / Resistant1. Additionally, these results may reflect the different substrate affinities presented by different efflux pumps1 and the different final effect on minimum inhibitory concentration (MIC) levels related to each P. aeruginosa efflux pump.The present data demonstrate an inverse relationship between multidrug resistance (MDR) and EPO  of isolates displayed amino acid changes or deletions which did not affect the OprD frameshift. Thus, 7 isolates showed sequences identical to the P. aeruginosa PAO1 strain, 35% (21/60) isolates showed punctual mutations, and 53% (32/60) of the isolates displayed the amino acid deletions S373/G383. These amino acid deletions were presented in addition to several punctual mutations including V127L, E185Q, P186G, V189T, E202Q, I210A, E230K, S240T, N262T, T276A, A281G, K296Q, Q301E, R310E/G, G312R, A315G, K347M, V359L, S403A, Q424E as well as a series of changes between amino acid 372 and 383 (372V-DSSSSYAGL-383). Eighteen additional isolates presented these mutations and also possessed relevant modifications. Overall, punctual mutations were presented conforming sets , V359L, (±Q424R) + 372V-DSSSSYAGL-383 , T103S, K115T, F170L, E185Q, P186G, V189T, R310E, A315G, (±G425A)  and V127L, E185Q, P186G, V189T, E202Q, I210A, E230K, S240T, N262T, T276A, A281G, K296Q, Q301E, R310E, (±G312R), A315G, L347M, S403A, Q424E + 372V-DSSSSYAGL-383  were the most frequently detected. It has been described that these types of mutations have no effect on the development of resistance to carbapenems, including several of the most frequently found in present isolates such as T103S, K115T, F170L, E185Q, P186G, V189T, R310E, A315G, G425A (15 isolates) or T103S, K115T, F170L (4 isolates) in OprD18. Furthermore, the presence of the alteration 372V-DSSSSYAGL-383, shortening loop L7, has been related to increased susceptibility to meropenem20; therefore, these types of alterations were classified as “irrelevant modifications” when presented alone. The presence of a potential association has been suggested between specific amino acid substitutions in OprD and MLST profiles, observing a series of amino acid deletions plus a set of punctual mutations on analysing 12 isolates belonging to the ST11121. This pattern of OprD amino acid substitutions was almost concordant with the present pattern C, having also strong similarities with pattern A. Therefore, despite the absence of specific MLST determinations, the present results suggest the relevant presence of this high risk P. aeruginosa clone in the area. In a previous study22, the clonal relationships among these isolates was established, observing a high diversity . Nonetheless, 72.2% (13/18) of the isolates classified within pattern C, suggestive of belonging to ST111 were from HCNH while the remaining 27.7% (5/18) were from HAL, accounting for 26% and 12.5% of the isolates analysed from each hospital. This finding is in accordance with Kim et al.21 who described differences in the prevalence of ST111 between different hospitals from the same area. In addition, another common set of amino acid substitutions (pattern B) very similar to those reported by Kim et al. 21 for P. aeruginosa ST298 and ST308 was also detected in 19 isolates.The p < 0.0001) . In this sense the presence of disruption of the oprD gene by different ISs including the ISPa27, ISPa45, ISPa46, ISPa47, ISPa133, ISPa1328, ISPa1635, ISPst12 or ISPpu21 has previously been shown24.On the other hand, alterations affecting porin functionality  were classified as “relevant modifications”. Overall, 33% (30/90) of the isolates showed relevant modifications; 40% 12/30) presenting frameshifts by base pair insertions and 47% (14/30) possessing premature stops in amino acid codons 65 and 49, and in four isolates no PCR amplification was obtained. Overall, the relevant modifications were strongly associated with carbapenem non-susceptible isolates  of the isolates showed punctual mutations leading to amino acid changes, being V126E [98%(41/42)] the most frequent; 43% (39/90) of the isolates did not have any modification, and the remaining 10% (9/90) of isolates repeatedly did not amplify by PCR assay. Difficulties in PCR amplification may have been due to the presence of polymorphisms in the primers annealing regions, or the presence of internal DNA sequence modifications resulting in specific DNA conformation which impaired PCR amplification. However, the non amplification of the mexR gene was probably associated with the presence of a disrupting IS, such as IS21, which has previously been described as breaking mexR and leading to an increased transcription of the mexAB-oprM operon25.Three MexAB-OprM regulators were analysed. Regarding the nalC gene, 87% (77/90) of the isolates showed punctual mutations, being G71E (76/77) and S209R (67/77) the most frequent. In addition, one isolate showed a 10 base pair deletion from C234 to G243. Regarding nalD, 71% (64/90) of the isolates did not show modifications, and 20% (18/90) showed relevant modifications, being two base pair deletions (Δnt397–398) the most frequent in 39% (7/18) of the isolates  of the isolates displayed relevant modifications in the mexR, nalC or nalD genes which were significantly associated with MDR (p < 0.0001), carbapenem non-susceptible isolates (p < 0.0001) and relevant modifications of the oprD gene (p < 0.0001). In addition, these relevant modifications were significantly associated with strong biofilm producer (SBP) isolates (p = 0.006) . The remaining 5.5% (5/90) of the isolates did not amplify the mexT gene. Meanwhile, 59% (53/90) of the isolates showed N249 in the mexS gene, being identical to the sequence of PA14. Only 3 of these isolates carried a single amino acid change in positions V73A (2) and G224S (1). All were considered as fully functional and able to inhibit the expression of MexEF-OprN (Table 15. The remaining 41% (37/90) of the isolates did not amplify the mexS gene  the mexS and mexT genes identical to those of PA14 (47 isolates), and ii) the non amplified mexS gene and mexT gene identical to that of PA14 (34 isolates). The remaining scenarios were not further analysed due to the small numbers of isolates presenting the required characteristics (9 isolates). The second combination was significantly associated with the presence of pigment (p = 0.004)  Fig. .mexS and mexT were classified as irrelevant modifications. Nonetheless, a direct effect of the amino acid substitutions detected on the functionality of these regulators cannot be ruled out15. Thus, the analysis of isolate 1096 presenting the amino acid change G224S showed mexE expression levels of 0.13, suggesting an enhancement of the inhibitory activity of MexS instead of a loss of MexS function. Meanwhile, in isolate 1088 carrying amino acid changes in MexT , the mexE expression values were of 0.64, within the range of isolates 1083, 1090, 1092,1093, 1099 which did not carry alterations in both MexS and MexT.As mentioned above, amino acid changes both in mexE gene expression analyses showed that no amplification of mexS only correlated with mexE overexpression in 2 isolates (1087 and 1095). In isolate 1095, in which the expression levels of mexE were of 8.92, both N- and C-terminal regions were amplified while in isolate 1087 (mexE expression levels of 1.98) a PCR product was only obtained by amplifying the N-terminal region. In both cases, this may have occurred due to the presence of an IS disrupting the mexS gene27. Meanwhile, in other 6 isolates  no deregulation of mexE was observed (mexE expression levels ranging from 0.53 to 1.16), despite the presence of a fully functional mexT, thereby suggesting the presence of polymorphisms in mexS primer annealing regions and/or specific internal DNA conformation hampering PCR amplification, although a possible impairment of MexT activity in isolate 1088 related to specific amino acid changes cannot be ruled out. Nonetheless, it should be mentioned that a similar scenario of non mexE overexpression in the presence of fully functional MexT and inactive MexS has previously been described12. In the remaining isolate analysed (1091) with relevant alterations in mexS, the mexE expression levels of 0.37 were concordant with the absence of mexT amplification, which as mentioned above might be related to the presence of a disrupting internally inserted sequence  expression of mexE in which it was only possible to amplify the initial 462 bp of mexT, suggesting major mutations affecting this region26. Finally, in isolates 1086 and 1100 a clear overexpression of mexE (expression levels of 7.92 and 4.81 respectively) was observed, despite the mexS and mexT genes being identical to those of PA14  when mexS was wild type, and therefore able to exert a negative regulation effect on the expression levels of MexEF-OprN. Furthermore, in 2 out of 3 isolates (isolates 1095 and 1100) in which the presence of relevant modifications in the mexAB-OprM regulators did not result in mexA overexpression (expression levels of 1.04 and 0.8 respectively), the expression levels of mexE were of 4.81 and 8.92 (Table Previous studies have reported that C4-HSL induces the expression of the t p = 0.09 when memexA (expression levels of 5.10) and mexE (expression levels of 7.92)  Table , which amexS amplification, highlighting the modulatory effect of mexEF-OprN overexpression on the final resistance phenotype.Overall, the present data showed a relevant role of modifications leading to the loss of MexR, NalC and NalD functionality in the clinical isolates analysed, which were associated with higher levels of antibiotic resistance and different bacterial virulence including biofilm formation. The effect of these modifications on multidrug resistance levels was significantly higher in the presence of P. aeruginosa from clinical samples of patients attended at the HAL (78 isolates) and the HNCH (112 isolates) in Lima (Peru), from December 2012 to June 2013. In all cases only non-duplicated isolates from different patients were included in the study. The isolates were stored at −70 °C in skim milk medium  until use. The clonal relationships, carbapenem susceptibility and MDR levels, biofilm formation, swarming motility and pigment presence were determined in a previous study22. In all cases antibiotic susceptibility was classified, according CLSI breakpoints39. MDR was defined as resistance to three or more unrelated families of antibiotics . The isolates intermediate or resistant to both imipenem and meropenem were classified as “carbapenem resistant”. Throughout the text the term “resistance” englobes resistant and intermediate isolates.We studied a total of 190 isolates of 39 both with (MICPAβN) 20 µg/ml and without (MICI) of PAβN. An EPO phenotype was defined when MICI/MICPABN was >2 as previously described40. The effect of this concentration of PAβN on the viability of microorganisms was also assessed.EPO was established by determining the effect of the EPI PAβN  on the MICs of LVX. Thus, the MIC of LVX was established by the agar dilution methodoprD gene was performed by PCR .Amplification of the CR Table 41. NegatmexR, nalC, nalD, mexT and mexS were designed by Solé et al.9 with slight modifications of the annealing conditions . However the mexT and mexS genes were analysed according to the full functional MexS and MexT of P. aeruginosa PA14 (GenBank: CP000438), because PAO1 lacks the functionality of those genes related to the presence of 8 bp insertion resulting in a frameshift in MexT and D249 in MexS15. To determine the presence of undetected insertions within to the mexS gene, a PCR strategy was designed. Briefly, in those isolates in which PCR product was repeatedly not obtained, two new PCR reactions were designed in order to amplify the N- and C-terminal regions, respectively  or PA14 (mexE).The expression levels of ed Table 13,42. In2 (Chi square test) was used to determine the presence of significant differences which were considered with a p value of ≤0.05. R studio version 3.4.0.was used for all statistical analyses. Resistant and intermediate isolates were classified together as “non-susceptible” for statistical analyses.The χThe study was approved by the Ethical Committee of the Universidad Peruana Cayetano Heredia  and by the Ethical Committee of Hospital Clinic , and all experiments were performed in accordance with relevant guidelines.All samples were obtained within routine clinical practice; no personal data was requested or available to researchers.


