Mesenchymal stem cells (MSC) are versatile in stem cell therapy and regenerative medicine, due to its capacity of in-vitro expansion and multilineage differentiation. However, how the MSC’s functions and the underlying epigenomic features are affected by aging and cellular senescence remains poorly understood. Here we used culture expanded human MSC isolated from umbilical cord tissue as an ex vivo model to characterize the chromatin 3D conformational changes associated with aging and senescence and correlate these changes to the changes in MSC stem cell functions during aging. By comparing late passage (P15) vs early passage (P5) MSC, no fundamental changes were found on the large-scale chromatin spatial structures including compartment switching and TAD boundaries. However, when focused on super enhancers, which act as major cis-regulatory hubs associated with lineage-specific functions, a large number of significant promoter-super enhancer interaction changes were discovered. Further examination revealed that YY1, a key regulator of promoter-enhancer looping, was highly enriched in these promoters and their corresponding super-enhancers. The expression of the genes involved in such changes is positively correlated with the variation of promoter-super enhancer interaction, indicating that the spatial communications between promoter and super-enhancer positively regulate gene expression. GO-term and TFBS enrichment analysis showed that these genes involved in cell cycle, adipocyte and osteoblast differentiation. Collectively, these results suggest that YY1-mediated promoter-super enhancer looping is an important regulatory mechanism for MSC multipotency during replicative aging.


Vaccines based on virus-like particles (VLPs) can induce potent B cell responses. Some non-chimeric VLP-based vaccines are highly successful licensed products . Chimeric VLPs are designed to take advantage of the VLP framework by decorating the VLP with a different antigen. Despite decades of effort, there have been few licensed chimeric VLP vaccines. Classic approaches to create chimeric VLPs are either genetic fusion or chemical conjugation, using cross-linkers from lysine on the VLP to cysteine on the antigen. We describe the principles that make these classic approaches challenging, in particular for complex, full-length antigens bearing multiple post-translational modifications. We then review recent advances in conjugation approaches for protein-based non-enveloped VLPs or nanoparticles, to overcome such challenges. This includes the use of strong non-covalent assembly methods (stick), unnatural amino acids for bio-orthogonal chemistry (click), and spontaneous isopeptide bond formation by SpyTag/SpyCatcher (glue). Existing applications of these methods are outlined and we critically consider the key practical issues, with particular insight on Tag/Catcher plug-and-display decoration. Finally, we highlight the potential for modular particle decoration to accelerate vaccine generation and prepare for pandemic threats in human and veterinary realms. The medical and commercial success of virus-like particle (VLP) vaccines against hepatitis B virus  and human papillomavirus  is attributed to their particulate nature , 2. The via major histocompatibility complex (MHC) cross-presentation . Given that many VLPs are icosahedral, the symmetry at the fusion site should match the symmetry of the intended fusion. Other important antigens are heteromultimeric, such as in herpesviruses.Escherichia coli , the Hytönen lab decorated norovirus VLPs Figure  45). tr. tr45). To leverage one of the strongest non-covalent interactions, VLNPs may be biotinylated chemically or using biotin ligase. Biotinylated VLNPs are subsequently recognized by a member of the avidin family Figure . Avidin Flock house virus coat protein was genetically fused to part of the human anthrax toxin receptor (ANTXR2). This fusion allows decoration with anthrax protective antigen, which binds with 170 pM dissociation constant . High tiCovalent reaction between the target antigen and particle brings the obvious advantage of stability. This includes not only stability during storage but also stability after injection, where the lower concentration and altered conditions  favor dissociation of non-covalently linked antigen from the particle. Covalent conjugation also facilitates analysis of decoration. A simple SDS-PAGE can quantify what proportion of particle subunits are coupled to antigen and how much non-covalently associated antigen remain . Covalenvia cell-free protein synthesis  to nanoparticles . SNAP-TaSortase is an enzyme from Gram-positive bacteria that covalently joins proteins with a C-terminal LPXTGX motif to other proteins with an N-terminal oligoglycine motif Figure  65). So. So65). via native chemical ligation  being joined by a peptide bond Figure  68). Sp. Sp68). ligation . Split iligation . Some ofgens see . HoweverElectrostatic interactions have been utilized to align cysteine residues to form preferred disulfide bonds, yielding EILs . Attemptvia side chains, so