Butyrophilins (BTNs) are a group of the moonlighting proteins, some members of which are secreted in milk. They constitute a large family of structurally similar type 1 transmembrane proteins from the immunoglobulin superfamily. Although the founding member of this family is related to lactation, participating in the secretion, formation and stabilization of milk fat globules, it may also have a cell surface receptor function. Generally, the BTN family members are known to modulate co-stimulatory responses, T cell selection, differentiation, and cell fate determination. Polymorphism of these genes was shown to be associated with the pathology of several human diseases. Despite their biological significance, structural information on human butyrophilins is rather limited. Based on their remarkable multifunctionality, butyrophilins seem to belong to the category of moonlighting proteins, which are known to contain intrinsically disordered protein regions (IDPRs). However, the disorder status of human BTNs was not systematically investigated as of yet. The goal of this study is to fill this gap and to evaluate peculiarities of intrinsic disorder predisposition of the members of human BTN family, and to find if they have IDPRs that can be attributed to the multifunctionality of these important proteins. BTN genes encodes 14 proteins that include seven BTNs grouped into three subfamilies , five BTN-like (BTNL) proteins , an erythroid membrane-associated protein (ERMAP), and a myelin-oligodendrocyte glycoprotein (MOG) [BTN genes are distributed within the juxta-telomeric region of the major histocompatibility complex class I (MHCI), which contains a single copy of the BTN1A1 gene and the BTN2 and BTN3 genes that have undergone tandem duplication resulting in three copies of each type of gene, giving rise to BTN2A1, BTN2A2, and BTN2A3, and BTN3A1, BTN3A2, and BTN3A3 genes, respectively [Butyrophilins (BTNs) are transmembrane glycoproteins found in the milk fat globules (MFGs) ,2. They in (MOG) . Interesin (MOG) ,6. Humanectively ,7. It isectively ,7.BTNs are expressed primarily by the secretory and intestinal epithelium. Being the major protein component of milk fat droplets, they are necessary for the droplet secretion. In fact, BTN1A1 is known to regulate the secretion of milk lipid droplets, whereas BTN2A2 plays a role in lipid, fatty acids, and sterol metabolism. Additionally, BTNs can regulate T cell activation and proliferation, and have been reported to bind to xanthine-oxidase and form a complex with other proteins on the cytoplasmic part of the fat globule .BTN1A1 gene (or Btn1a1 gene in mouse) is highly expressed in the secretory epithelium of the mammary gland during lactation [BTNL2 and ERMAP) are predominantly expressed in skeletal muscle [BTN2A1, BTN2A2, BTN3A1, BTN3A2, and BTN3A3, are characterized by the abundant expression in many tissues [The human actation ,10,11. Tl muscle  and in tl muscle , respect tissues ,15, sugg tissues .The goal of this article is to apply the strategy we developed earlier for the analysis of functional intrinsic disorder in milk proteins ,18,19,20Being modular membrane proteins, members of the BTN family are characterized by similar domain organization, with classical butyrophilin consisting of three domains: two extracellular Ig-like domains, IgV and IgC, one transmembrane domain (TMD), and a cytoplasmic B30.2/SPRY domain . One shomean) values. Furthermore, for each protein, FIT values based on the PONDR® FIT analysis.First, intrinsic disorder predispositions of the members of human butyrophilin family were analyzed by a series of common disorder predictors . Results of this multi-tool disorder predisposition analysis are summarized in mean and PPIDRFIT values for individual butyrophilins. Overall, this analysis revealed that the members of human butyrophilin family are characterized by different degrees of intrinsic disorder, which range from 11.2% for BTNL2 to 21.4% for BTN1A1, as judged by their PPIDRmean values. One should keep in mind though that both PONDR® FIT and PPIDRmean analyses represent very conservative approaches that, likely, provide the lower estimate of disorder levels in query proteins.Typically, two arbitrary cutoffs for the levels of intrinsic disorder are used to classify proteins as highly ordered (PPIDR < 10%), moderately disordered (10% ≤ PPIDR < 30%), and highly disordered (PPIDR ≥ 30%) . Therefo® VLXT [® VL3 [® VSL2 [® FIT [® VLXT , PONDR® T [® VL3 , PONDR®  [® VSL2 , and PON2 [® FIT . Figure https://www.ebi.ac.uk/Tools/msa/clustalo/) [Furthermore, ustalo/) ,27,28. TN-terminal regions of the human butyrophilin family members containing extracellular Ig-like domains are typically more conserved than their cytoplasmic domains; i.e., regions containing long IDPRs.These observations are in line with the known fact that the multiple sequence alignment cannot be used to identify the different butyrophilin members, and an appropriate phylogenetic analysis can be done only in the domain by domain manner . Here, tHomo sapiens), mouse (Mus musculus), and Tasmanian devil (Sarcophilus harrisii). Results of this analysis are presented in http://www.timetree.org/) [Next, to see if specific features found in the disorder profiles of the members of the human butyrophilin family are evolutionary conserved, we compared per-residue disorder propensities of some of their orthologues, using BTN1A1, BTN2A2, ERMAP, and MOG as illustrative examples, since sequences of these proteins are known for evolutionary distant species, such as human (ee.org/) ) and theAmong major disorder-based functions ascribed to various proteins are protein-protein interactions and molecular recognition ,40,41,42Chibi algorithm that predicts MoRFs relying on the local physiochemical sequence properties [Many IDPs/IDPRs contain disordered-embedded order-prone regions capable of undergoing the disorder-to-order transitions induced by binding to a specific partner. These disorder-based binding regions are known as Molecular Recognition Feature (MoRF), and can be computationally identified by various means, such as α-MoRF predictor that indicates the presence, within a largely disordered sequence, of a relatively short, loosely structured helical region that can undergo a disorder-to-order transition induced by binding to partners ,47,48, ooperties , or DISOoperties , or MFSPoperties .WebChibi_, to human butyrophilins are outlined below. ANCHOR algorithm (http://anchor.enzim.hu/) generates a list of predicted disordered binding regions  are not predicted to contain MoRFs, whereas other members of this family are expected to have at least one disorder-based binding site  [Note that to be considered as a MoRF, a region of a query protein should be longer than six residues and show MoRF probability exceeding 0.5 for its entire length ,53. Althee.org/) ).2P2 database (http://d2p2.pro/) [p2.pro/) . UnfortuThe fact that disordered regions of human BTNs contain numerous phosphorylation, acetylation, and ubiquitination sites is in agreement with the well-known notion that phosphorylation , as wellResults of the analysis of interactivity of the members of human butyrophilin family by STRING and BioGrid computational platforms are summarized in Salmonella typhimurium, acts as a component of the FTS/Hook/FHIP complex, and has several other functions), Xanthine dehydrogenase ; Hook homolog 1 , Hook homolog 2 , Ras-related protein Rab-7A , Ras-related protein Rab-9A , Ras-related protein Rab-11A , Tumor necrosis factor receptor superfamily member 10d , Tumor necrosis factor receptor superfamily members 10b and 10a , Perilipin 2 , RAN binding protein 9 , and Natriuretic peptide A . This interactome insures involvement of BTN1A1 in a wide array of biological processes, such as lysosome organization, early endosome to late endosome transport, vacuolar transport, endosome to lysosome transport, endosomal transport, endosome organization, single-organism intracellular transport, organic substance transport, Rab protein signal transduction, cellular response to mechanical stimulus, protein processing, activation of cysteine-type endopeptidase activity involved in apoptotic process, cellular response to external stimulus, vesicle-mediated transport, activation of NF-kappaB-inducing kinase activity, protein transport, and cytoplasmic transport. In this way, BTN1A1 is controlling five biological pathways, namely apoptosis, cytokine-cytokine receptor interaction, influenza A and measles infection, and natural killer cell mediated cytotoxicity. Similarly, a list of proteins interacting with human ERMAP includes trans-2,3-enoyl-CoA reductase , Wolf-Hirschhorn syndrome candidate 2 , TH1-like , Cofactor of BRCA1 , Nuclear cap binding protein subunit 1 , Phosphoglucomutase 1 , Arginine/serine-rich coiled-coil 1 , Tryptophan hydroxylase 1 , WD repeat domain 48 , NAD kinase domain containing 1 , Nuclear receptor corepressor 1 , COP9 constitutive photomorphogenic homolog subunit 3 , and Structure specific recognition protein 1 . In this way, human ERMAP participates in multiple biological processes, such as positive regulation of viral transcription, transcription elongation from RNA polymerase II promoter, viral process, transcription from RNA polymerase II promoter, cellular biosynthetic process, and organic substance biosynthetic process. Finally, according to the STRING-based analysis, among the members of the human butyrophilin family, MOG is the most advanced regulator affecting 41 signaling pathways, such as african trypanosomiasis, allograft rejection, amoebiasis, antigen processing and presentation, asthma, autoimmune thyroid disease, cell adhesion molecules (CAMs), Chagas disease (American trypanosomiasis), cytokine-cytokine receptor interaction, cytosolic DNA-sensing pathway, Fc epsilon RI signaling pathway, graft-versus-host disease, hematopoietic cell lineage, hepatitis B, herpes simplex infection, and HTLV-I infection, inflammatory bowel disease (IBD), influenza A, intestinal immune network for IgA production, Jak-STAT signaling pathway, legionellosis, leishmaniasis, malaria, measles, natural killer cell mediated cytotoxicity, NF-kappa B signaling pathway, osteoclast differentiation, pertussis, primary immunodeficiency, rheumatoid arthritis, RIG-I-like receptor signaling pathway, Salmonella infection, Staphylococcus aureus infection, systemic lupus erythematosus, T cell receptor signaling pathway, toll-like receptor signaling pathway, toxoplasmosis, transcriptional misregulation in cancer, tuberculosis, type I diabetes mellitus, and viral myocarditis.For example, according to the results of STRING analysis shown in Therefore, butyrophilins are among the cellular multitasking proteins involved in protein-protein interaction networks (PPI), where they have multiple links and serve promiscuous binders or hubs that play crucial role in the temporal structure of the PPI networks. It is known that depending on timing of their interactivity, such highly connected proteins can be classified as “party hubs”  and “daBroad functionality of the members of BTN family that ranges from the crucial roles in the formation, secretion, and stabilization of milk fat globules to regulation of the immune response , associaResults of the multifactorial bioinformatics analysis of the members of human butyrophilin family conducted in this study are in line with these considerations. In fact, we are showing here that human BTNs are characterized by diversified disorder propensity and possess IDPRs that can be attributed to the multifunctionality of these important proteins. To provide some support to the findings of this study, sections below describe structural organization of the human butyrophilin family members and also disclose some evidence of the multifunctionality of human butyrophilins and discuss the therapeutic potentials of these important proteins.ERMAP in the intestine and erythroid cells [Butyrophilins are known as modular transmembrane proteins containing several functional domains. For example, human BTN1A1 (UniProt ID: Q13410) has five functional regions, such as a signal peptide (residues 1–26), two immunoglobulin-like (Ig-like) domains, IgV and IgC , a transmembrane domain (residues 243–269), and a B30.2/SPRY domain (residues 285–479). In mature protein, these functional regions constitute three topological domains, extracellular, transmembrane, and cytoplasmic . It was pointed out that although this domain organization was used for identification of homologous proteins, not all members of the human butyrophilin family have such domain structure. In fact, MOG does not have a B30.2 domain, whereas ERMAP does not possess the IgC domain , BTNL2 iid cells , suggestSince butyrophilin genes have mosaic structure , it is not surprising that some of these proteins might have multiple isoforms generated by alternative splicing . ThDespite the significant attention of researchers to butyrophilins (as of 1 January 2018 there were 2155 publications dedicated to these proteins in PubMed), the currently available structural information about human proteins is rather finite. As a matter of fact, structural characterization of human butyrophilins is limited to the presence of crystal structures of several isolated domains of some members of human butyrophilin family, whereas no structure of any full-length butyrophilin was determined as of yet. For example, for BTN3A1 , crystal structures are known for the Ig-like V-type 1 domain (residues 28–143 PDB ID: 4JKW), the ectodomain , and the intracellular B30.2/SPRY domain . Also, crystal structures (PDB ID: 4F8Q and 4F8T) were solved for ectodomains of human BTN3A2 and BTN3A3 . It is likely that the lack of the success in crystallization of the full-length butyrophilins can be, at least in part, explained by the presence of flexible  regions. Careful analysis of the structural information currently available for human BTN3A1 provides support to this hypothesis. In fact, although structures were solved for the isolated domains of this protein, it seems that almost entire sequence  was structurally characterized.However, although the whole cytoplasmic domain (residues 272–513) was used in crystallization experiments, no coordinates have been determined for the 272–322 region of this domain, indicating its high conformational dynamics (PDB ID: 5HM7). Similarly, regions 215–215 and 242–245 of the BTN3A1 ectodomain are likely to be intrinsically disordered, since they represent regions of missing electron density and no coordinates have been determined for them (PDB ID: 4F80) .N-terminal Ig-V like domain and a C-terminal Ig-C type domain) were highly similar, differing from each other by the backbone Cα RMSD of no more than 0.9 Å2 [These observations are in agreement with an earlier structural and functional comparison of the extracellular domains of the three BTN3A isoforms that clearly showed that structurally, these recombinant domains  ,120, (E)Combined NMR and SAXS analysis revealed that binding of HMBPP to the full-length intracellular domain not only affect the B30.2 (PRY/SPRY) domain, but also introduce perturbation of residues in the JM region ,128. In BTN, being one of the milk protein responsible for the fat transport, is considered structurally as a type I membrane protein from the immunoglobulin V domain superfamily. In addition, the extracellular domains of BTNs share structural similarity with the family of the B7 costimulatory molecules that includes numerous signaling molecules playing principal roles in the T-cell regulation, activation, and tolerance. This family includes receptors and ligands implicated in T-cell co-stimulation, such as B7.1 (CD80) and B7.2 (CD86) and their receptors, as well as immune-checkpoint molecules of T-cell, such as PD-L1 (CD274) and PD-L2 (CD273) and their receptors, such as PD-1 (CD279) ,117,129.Mycobacterium bovis, Mycobacterium tuberculosis, Listeria monocytogenes, as well as certain species of malaria protozoa) for the γδ T-cells [BTN3A1 (CD277) is acting as a strong antigen-presenting molecule of both, IPP  ,120 and  T-cells . The cry T-cells . Curious T-cells . It was  T-cells . Therefo T-cells ,92,127.  T-cells . Further T-cells .Plasmodium falciparum [M. tuberculosis [+ T-cells to PAgs was revealed to be dependent not only on the expression of the BTN3A molecules by antigen-presenting cells, but also on the presence of a product(s) of the Chr6 gene required for sufficient PAg-mediated activation of Vγ9Vδ2 T lymphocytes [The Vγ9Vδ2 (and Vγ2Vδ2 as well) T-cells are a subset of the human γδ T-lymphocytes expressing TCR γ9 that are HLA-unrestricted and CD1-independent. They are able to recognize both protein and the non-peptide molecules (BTN3A1/CD277 and phosphoantigens (PAgs), respectively). These T-cells constitute about 65–90% of the γδ T-lymphocytes and 1–10% of the total blood T-lymphocytes. They circulate in peripheral blood of human and non-human primates only . Howeverlciparum ,137,138 rculosis ,140, andrculosis ,142. Therculosis ,143. Thephocytes ,144,145.Many reports have established that BTN molecules play a set of crucial roles in controlling the biological activity of T-lymphocytes via regulating their proliferation and activation through anti-CD3 ,147. FurE.coli (ETEC) strains and prevented attachment of this bacteria to the neonatal porcine intestinal enterocytes in vitro, suggesting that utilization of this protein may reduce the diarrhea in piglets [+ T-cells by IPP or HMBPP antigens was dependent on the BTN. It is known that both antigens are secreted by certain microbial pathogens. Therefore, the butyrophilin/ Vγ9Vδ2+ T-cells may play a crucial role not only in the defense against M. tuberculosis, M. bovis, and L. monocytogenes during primary infection, but also, they mount the recall expansion in systemic infection or in lung compartments after reinfection with M. tuberculosis or other related pathogens [Purified BTN was shown to interact with the F4ac molecules of the enterotoxigenic  piglets ,151. As athogens . This adathogens . This reThe autoantibodies raised against nine different neuron-specific antigens were detected in sera of autistic children. Those autoantibodies were able to significantly cross-react with the BTN peptide 89–109 and some other neurological antigens . Is thisThe binding of PAgs to the extra- or intracellular domains of the cell surface membrane molecule BTN3A1 may lead to a novel structure or conformation that seems to be needed and subsequently detected by the T-lymphocytes expressing the Vγ9Vδ2 TCR, thereby triggering their cytokine production and/or cytotoxicity ,156. ThiTwo experimental models were designed by Werter et al. to eliminate cancer cells via Vγ9Vδ2 T-cell/BTN3A1, which is working as an antigen presenting cell for the invariant natural killer T-cells (iNKT) . Both apAlthough a few studies have explored the association between human BTN3 molecules and ovarian cancer ,92,168, BTN2A1 gene allele was significantly associated with the myocardial infarction [In a series of reports from the research group of Prof. Yoshiji Yamada at Inabe General Hospital (Japan) a link has been found between the polymorphisms of the BTN2A1 gene and several medical symptoms and deficiencies ,178,179.farction ,170, disfarction ,172,173,farction ,175,176,farction ,178,179,farction , and metfarction  in JapanMus musculus), and Tasmanian devil (Sarcophilus harrisii). The analyzed mouse proteins were Btn1a1 (UniProt ID: Q62556), Btn2a2 (UniProt ID: A4QPC6), Ermap (UniProt ID: Q9JLN5), and Mog (UniProt ID: Q61885). The analyzed Tasmanian devil proteins were Btn1a1 (UniProt ID: G3VLA4), Btn2a2 (GI ID: XP_012404130.1), Ermap (GI ID: XP_023356020.1), and Mog (GI ID: XP_012404306).In this article, we analyzed fourteen members of the human butyrophilin family, BTN1A1 (UniProt ID: Q13410), BTN2A1 (UniProt ID: Q7KYR7), BTN2A2 (UniProt ID: Q8WVV5), BTN2A3 (UniProt ID: Q96KV6), BTN3A1 (UniProt ID: O00481), BTN3A2 (UniProt ID: P78410), BTN3A3 (UniProt ID: O00478), BTNL2 (UniProt ID: Q9UIR0), BTNL3 (UniProt ID: Q6UXE8), BTNL8 (UniProt ID: Q6UX41), BTNL9 (UniProt ID: Q6UXG8), BTNL10 (UniProt ID: A8MVZ5), ERMAP (UniProt ID: Q96PL5), and MOG (UniProt ID: Q16653). Sequences of these proteins in FASTA format were extracted from the UniProt database . We alsohttps://www.ebi.ac.uk/Tools/msa/clustalo/) [Amino acid sequence of the members of human butyrophilin family were aligned using the CLUSTAL Omega (1.2.4) computational platform for multiple sequence alignments  ,27,28, u® VSL2 [http://mobidb.bio.unipd.it/) [mean). The corresponding PPIDRmean values were calculated by averaging outputs of the DisEMBL-465, DisEMBL-HL, ESpritz-NMR, ESpritz-XRay, GlobPlot, IUPred-long, IUPred-short and PONDR® VSL2.In this study, we utilized a multi-tool disorder predisposition analysis and used ESpritz-NMR and ESpritz-XRay , IUPred-® VSL2 ,185), acipd.it/) ,187. For® FIT analysis [® FIT is a metapredictor that combines six individual predictors, which are PONDR® VLXT [® VSL2 [® VL3 [® FIT is moderately more accurate than each of the component predictors [For each protein, we also used PONDRanalysis . PONDR® DR® VLXT , PONDR®  [® VSL2 , PONDR® 2 [® VL3 , FoldInd2 [® VL3 , IUPred 2 [® VL3 , TopIDP 2 [® VL3 . It was edictors .