each partner can react independently of the location within a protein, i.e., either at the N- or C-terminus, or internally within a protein sequence  and a B cell epitope . Linking antigen to scFv via SpyTag/SpyCatcher allowed targeting of antigen to DEC205-positive DCs for cytotoxic T cell stimulation (The first application of Catcher/Tag technology toward immune modulation was the development of SpyAvidins—variants of streptavidin bearing either SpyTag or SpyCatcher on specific subunits of the tetramer. SpyAvidin-MHC eicosamers (bearing 20 streptavidin subunits) surpassed conventional MHC tetramers in stimulating T cell activation . In anotmulation . The sammulation .Plasmodium falciparum blood-stage CIDR antigens or the transmission-blocking ookinete antigen Pfs25 .There is a shortage of side-by-side comparisons of vaccine platforms in the literature. As an initial test of different immunization platforms, mice were immunized with Pfs25 displayed on (i) SpyCatcher-AP205 VLPs using plug-and-display conjugation, (ii) Pfs25-VLPs from chemical decoration of Qβ, or (iii) genetic fusion of Pfs25 to the nanoparticle IMX313. Qβ display generated the highest anti-Pfs25 titer. However, SpyCatcher-VLP display gave the highest antibody quality, assessed by the ability of a given concentration of antibody to block malaria transmission to mosquitoes, using a standard membrane feeding assay Figure , 55. GivThrane and co-workers demonstrated fusion of SpyTag to the N- and C-termini of the AP205 coat protein. Spy-VLPs  were conjugated to a range of antigens from diverse viral and bacterial pathogens (up to 118 kDa) . Spy-VLPThe range of antigens and platforms linked using Tag/Catcher at the time of submission is summarized in Table Decoration of VLNPs with multiple ligands is technically difficult, but may be important for protecting against different strains of a pathogen or even against distinct diseases . For claP. falciparum transmission-blocking antigens, enhancing the response compared with monomeric antigen  coat protein VP1  in vivo . Upon im in vivo .Here, we describe some key issues for modular decoration, moving from proof of principle toward real world application. We illustrate these challenges for SpyTag/SpyCatcher-based assembly, but many issues may be generalized to other decoration technologies.Catcher/Tag decoration appears feasible for a range of non-enveloped VLPs , 91, 97.Yields for multimeric scaffolds of Catcher/Tag-VLPs (AP205 or IMX313) are still at the research scale of 1–20 mg/L using shaking-flask culture , 82, 83.E. coli-expressed Spy-VLPs has been demonstrated, important for well-controlled comparison between immunogens  Figure C 74); t; t74); tChimeric VLPs such as RTS,S  will contain immunologically relevant sequences other than those of the pathogen of primary interest. It is preferable that such sequences are not of human origin, to minimize the risk of autoimmune disease. For RTS,S, one vaccine construct raises antibodies against HBV and malaria. Would pre-existing antibodies against the platform prevent a new vaccine providing protection? For RTS,S in a typical three dose treatment, booster doses do improve the malaria protection even though anti-HBsAg antibodies are already present . SimilarThere is limited information whether SpyCatcher or SpyTag contains MHC class II epitopes, which might contribute to B cell memory. Deletion of the N-terminal region of SpyCatcher to give ΔN1SpyCatcher  reduced antibody titers against SpyCatcher . We showE. coli and Salmonella Typhimurium displaying SpyCatcher-linked pneumococcal antigens  are spherical membranous nanoparticles released from the outer membrane of Gram-negative bacteria. OMVs can be produced at large scale, displaying proteins of interest on their surface or packaging proteins stably inside. SpyTag was first used for OMV display through expression of a SpyTag-OmpA fusion, allowing conjugation of an enzyme for detoxification of nerve agents . SpyTag antigens . In addiantigens . The samantigens .In the future, Tag/Catcher display may facilitate comparison between other non-protein vaccination platforms. Such platforms are beyond our scope to cover in detail, but include emulsions, inorganic NPs, immune-stimulating complexes, and lipid-based NPs  , 111.Virus-like particles are pre-eminent vaccine platforms for inducing antibody responses; a central challenge for the field is to develop platforms giving potent T cell and B cell responses, important for the most intractable pathogens such as tuberculosis or HIV , 113. ThWhere VLNPs may not be able to provide a sufficient T cell response, viral platforms are leading the way at inducing cytotoxic T cell responses. SpyTag fusion has recently enabled modular modification of certain virus models, although immune stimulation has not been tested. The envelope protein GP64 from baculovirus was fused with SpyTag and labeled with SpyCatcher-labeled quantum dots for tracking and visualization in insect cells . SpyTag Beyond infectious diseases, VLP platforms may be adopted for therapeutic use. Allergens on VLPs may suppress allergic immune responses, while avoiding side-effects from direct activation of mast cells . New theIn all fields of science, progress can come faster when one can move from patient crafting of success to a reproducible assembly line –126. VacKB and MH conceived and wrote the manuscript.via spontaneous amide bond formation (EP2534484) and co-founded SpyBiotech Ltd.KB is a consultant and co-founder of Genie Biotech Ltd. MH is an inventor on a patent concerning peptide targeting