® VLXT [® VL3 [® VSL2 [® FIT [® VLXT is not among the most accurate predictors, this tool has a high sensitivity to local sequence peculiarities, which are often associated with disorder-based interaction sites [in silico predictors of intrinsic disorder [® VSL2 [® VL3 is known to perform well for finding long IDPRs [® FIT metapredictor [We further characterized the per-residue intrinsic disorder predispositions butyrophilins in a form of the PONDR-based disorder profiles. To this end, each human butyrophilin was analyzed by PONDR® VLXT , PONDR® T [® VL3 , PONDR®  [® VSL2 , and PON2 [® FIT . These ton sites . Accordidisorder ,192, PON [® VSL2  is one ong IDPRs , and advredictor  were alrChibi algorithm was used that predicts MoRFs relying on the local physiochemical sequence properties [To find disordered regions of human butyrophilins that have a potential to fold at interaction with binding partners and therefore can serve as disorder-based binding sites known as molecular recognition features (MoRFs), MoRFoperties .Potential binding sites in disordered regions of human butyrophilins were also identified by the ANCHOR algorithm ,53. This2P2 database (http://d2p2.pro/), which is a community resource of pre-computed disorder predictions for proteins from completely sequenced genomes [2P2 database is in its use of the outputs of several disorder predictors  combinehttp://www.thebiogrid.org) [The interactivity of human butyrophilins was further evaluated by STRING , which is the online database resource, that provides both experimental and predicted interaction information . STRING rid.org) .Despite the fact that human butyrophilins have a multitude of important biological functions and are engaged in the pathogenesis of several diseases, structural information about these modular transmembrane proteins is rather limited. Furthermore, no systematic in silico analysis was ever conducted to evaluate the intrinsic disorder propensity of human butyrophilins. The goal of this article was to show how intrinsic disorder is encoded in the sequences of fourteen members of the human butyrophilin family, and to look at the peculiarities of functional intrinsic disorder in these proteins. Our analyses revealed that although all members of human butyrophilin family contain noticeable levels of intrinsic disorder, they are characterized by highly diversified disorder propensities and possess very different disorder profiles. We show also that although disorder predisposition is highly diverged between the family members, disorder profiles of these proteins in different species are characterized by the remarkable similarity, indicating that the disorder predisposition peculiarities in individual butyrophilins are evolutionary conserved at least since the time of marsupial divergence 160 million years ago and therefore may be related to the functionality of these proteins. Furthermore, since disorder profiles are well preserved among the individual butyrophilins, but are highly diversified in the different members of the butyrophilin family, one can assume that such diversification of the intrinsic disorder propensity represents a molecular means for the functional diversification of these proteins. In fact, our study revealed that IDPRs in butyrophilins can be used for interaction of these proteins with various binding partners and serve as primary targets for various PTMs. Curiously, we found that the individual members of the BTN family are characterized by a remarkable similarity of their MoRF profiles, which represent the sequence distributions of the probability for being engaged in the disorder-based interactions. Therefore, findings of this study clearly indicate that intrinsic disorder plays important roles in function and evolution of the butyrophilin family and therefore should be taken into account in structural and functional studies of these proteins.


Background: POU1F1 is an essential transcription factor for the differentiation, proliferation and survival of somatotrophs, lactotrophs, and thyrotrophs. Mutations in the POU1F1 gene are characterized by growth hormone (GH), thyrotropin, and prolactin deficiencies, commonly presenting with growth retardation and central hypothyroidism. Since the first report in 1992, more than 25 mutations have been identified in POU1F1.Case Description: We describe a 17-year-old male who presented to our Pediatric Endocrinology clinic with extreme short stature , cognitive impairment, deaf-mutism, and neurological disabilities. L-thyroxine supplemental therapy, which had been initiated at the age of 6 months but ceased due to non-compliance, was reintroduced at presentation. GH therapy was initiated at 19 years of age, resulting in 42 cm linear growth, to a final height of 124 cm. Sequencing of POU1F1 revealed a previously described homozygous insertion mutation—c.580_581insT, p (Thr194Ilefs*7)—in exon 4 causing a frameshift that introduces a stop codon 7 amino acids downstream, leading to a severely truncated protein lacking the homeodomain.Conclusion: This case report sheds light on the natural history of untreated patients with POU1F1 mutations and raises awareness for early diagnosis and adequate treatment of central congenital hypothyroidism and GH deficiency. PROP1, POU1F1 (PIT1), LHX3, GLI2, HESX1, LHX3, LHX4, OTX2, SOX2, ROBO1, and SOX3 (Combined pituitary hormone deficiency (CPHD) is a rare heterogeneous condition in which there are at least two anterior pituitary hormone deficiencies. Anterior pituitary-derived hormones require multiple transcription factors for pituitary morphogenesis, differentiation and hormones synthesis. Mutations in the genes and SOX3 , which ePOU class 1 homeobox 1 (POU1F1), also known as pituitary transcription factor 1 (PIT-1), was the first pituitary-specific transcription factor to be identified in humans and mice , 7. It iPOUIFI gene (OMIM 601538) is located on chromosome 3p11, consists of 6 exons and encodes a 317-amino acid protein (NM_001122757) that has three functional domains: a transactivation domain, a POU-specific (POU-S) domain and a POU-homeo (POU-H) domain. The N-terminal part of POU1F1 is involved in the transcriptional activation of several pituitary-expressed genes ; these mutations are responsible for dominant or recessive CPHD, with 3 small insertion mutations among them (POU1F1 mutations include severe growth retardation in infancy due to GH deficiency (GHD) with distinctive facial features characterized by prominent forehead, marked mid-facial hypoplasia, and depressed nasal bridge. In addition, central hypothyroidism of variable severity has been reported, and brain magnetic resonance imaging (MRI) demonstrates either normal or hypoplastic anterior pituitary with unaffected posterior pituitary.The human  in 1992 , involviong them  The clinPOU1F1 gene (4) therapy. This description extends the recognized phenotype of POU1F1 mutations, shedding light on the severity of presentation in the untreated state.Here we describe a 17-year-old male who presented with extreme short stature, mental retardation, deaf-mutism, neurological impairment, and dysmorphic features, in whom CPHD  caused by a previously described homozygous frameshift mutation of the 1F1 gene  was iden4) , indicating central hypothyroidism. Further investigation demonstrated mild bilateral frontal and interhemispheric cortical atrophy (on brain computed tomography (CT) scan and sensorineural (SN) hearing loss (Brainstem Auditory Evoked Response test). Comprehensive metabolic investigations were normal, as were echocardiography and ophthalmic examination. The karyotype was 46, XY, and no chromosomal abnormalities were identified. LT4 treatment was initiated at a dose of 30 μg daily, but was discontinued by the parents after 2 months. At the age of 4 years, he was diagnosed with severe mental retardation and was fully disabled. At 5 years of age, the proband presented to a different Endocrine clinic for investigation of severe growth retardation. At that time, TSH was 0.01 mU/L, FT4 1.34 pmol/L and FT3 < 0.44 nmol/L . Thyrotropin-releasing hormone (TRH) test confirmed the diagnosis of central hypothyroidism  and insulin growth factor binding protein-3 (IGFBP-3) levels were undetectable with no GH response to arginine stimulation test , IGF-1 < 3.25 ng/mL, and IGFBP-3 < 500 ng/mL. Cortisol and adrenocorticotropic hormone (ACTH) levels were within normal limits and gonadotropin levels were pre-pubertal  and growth velocity. Gonadotropin-releasing hormone (GnRH) analog treatment (3.75 mg every 28 days) was initiated at the onset of puberty  to facilitate maximal linear growth. Calcium supplement and vitamin D3 were added due to low serum levels of vitamin D. In addition, physiotherapy and speech therapy were implemented. At the age of 24 years, growth velocity decreased to <2 cm per year and therefore GH and GnRH analog therapy were terminated. At 28 years of age, height had increased by 42.3 cm (+3 SD) to a final adult height of 124 cm  of Arab descent, was born after pregnancy with polyhydramnios and delivered at term, weighing 2,900 g and his length was 45 cm. Both parents were healthy; paternal height was 170 cm and maternal height 160 cm. There was one healthy brother of normal stature. There was no family history of short stature, hypothyroidism, deafness, or neurological impairment. In the first week of life, the proband had recurrent vomiting and prolonged neonatal jaundice with indirect hyperbilirubinemia of 15 mg/dL that resolved spontaneously. At age 6 months, severe failure to thrive and dehydration were diagnosed, resulting in hospital admission. Delayed psychomotor development, growth retardation, and dysmorphic facial features were noted. On physical examination, he had enlarged fontanelle, severe hypotonia, umbilical hernia, and dry skin consistent clinically with hypothyroidism. Laboratory evaluation revealed low TSH  and free thyroxine , formerly c.502insT (p.Thr168IlefsX7) as previously described by our group . CPHD is classically divided into two subgroups according to the transcription factor gene types affected: early expressed pituitary transcription factor genes, defined as syndromic CPHD, and late expressed pituitary developmental genes, defined as non-syndromic CPHD. Whereas, mutations in early developmental genes are commonly involved in organogenesis and therefore present clinically with brain, ophthalmic, and/or skeletal anomalies, mutations in late developmental genes generally present with isolated pituitary hormone deficiencies in the absence of other anomalies , who presented with extreme short stature, deaf-mutism, and neurological disabilities  with a homozygous nomalies . POU1F1 ated GHD .4 therapy was ceased within 2 months of the initial diagnosis, briefly reinitiated at 5 years of age, but ceased again shortly thereafter until presentation to our clinic at age 17.5 years.Neonatal thyroid screening in Israel aims to identify primary congenital hypothyroidism (CH), such that only abnormally elevated levels of TSH are reported. Central hypothyroidism (in which TSH is low) is thus not reported on neonatal screening, potentially resulting in delayed diagnosis. Furthermore, TSH is also frequently relied upon for screening when hypothyroidism is suspected later in life (with FT4 measured only if it is elevated), such that a diagnosis of central hypothyroidism may again be missed. In this case, diagnosis was delayed until 6 months of age with limited subsequent treatment compliance. LT4 treatment. Mental retardation, microcephaly, sensorineural deafness, and severe prenatal and postnatal growth failure were described in patients with IGF-1 mutation  (POU1F1 mutations .POU1F1, revealed a homozygous mutation c.580_581 insT p.(Thr194llefs*7) and heterozygous parents. This mutation abolishes the transactivation properties of POU1F1 on three target promoters, resulting in severe TSH, PRL and GH deficiencies (Sequencing of the candidate gene, ciencies  which maPOU1F1 gene illustrates the consequences of inadequate CPHD treatment. The remarkable presentation of this patient, who looked 2 years old at age 17.5 years, and suffered from deafness and extensive neurological impairment, extends the recognized phenotype of POU1F1 mutations and emphasizes the great importance of early and adequate thyroid and GH supplemental therapy.This case of a 17-year-old male with a frameshift mutation in the No datasets were generated or analyzed for this study.This study was approved by the Institutional Review Board of Ha'Emek Medical Center. The patient is described anonymously and gave written informed consent with the publication.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hepatoblastoma (HB) is the most common form of primary liver malignancy found in pediatric populations. HB is considered to be clonal and arises from hepatoblasts, or embryonic liver progenitor cells. These less differentiated tumor-initiating progenitor cells, or cancer stem cells (CSCs), may contribute to tumor recurrence and resistance to therapies, and have high metastatic abilities. Phenotypic heterogeneity, undesired genetic and epigenetic alterations, and dysregulated signaling pathways provide CSCs with a survival advantage over current therapies. The molecular and cellular basis of HB and the mechanism of CSC induction are not fully understood. The Wnt/beta-catenin pathway is one of the major developmental pathways and is believed to play an important role in the pathogenesis of HB and CSC formation. This review summarizes the cellular and molecular characteristics of HB with a specific emphasis on CSCs and Wnt/beta-catenin signaling. Hepatoblastoma (HB) is the primary liver cancer found in children generally under three years of age . The terIGF-2), delta-like 1 homolog (DLK1), transforming growth factor beta 1 (TGF beta-1), mitogen-inducible gene 6 (MIG-6), and non-coding RNA MALAT1 are induced in HB but not in HCC [HB and HCC (hepatocellular carcinoma) are common liver malignancies that affect different age groups. Even though there are similarities in their tumorigenic characteristics, recent studies have shown that HB differs from HCC not only in its origin, but also in various molecular signatures associated with each malignancy . Global t in HCC . On the t in HCC . The FXRt in HCC .CTNNB1 gene, which encodes beta-catenin, is the most frequently mutating gene in exon 3 in HB; consequently, an adenomatous polyposis coli (APC) germline mutation in HB has been associated with a familial adenomatous polyposis history. Genomic analysis has revealed a CTNNB1 mutation rate of ≈68% in HB [The presence of various fetal and embryonic liver progenitor markers strengthens the hypothesis that HB originates from transformed hepatoblasts. HB tumors are categorized into two distinct subtypes: C1 and C2 . The C1 8% in HB ,20,21,22AFP, TACSTD1, DLG7, CDC2, BUB1, AURKB, IGF2, DLK1, PEG3, PEG10, BEX1, MEG3, NDN, BIRC5, NPM1, and HDAC2, and the activation of Myc signaling have been associated with the HB C2 subtype, which has embryonal characteristics and is enriched with CSCs [GLUL, RHBG, CYP2E1, and CYP1A1. It is also important to note that different sets of Wnt/beta-catenin-targeted genes are overexpressed in the two HB subtypes [NFE2L2 , which encodes a transcription factor involved in the antioxidant response pathway [NRF2 mutations are correlated with the activation of nicotinamide adenine dinucleotide phosphate dehydrogenase quinone 1 (NQO1), which has high expression levels in HB and has been found to be associated with invasive and metastatic C2 subtype HB tumors [Overexpression of genes, such as ith CSCs . On the subtypes . The HB subtypes . Another pathway . NRF2 muB tumors . ComerfoB tumors . This moAdditionally, cytoplasmic activation/proliferation-associated protein-2 (CAPRIN2), a known oncogene, and tumor suppressors, such as speckle-type POZ protein (SPOP), olfactory receptor-511 (OR5I1), and cell division cycle 20B (CDC20B), have been found to influence HB cell growth. CAPRIN2 is known to activate Wnt signaling via low-density lipoprotein receptor-related protein (LRP) 5/6 phosphorylation , and gaiHB differs in its developmental origin from HCC in adults. HB occurs in the very early stages of life, while HCC frequently develops on a background of chronic liver diseases. Studies have demonstrated that HB tumors consist of heterogenous populations of stem/progenitor cells ,13,28. TCSCs express numerous progenitor/stem cell-specific genes and share many characteristics with normal stem cells. HB tumors have been found to express progenitor markers, such as clusters of differentiation 133 (CD133), EpCAM , CD44, CD24, CD90, oval cell antigen-6 (OV6), and aldehyde dehydrogenase1 (ALDH1) ,30. CD13EpCAM is another potential CSC marker in the liver. In a normal liver, EpCAM is expressed in the bile duct cells at low levels. EpCAM is a transmembrane protein induced by Wnt/beta-catenin signaling and expressed in a variety of progenitor and stem cells , includiCD90 and CD44 have also emerged as markers of liver CSCs. Yang et al. found that the expression of CD90 correlated with tumorigenic potential in liver cancer cell lines. The presence of CD90-positive and CD44-positive cells was found to correlate with tumor metastasis. Blockade of CD44 by a neutralizing antibody induced apoptosis of CD90-positive cells and prevented tumor formation in mice .Epithelial–mesenchymal transition (EMT) is a cellular reprograming process whereby epithelial cells undergo transformation into cells with mesenchymal characteristics. In EMT-activated cells, intercellular junction proteins, such as E-cadherin, are downregulated, while mesenchymal-related proteins, such as N-cadherin, fibronectin, and vimentin, are upregulated. When primary tumor cells undergo EMT, polarized cells become highly migratory and are capable of invasion, leading to distant metastatic colonization ,51. SeveCTNNB1 or mutations prevent beta-catenin phosphorylation, and thus its degradation leads to an increase in both cytoplasmic and nuclear beta-catenin protein levels, which results in the constitutive activation of the Wnt/beta-catenin pathway  are a family of small, endogenous, non-coding RNAs, approximately 19–25 nucleotides in length. MicroRNAs regulate key signaling pathways in the pathogenesis of hepatoblastoma and are known to regulate CSCs ,74. HB tTargeting CSCs is challenging due to their high degree of heterogeneity and the intrinsic survival mechanisms that they develop over time to bypass apoptotic signals. At present, conventional therapies, such as chemotherapy, radiotherapy, and immunotherapy, can effectively suppress the growth of differentiated tumor cells; however, they potentially lag behind in inhibiting CSC proliferation . AlthougCTNNB1 [Several basic and clinical studies have demonstrated the critical role for Wnt/beta-catenin signaling in HB. The greatest challenge in targeting Wnt/beta-catenin signaling is its complexity, as it consists of several components regulated by protein post-translational modifications at various levels, as well as extensive signaling crosstalk with other cell-signaling pathways. Targeting nuclear beta-catenin and/or its nuclear regulators responsible for the transcription of downstream Wnt/beta-catenin genes could be a plausible approach to target CSCs. Beta-catenin interaction with cyclic adenosine 3′,5′-monophosphate response element-binding protein (CBP) promotes stem cell renewal, while interaction with P300 enhances progenitor cell differentiation. Therefore, targeting the beta-catenin-CBP interaction is a promising strategy, and small-molecule drugs, such as ICG-001, have been found to effectively target cancer stem cells ,86,87,88CTNNB1 . ActivatCTNNB1 . Based oEven though several cancer therapeutic approaches are available at present, tumor recurrence is a growing challenge in modern medicine. Recent evidences strongly suggest that CSCs that resist current therapies are the basis of cancer recurrence. Better understanding of the ways in which they originate from normal progenitor/stem or hepatoblast cells, their intrinsic self-renewal and survival mechanisms, and differential drug-metabolizing abilities compared to normal and differentiated cancer cells is critical for developing CSC-targeting therapies.