Myeloid-derived suppressor cells (MDSCs), one heterogeneous population of immature myeloid cells, have suppressive function on immune response during tumor, inflammation, infection and autoimmune diseases. The molecular mechanism underlying expansion and function of MDSCs is becoming appreciated to manipulate immune response in the diseases. MicroRNA (miRNAs) as one short noncoding RNAs, are involved in regulating cell proliferation, differentiation and maturation. However, it needs to be further studied how miRNAs mediate the development and function of MDSC in association with cancer and other diseases. In the review, we report and discuss recent studies that miRNAs networks regulate the differentiation, expansion and suppression function of MDSCs in tumor microenvironment or other diseases through different signaling pathways. Those studies may provide one novel potential approach for tumor immunotherapy. In tumor bearing-mice, MDSCs are generally characterized as GR-1miRNAs are the abundant small, single-stranded, non-coding RNA of about 22 nucleotides. miRNAs base-paired with the complementary sequence within target mRNAs to mediate post-transcriptional gene repression or target mRNA degradation Recent reports demonstrated that miR-494 expression in tumor MDSCs was dramatically induced by tumor-derived factors, such as TGF-β1 to regulate the accumulation and activity of MDSCs by targeting of phosphatase and tensin homolog (PTEN) and activation of the Akt pathway miRNAs also negatively mediated the differentiation and activity of tumor MDSCs. The overexpression of miR-17 family members such as miR-17-5p, miR-20a and miR-106a in human progenitor cells repressed AML1 by binding to its promoter, which resulted in the down-regulation of M-CSFR, thus limiting MDSC differentiation Extracellular vesicles (EVs) were involved in miRNAs regulation on MDSCs. Cancer cells secreted EVs, which were involved in the intercellular transfer of proteins, lipids, and genetic material (such as miRNAs). Those tumor-associated EVs represented an ideal candidate due to their ability to recirculate in body fluids during the process of MDSC generation from bone marrow in tumor microenvironment Exosomes derived from tumor (such as gliomas) were also involved in MDSC differentiation. In glioma-bearing mice, glioma-derived exosomes (GDEs) facilitated the expansion and function of MDSCs. Hypoxia promoted the upregulation of miR-10a and miR-21 expression in GDEs to induce MDSC activation by targeting the IκBα/NF-κB and PTEN/PI3K/AKT pathways. The reduced numbers of MDSCs were observed in the spleens of mice bearing miR-10a or miR-21 knockout glioma cells, compared with those in bearing glioma cells We ever discussed the findings about the relationship between miRNAs and JAK/STAT3 in cancer Recent data demonstrated that four members of miR-17 family  played the inhibitory roles in the function of tumor MDSCs. In tumor microenvironment, tumor-associated factors downregulated the expression of miR-17-5p and miR-20a and promoted the Stat3-associated suppressive function of MDSCs The enhanced expression of miR-142-3p reduced the immunosuppressive activity of tumor BM-MDSCs, restoring CD8+ T cell proliferation through inhibiting C/EBPβ/STAT3 pathway The regulatory roles of miRNAs on tumor MDSCs are positively involved in STAT3 pathway. miR-200c promoted suppressive potential of tumor MDSCs by targeting PTEN/friend of Gata 2 (FOG2), which led to STAT3 and PI3K/Akt activation The immunotherapy of checkpoints PD-L1/PD-1 on tumor have been broadly applied and those checkpoints were also associated with the tumor MDSCs miRNAs networks regulate the function of MDSCs through other target genes. Recent studies demonstrated that EL-4 tumor-elicited MDSCs showed increased expression of miR-690 with attenuated C/EBPα expression MDSCs also play an important role in other pathological conditions, including inflammation, infection and autoimmune diseases Recent studies demonstrated that MDSCs were involved in autoimmune diseases. In experimental autoimmune encephalomyelitis (EAE), MDSCs can suppress T cell activities, in which miR-223 downregulate the number and function of MDSCs via STAT3. In miR-223 knockout mice, MO-MDSCs suppressed T cell proliferation in vitro and EAE in vivo more than wild-type MO-MDSCsin vivo to act on MDSCs on key signaling pathway. In addition, most of research data about miRNA function on tumor MDSCs were gained from murine studies, even though there are a few miRNA data from human patients with cancer. The significant and application of miRNAs for the expansion and function of MDSCs in patients with cancer need be further investigated. Therefore, the interaction of dysregulated miRNAs on MDSCs with transcription factors, cofactors and chromatin modifiers may target specific miRNA-regulated pathways to provide novel ways to treat MDSCs in tumor microenvironment.MDSCs are one of important immune suppressive cells in tumor microenvironment and may be next breakthrough target for tumor immunotherapy