Obesity is a global disease that causes many metabolic disorders. However, effective agents for the prevention or treatment of obesity remain limited. This study investigated the anti-obesity effect and mechanism of chitosan oligosaccharide capsules (COSCs) on rats suffering from obesity induced by a high-fat diet (HFD). After the eight-week administration of COSCs on obese rats, the body weight gain, fat/body ratio, and related biochemical indices were measured. The hepatic expressions of the leptin signal pathway (JAK2-STAT3) and gene expressions of adipogenesis-related targets were also determined. Our data showed that COSCs can regulate body weight gain, lipids, serum alanine aminotransferase, and aspartate aminotransferase, as well as upregulate the hepatic leptin receptor-b (LepRb) and the phosphorylation of JAK2 and STAT3. Meanwhile, marked increased expressions of liver sterol regulatory element-binding protein-1c, fatty acid synthase, acetyl-CoA carboxylase, 3-hydroxy-3-methylglutaryl-CoA reductase, adiponectin, adipose peroxisome proliferator-activated receptor γ, CCAAT-enhancer binding protein α, adipose differentiation-related protein, and SREBP-1c were observed. The results suggested that COSCs activate the JAK2-STAT3 signaling pathway to alleviate leptin resistance and suppress adipogenesis to reduce lipid accumulation. Thus, they can potentially be used for obesity treatment. Obesity is a chronically trophic metabolic disease mainly caused by an energy imbalance that leads to excess body fat accumulation. It has caused increasing concern in recent years. Obesity contributes to type 2 diabetes (T2D), hyperlipidemia, hypertension, cerebrovascular incidents, and cancers ,2. RecenN ≤ 1000 Da, COST) is a small molecular derivative of chitosan (CTS) with 2–10 polymerization degrees [Chitosan oligosaccharide , is an important hormone for weight management via the homeostatic control of energy equilibrium, thus facilitating the prevention of obesity ,18. LeptIn addition, Inoue et al.  revealedAdiponectin, an endogenous bioactive polypeptide secreted by adipocytes, is associated with the counteracting development of obesity and its related diseases. The gene expression and circulating levels of adiponectin are reported to be inversely correlated with obesity ,25,26. SThe hyperplasia and hypertrophy of adipocytes facilitate the development of obesity and its complications. Therefore, decreasing lipogenesis may benefit obesity prevention or treatment. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer binding protein α (C/EBPα) are two primary modulators of lipogenesis and regulate the proliferation, differentiation, and fat accumulation of adipocytes in WAT ,34. AdipIn this study, COST capsules (COSCs) were first prepared in capsule form due to several advantages, including the enhanced stability of COST manifested through strong hygroscopicity and oxidability, and the accelerated action rate of COST via the rapid dissolution, dispersion, and assimilation of the capsule in the gastrointestinal tract. Also, the capsule form can be easily identified and taken by most people. Subsequently, the anti-obesity effects of COSCs in a rat model exhibiting obesity induced by a high-fat diet (HFD) were investigated. Significantly ameliorated effects of COSCs on the development of obesity and its related complications were observed. To explore the anti-obesity mechanisms, the liver LepRb, JAK2, and STAT3 expressions in the leptin metabolic JAK2-STAT3 signaling pathway were evaluated by Western blotting, which suggested an improved leptin-resistant state via activating the JAK2-STAT3 signaling pathway. Moreover, the lipogenesis-related genes in the liver  and in adipose tissues  were also determined using quantitative RT-PCR analysis, indicating the adipogenesis suppression action of COSCs. These studies suggested that COSCs may be a prospective agent for obesity prevention or treatment.To evaluate the anti-obesity effects of COSCs, food intake, body weight, and the body weight gain of the rats were measured over an eight-week period, the results of which are shown in For the body weight gain, the average body weight of the HF group increased by 41.22% after eight weeks of feeding with HFD. Meanwhile, the eight-week administration of Orlistat, high (COSC-H), middle (COSC-M), and low dose of COS (COSC-L) significantly decreased body weight gain by 37.21%, 33.52%, 29.62%, and 16.61%, respectively b,c. AddiRat serum lipid levels after the eight-week administration were determined and are displayed in Various studies have indicated that leptin resistance, manifested by high levels of circulating leptin, is an important characteristic of nutritional obese patients . In the The liver morphology, lipids, AST, and ALT levels are shown in Meanwhile, the liver morphology g showed p < 0.05), suggesting that COSCs play an important role in hepatoprotective function.Liver injury, or hepatotoxicity, is the main relative factor of hyperlipidemia and obesity . To checAll data demonstrated that COSCs exert a hepatoprotective effect via decreasing the liver index, enhancing liver function, and relieving fatty liver, which contribute to the anti-obesity effects.p < 0.05) and fat/body ratio (p < 0.05) of rats dose-dependently to the normal range. Moreover, the inhibiting effects of COSC-H on HFD-induced elevated fat pad and fat/body ratio were slightly superior to that of Orlistat.After the eight-week administration, the epididymal and perirenal WAT weights were measured to calculate the wet weight of the fat pad and fat/body ratio . CompareIn addition, the histopathological results of epididymal e and perLeptin resistance, characterized by elevated circulating leptin levels and decreased leptin sensitivity, is the central mechanism for the development of obesity. Attenuation of the JAK2-STAT3 signaling transduction pathway can be considered as the crucial risk factor for leptin resistance. Therefore, the improvement of leptin resistance by regulating the JAK2-STAT3 signaling pathway effectively exerts anti-obesity activity ,38. In tSREBP-1c, FAS, ACC, and HMGCR, as well as the WAT lipogenesis-related genes, adiponectin, PPARγ, C/EBPα, ADRP, FAS, and SREBP-1c, were also measured  ,39. ObesSeveral studies by others and our group have demonstrated that COST has anti-obesity effects and is a promising anti-obesity agent ,16,41. IThe obese animal model used in the experiment showed obviously increased body weight gain and lipid accumulation as well as elevated organ index, as reported before the anti-obesity treatment ,43. The Hepatic steatosis (fatty liver), induced by excessive lipid accumulation in the liver, is highly relevant to obesity and its related complications ,45. In tLeptin resistance, characterized by elevated circulating leptin levels and decreased leptin sensitivity, is the central mechanism for the development of obesity. Attenuation of the JAK2-STAT3 signaling transduction pathway is a crucial risk factor for leptin resistance ,20. ChitSREBP-1c, FAS, ACC, and HMGCR are the crucial factors that influence lipid metabolism in the liver. SREBP-1c increases the activity of the key enzyme FAS and regulates the synthesis of TC and TG when free fatty acids are released into hepatocytes . Acetyl-In addition, the expressions of genes involved in WAT were also determined to evaluate the influences of COSCs on adipogenesis in WAT. PPARγ, together with C/EBPα, can promote the proliferation and differentiation of adipocytes and control lipid storage in WAT ,46; ADRPIn summary, COSCs could be used as a drug candidate with a good ability to control obesity. Chitosan oligosaccharide capsules can activate the JAK2-STAT3 signaling pathway to alleviate leptin resistance and inhibit adipogenesis by regulating the relevant adipocytokines. Therefore, COSCs may serve as a natural product prospective agent for obesity prevention and treatment.N ≤ 1000 g/mol; deacetylation degree, 95.6%) was obtained from Laizhou Haili Biological Products Co. Ltd. Laizhou, Shandong, China. The COSCs used in this work were prepared in our laboratory. The Orlistat capsules were provided by Chongqing Fortune Pharmaceutical Co. Ltd., Chongqing, China. Total cholesterol, TG, HDL-C, LDL-C, AST, and ALT kits were provided by BioSino Biotechnology and Science Inc., Beijing, China. The BCA protein quantified assay kit (Cat. No. P0012) was provided by Beyotime Biotechnology Inc., Shanghai, China. The rabbit polyclonal anti-LepRb (Cat. No. 20966-1-AP), anti-JAK2 (Cat. No. 17670-1-AP), and anti-STAT3 (Cat. No. 10253-2-AP) antibodies were purchased from Proteintech, Inc., Wuhan, China. The rabbit monoclonal anti-JAK2 (Cat. No. ab219728) and anti-STAT3 (Cat. No. ab76315) phosphorylated antibodies were purchased from Abcam Co. Ltd., Cambridge, UK. The rabbit anti-β-actin (Cat. No. bs-0061R) and Goat anti-rabbit IgG/HRP (Cat. No. bs-0295G-HRP) antibodies were purchased from Biosynthesis Biotechnology Co., Ltd., Beijing, China. The reverse transcription kit (PrimeScript™ RT reagent kit with gDNA Eraser (Cat. No. RR047A)) and the PCR kit (SYBR Premix Ex Taq™ kit (Cat. No. RR420A)) were all supplied by TaKaRa Inc. . All of the other reagents were of analytical grade and were used without further purification.Chitosan oligosaccharide  Sprague–Dawley (SD) rats  were provided by the Guangdong Medical Laboratory Animal Center  and were maintained in an SPF room . Water was given to rats freely throughout the experiments. The welfare of the animals in the in vivo experiments was handled strictly following “Guidelines on kindly treatments for experimental animals” by the Science and Technology Ministry of China (2006) 398. All protocols were approved by the Institutional Animal Ethics Committee of Guangdong Pharmaceutical University . All animals were fed with the normal diet  for one week. Subsequently, the 95 rats were divided into two groups: the normal diet group (NF) with 10 rats fed the normal diet, and the HFD group (HF), with 85 animals fed the HFD to obtain the obese model.The HFD was comprised of 54% basic feed, 15% lard, 15% sucrose, 4% milk powder, 3% peanut, 5% egg yolk powder, 1% sesame oil, 2% salt, 0.6% dicalcium phosphate, and 0.4% mountain flour in SPF packaging (provided by GMLAC (No. 20150925)). After two weeks of feeding with HFD, two thirds of the rats were sorted by body weight gain. The excluded one third of the rats that gained less weight were kept on the HFD for another six weeks. The obese model was regarded as being accomplished when the average weight of the rats fed with HFD surpassed 20% of that of the basic fed rats.The 50 obesity-sensitive rats were then randomly and equally divided into five groups as follows: (1) HFD group fed with HFD ad libitum only (HF); (2) HFD group treated with Orlistat (75 mg/kg·day) (Orlistat); (3–5) HFD group supplemented with low , middle , and high  dose COSCs. Because the capsules and COST were all water-soluble, the COST capsules were consequently dissolved in distilled water, which formed the COST solution with a constant concentration of 600 mg/mL. The corresponding samples to be tested should be solubilized in distilled water and administered by oral gavage daily with a dose of 1 mL/100 g (the rats’ body weight) at the same time for eight weeks until the experiment ended. The rats in the HF and NF groups were simultaneously administered equal amounts of distilled water in the same way as described above. All rats obtained food and water freely during the experimental period, and were sacrificed at the age of 22 weeks.The 24-h food intake levels of rats were recorded daily, while body weight, body length, and abdominal girth were determined every week during the animal study.The rats were fasted for 16 h and then subjected to 1% sodium pentobarbital (0.5 mL/100 g body weight) anesthesia, and their blood was collected from the aorta abdominals. The serum was obtained via centrifuging at 3500 rpm for 30 min at 4 °C, and the serum lipid and leptin levels were then measured. The concentrations of TC, TG, HDL-C, LDL-C, AST, and ALT were measured with commercial assay kits using an automated biochemistry analyzer BC200 instrument. The concentration of leptin were measured using an enzyme-linked immunosorbent assay (ELISA) kit , and the absorbance was detected at 450 nm using a multifunctional Berthold Mithras LB940 microplate reader .Rats were subjected to ether anesthesia, sacrificed, and necropsied after serum preparation. Then the liver, epididymal WAT, and perirenal WAT were quickly stripped and weighed by an electronic scale at 4 °C. After a picture was taken of the whole liver from each animal, the total wet weight of liver, epididymal fat, and perirenal fat were used to determine the liver index, fat pad, and fat/body ratio, respectively. The tissues were promptly stored at −80 °C for further analysis following their removal.A piece (approximately 0.1 g) of liver tissue was homogenized in 0.9 mL 0.9% sodium chloride, and the supernatant was collected after centrifuging at 2500 rpm for 10 min at 4 °C. The hepatic TC, TG, AST, and ALT levels were measured with commercial assay kits, as was performed for serum lipids previously.3, then were rinsed with normal saline and put in a tissue cassette. The cassettes were marked with a pencil and then placed into a 12% formaldehyde solution for 24 h to fix the tissue. After the residual fixative was cleaned with distilled water, the tissues were firstly dehydrated via 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, then embedded in paraffin  and lastly cut into 5-μm-thick sections using a Leica RM2235 microtome . The tissues were stained with hematoxylin and eosin (H&E) and observed under a microscope at 200× magnification.The liver, white epididymal, and perirenal WAT were cut into 0.6 cmg for 30 min at 4 °C. The protein concentrations were quantified by the Beyotime BCA protein assay kit. The isolated proteins were diluted to the same protein concentrations. An equal amount of each protein lysate (35 μg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels. After electrophoresis, the gels were then transferred to polyvinylidene fluoride membranes . After blocking with 5% bovine serum albumin (BSA) in TBST buffer  for two hours, the membranes were correspondingly incubated for 2 h with primary antibodies, including rabbit polyclonal anti-LepRb, anti-JAK2, and anti-STAT3 antibodies, and rabbit monoclonal anti-JAK2 and anti-STAT3 phosphorylated antibodies, as well as rabbit anti-β-actin antibody, in 5% skim milk. The secondary antibody (Goat anti-rabbit IgG linked HRP) was incubated with the membranes for another 1 h after washing with TBST buffer. The membranes were developed with Pierce’s West Pico chemiluminescence substrate  after washing with TBST buffer. The chemiluminescence imaging system  and its Lane 1D gel image software were applied to quantify the grayscale of the protein bands with the value of the β-actin band as an internal reference.Total protein was isolated from the liver tissues (25 mg) with 0.50 mL of cold RIPA Lysis Buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease and phosphatase inhibitor), followed by centrifugation twice at 12,000 Total RNA was isolated from rat livers or white epididymal adipose tissues using the TRIzol reagent . Single-stranded cDNA was generated from 1 μg of total RNA using TaKaRa PrimeScript™ RT reagent kit. The cDNA products were amplified by real-time RT-PCR using TaKaRa SYBR Premix Ex Taq™ kit and the Bio-Rad IQ5 real-time PCR system, and its analysis software  was used for data collection and analysis. The primer sequences  used for−ΔΔCt method.The PCR protocols were performed as follows: 95 °C for 30 s , followed by 39–40 cycles at 95 °C for 5 s and 60 °C for 30 s, after which the amplified products were heated from 65 °C to 95 °C at 0.5 °C steps for 5 s. The relative quantification of mRNA expression was analyzed using the 2p < 0.05 was considered statistically significant.All data are expressed as the means ± standard error (SE) for each group, and the differences between the groups were compared with a two-way ANOVA test using SPSS for Windows, version 19.0 . The significant differences among the means were determined using Student–Newman–Keuls multiple range tests, and


Xanthomonas citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo). In particular, the compounds 5a, 5f, 5g, 5h, 5i, and 5l, with EC50 values of 11.5–27.3 μg/mL, showed potent antibacterial activity against Xac that was better than the commercial bactericides Bismerthiazol (34.7 μg/mL) and Thiodiazole copper (41.1% μg/mL). Moreover, the in vivo antiviral activities against tobacco mosaic virus (TMV) of the target compounds were also tested. Among these compounds, the curative, protection, and inactivation activities of 5g were 49.9, 52.9, and 73.3%, respectively, which were better than that of the commercial antiviral Ribavirin . This study demonstrates that myricetin derivatives bearing amide, thioether, and 1,3,4-thiadiazole moieties can serve as potential alternative templates for the development of novel, highly efficient inhibitors against plant pathogenic bacteria and viruses.A series of myricetin derivatives containing amide, thioether, and 1,3,4-thiadiazole moieties were designed and synthesized, and their antiviral and antibacterial activities were assessed. The bioassays showed that all the title compounds exhibited potent in vitro antibacterial activities against  In recent years, pathogenic bacteria and plant viruses targeting agricultural crops have become increasingly difficult to control, leading to enormous losses in worldwide crop production each year. As such, the development of environmentally friendly pesticides with high selectivity, high efficiency, low toxicity, and suitable degradation are important challenges to both chemists and biologists . NaturalMyricetin  belongs The compound 1,3,4-thiadiazole contains an important nitrogenous pharmacophore and exhibits different biological activities, such as antimicrobial, antidepressant, and anti-tumor activities. The 1,3,4-thiadiazole ring is very weak due to the induction of sulfur atoms, which makes it easy for it to unlink in alkaline aqueous solutions; however, it is relatively stable in acidic aqueous solutions . The rinXanthomonas citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo). In addition, the ability of these compounds to inhibit tobacco mosaic virus (TMV) was also evaluated in vivo via the half-leaf method at 500 μg/mL, with the commercial antiviral Ribavirin serving as a positive control  was obtained according to the previously reported method  + 590.1262, found 590.1252.5a–5p against Xac , Rs , and Xoo  were evaluated based on the turbidimeter test [μL of NB containing bacterium Xac/Rs/Xoo was added to five mL of solvent NB containing the test compounds or TC/BT. The above test tubes were incubated at 30 ± 1 °C with continuous shaking at 180 rpm for 1–3 days. Bacterial growth was monitored with a spectrophotometer by measuring the optical density at 600 nm (OD600), given by corrected turbidity values = ODbacterium − ODno bacterium. The relative inhibitory rate (I%) of the circle mycelium compared with a blank assay was calculated as follows: I = (Ctur − Ttur)/Ctur × 100%. The Ctur represents the corrected turbidity value of bacterial growth in untreated NB (blank control), and Ttur is the corrected turbidity value of bacterial growth in treated NB. Similarly, the excellent antibacterial activities of the target compounds and TC/BT were further assessed against the bacteria Xac, Rs, and Xoo at five double-declining concentrations  to obtain their EC50 values, which were statistically determined using probit analysis within the software package SPSS 17.0. The listed EC50 values in The antibacterial activities of compounds ter test ,26,27. TThe curative, protection, and inactivation activities against TMV of the target compounds were tested using previously reported methods ,29. The Xoo, Rs, and Xoo. For example, compounds 5a, 5f, 5g, 5h, 5i, and 5l showed strong antibacterial activity against Xac, with the EC50 values of 11.5–27.3 μg/mL, which are significantly better than the commercial bactericides BT (34.7 μg/mL) and TC (41.1% μg/mL). An anti-TMV activity test for the target compounds was performed, and among these compounds, the curative, protection and inactivation activities of 5g were 49.9%, 52.9%, and 73.3%, respectively, which are better than that of the commercial antiviral RBV . It is noteworthy that most of the myricetin derivatives obtained by derivatization had better antibacterial and antiviral activities than those of myricetin. The above studies showed that myricetin derivatives containing amide, 1,3,4-oxadiazole, and thioether moieties could serve as promising candidates for the development of new and more efficient agricultural bactericides against bacterial pathogens and plant viruses.In conclusion, a series of myricetin derivatives containing amide, 1,3,4-oxadiazole, and thioether moieties were developed as potential antibacterial and antiviral agents, and sixteen novel compounds were synthesized. The bioassay results indicated that some of the target compounds possessed excellent antibacterial activities against the plant pathogens Rs, Xoo, and TMV by extending the linking group between 1,3,4-thiadiazole and myricetin.On the basis of the previous work , the hyd


The unfolded protein response (UPR) is an adaptive response that maintains the fidelity of the cellular proteome in conditions that subvert the folding capacity of the cell, such as those noticed in infection and inflammatory contexts. In immunity, the UPR sensor IRE1  has emerged as a critical regulator of the homeostasis of antigen presenting cells (APCs). In the past few years, it has become clear that IRE1 plays canonical and non-canonical roles in APCs, many of which intersect with key features of these cells, including the initiation of inflammation, antibody production, and antigen presentation. The aims of the present review are to provide recent insights on the mechanisms by which IRE1 regulates the diversity of APC functions and to highlight its relevance in the coordination of innate and adaptive immunity. Antigen presenting cells (APCs)—composed of dendritic cells (DCs), B cells, and macrophages—are chief sentinels of the immune system and major cellular sensors of infection and tissue malfunction. These cells detect cell-extrinsic or cell-intrinsic inflammatory cues and respond by coordinating key immunological processes. Macrophages are tissue-resident phagocytes with multiple functions, including the production of inflammatory mediators that induce immune cell mobilization to the affected area (M1-polarization) or production of growth factors to promote tissue repair (M2-polarization) ,4. ConcoDespite its canonical role in protein homeostasis, the relevance of the UPR in the biology of DCs, B cells, and macrophages stretches far beyond its classical function ,17,18. K2+ and a hub for lipid biosynthesis for cellular membranes [The ER plays a pivotal role in synthesis, folding, and maturation of up to 30% of proteins in the cell . This orembranes . Changesembranes . Additioembranes ,28. ER sembranes   [PERK is a type I transmembrane kinase that under ER stress oligomerizes and auto actor-2) . At the actor-2)   ,14. Remaactor-2) . In immuactor-2) . Activatactor-2) .trans-phosphorylates, activating its RNase domain, which catalyzes the excision of a 26-nt intron within the Xbp1 (X-box binding protein 1) mRNA sequence [Xbp1 spliced mRNA, allowing translation of the active transcription factor XBP1s [Xbp1 unspliced mRNA [IRE1 is the most conserved UPR sensor and the most studied in the immune system ,17,36. Isequence ,38,39,40or XBP1s ,42,43. Xor XBP1s ,45. Furtor XBP1s ,47  components, and proteins involved in lipid biogenesis ,27 Figu. NotablyFinally, beyond the dedicated tasks operated by each branch of the UPR, cooperative interactions between the UPR transducers has also been reported. XBP1 can form heterodimers with ATF6, which regulates an additional core of target genes involved in protein homeostasis ,69. Moreover, recent evidence indicates that the UPR is not only initiated by the accumulation of misfolded proteins, but it can also be activated by additional sources of stress. For instance, IRE1 and ATF6 are sensitive to lipid metabolism and can be activated upon changes in membrane lipid composition, regulating gene expression ,71,72,73+ and CD4+ T cells, respectively. For initiation of CD8+ T cell immunity, Ags of cytosolic origin are processed into peptides by the proteasome [+ T cells, Ag is phagocytosed and directed to early endosomes, which mature into late endosomes that recruits proteolytic enzymes , chaperones, and MHC-II molecules required for processing and loading of Ags into MHC-II molecules [+ T cells against tumors and virally-infected cells [While macrophages, DCs, and B cells differ in ontogeny and perform non-overlapping functions in the immune system, all these lineages can present Ag to T cells to elicit long-term immunity. Different routes of Ag presentation lead to the activation of CD8oteasome . These poteasome ,75. The olecules . Interesed cells . + T cells in steady state [+ T cell priming in pathological or inflammatory settings. On the other hand, prominent findings connect the IRE1/XBP1s axis with Ag presentation in MHC class I . Reportiewed in ,69,80. Iiewed in  , which were associated with tissue inflammation, obesity, and diabetes, induced ER stress and IRE1 signaling in these cells . SFAs inFinally, many studies have also demonstrated a relevant role of the UPR in cancer-associated immune cells and the topic has been extensively reviewed elsewhere . It has Chlamydia trachomatis synthetized IFN-I via a mechanism involving TLR4-IRE1 and PERK [As previously noticed in macrophages, monocytes also differentiate into ‘DC-like cells’ upon inflammation. These cells are referred to as ‘monocyte-derived DCs’ or MoDCs ,94. MoDCand PERK . From a and PERK . As suchand PERK , indicatin vitro [+ T cell activation. Accordingly, enforced expression of XBP1s rendered MoDCs more effective as vaccines in a melanoma setting, by promoting Ag presentation to CD4+ and CD8+ T cells [In cancer models, transmissible ER stress also promoted proinflammatory cytokine secretion in MoDCs, but intriguingly, it impaired the generation of surface MHC-I/peptide complexes and Ag cross-presentation, which facilitated tumor growth . It remain vitro . Inhibitin vitro . This ev T cells ,108. AltDCs are a highly heterogeneous population of leukocytes found in all organs that develop from hematopoietic precursors and that can be divided as plasmacytoid and conventional DCs  . pDCs ar+ T cells [+ T cells [cDCs comprised two major subtypes: cDC1s and cDC2s, which displayed specialized immune functions . cDC1s r T cells ,116 , Ag processing , MHC-peptide loading , vesicular trafficking (ergic3), ER sensors of Ca2+ depletion (stim1/2), chemokines (ccl22), integrins (cd18), and DC receptors (langerin), among others [tapasin and ergic3 were identified as RIDD substrates [XBP1 deletion in hematopoietic precursors resulted in lower numbers of cDC1s and cDC2s, demonstrating a role of the pathway in cDC development ,84. In rg others . As suchg others . On a fug others . While tbstrates .Beyond Ag cross-presentation, IRE1 RNase also regulated the survival of cDC1s in mucosal tissues . XBP1 deHowever, in cDC2s, XBP1 deficiency did not lead to phenotypical or transcriptomic changes . While ncDCs can be partially recapitulated in in vitro cultures of bone marrow cells containing FMS-like tyrosine kinase 3 ligand FLT3L . In thes-/- blastocysts, reconstituted with XBP1-/- embryonic stem cells, showed a role for XBP1 in plasma cell development [Early studies using rag2elopment . XBP1s oelopment  and was elopment ,123. In elopment . While Xelopment , these celopment . In factelopment . Additioelopment ,124. Furelopment . On a fuelopment . A recenelopment . In agreelopment , S729A melopment . Intriguelopment , support+2 sensors, which are required for Ag processing in DCs [In recent years, enormous progress has been made in elucidating the mechanisms by which IRE1 controls protein homeostasis. Remarkably, many of these processes may also be relevant for the APC function in a field that remains largely unexplored. In one respect, XBP1s directly control the expression of Cag in DCs ,128. Furg in DCs , which mg in DCs ,131. Morg in DCs , but it g in DCs , but theWe are just beginning to appreciate the multiple functions of IRE1 in the variety of functions of APCs. To date, the overall picture is far from complete. As such, a major long-term goal is to integrate current knowledge with novel insights on the role of IRE1 in APC biology for generating a comprehensive theoretical framework that leads to translational applications.