Biotic stresses do damage to the growth and development of plants, and yield losses for some crops. Confronted with microbial infections, plants have evolved multiple defense mechanisms, which play important roles in the never-ending molecular arms race of plant–pathogen interactions. The complicated defense systems include pathogen-associated molecular patterns (PAMP) triggered immunity (PTI), effector triggered immunity (ETI), and the exosome-mediated cross-kingdom RNA interference (CKRI) system. Furthermore, plants have evolved a classical regulation system mediated by miRNAs to regulate these defense genes. Most of the genes/small RNAs or their regulators that involve in the defense pathways can have very rapid evolutionary rates in the longitudinal and horizontal co-evolution with pathogens. According to these internal defense mechanisms, some strategies such as molecular switch for the disease resistance genes, host-induced gene silencing (HIGS), and the new generation of RNA-based fungicides, have been developed to control multiple plant diseases. These broadly applicable new strategies by transgene or spraying ds/sRNA may lead to reduced application of pesticides and improved crop yield. The arms race of plants and host-pathogens seems never to stop, and sometimes the race is very intense. During the evolutionary process, plants have had to evolve multiple immunity mechanisms to survive danger signals in extracellular and intracellular milieus. Plants are able to enhance disease resistance and increase the food security, as well as to balance the resource allocation between growth and development. The prevalent defense mechanisms are categorized into three defense layers: the preliminary defense, pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) , the secIt is well-known that PTI functions in basal defense. Using the cell surface-localized pattern recognition receptors (PRR), plants can detect the infection of invaders by recognizing the conserved microbe-associated or pathogen-associated molecular patterns (MAMPs or PAMPs) . Plant PNBS-LRR genes can detect or recognize the polymorphic, strain-specific pathogen-secreted virulence effectors, and then transfer the signals to the downstream of defense genes. Thus, ETI-pathways belong to the species-specific disease resistance, and rapidly co-evolve with their pathogens. Plant species in eudicots and dicots have lots of NB-LRR genes. According to the N-terminal features and functions, the NB-LRR proteins in plants can be termed into two classes with the terminal Toll/interleukin-1receptor (TIR) or coiled-coil (CC)/resistance to powdery mildew8 (RPW8) domains Arabidopsuding P2 ,129, P [uding P2 , and P1 uding P2 . One disuding P2  in rice,ematode) .Triticeae for example, the defense genes Sr31 and Sr50 [Secale cereale), can confer the resistance to stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt). Sr35 gene from Triticum monococcum confers the resistance to Ug99 Stem Rust Race Group [NBS-LRR genes can also provide the defense to pathogens. For example, Stb6 in wheat can directly interacted with the effector AvrStb6 that produced by wheat pathogen Zymoseptoriatritici [The disease resistance genes were abundant in the wild resource. In and Sr50  from cerce Group . In additritici . The X10tritici ,137.Sr50 gene were resistant to Puccinia graminis f. sp. tritici (Pgt), while lines without the transgene were susceptible [By introgression or transgene strategy, these defense genes confer the disease resistance in plants. For example, by overexpressing Pm3a/c/d/f/g in wheat, all tested transgenic lines showed the significantly more resistance than their respective non-transformed sister lines in field experiments . The T0 ceptible . Botrytis cinerea can deliver small RNAs (Bc-sRNAs) to plant cells to silence host immunity genes [B. cinerea are mostly produced by Dicer-like protein 1/2 (Bc-DCL1/2). In reverse, over-expressing sRNAs that target Bc-DCL1 and Bc-DCL2 in tomato and Arabidopsis, would silence Bc-DCL genes and inhibit fungal growth and pathogenicity. It exemplified bidirectional CKRI and sRNA trafficking between plants and fungi [It had been demonstrated that plasmodesmata sRNAs can presumably move from cell to cell, and they systemically travel through vasculature . Remarkaty genes . Such smnd fungi . The easVerticillium dahliae, cotton plants increase the dose of miR159 and miR166 in expression level and then export both to the fungal hyphae for specific silencing. Two genes encoding an isotrichodermin C-15 hydroxylase and a Ca2+-dependent cysteine protease, were targeted by miR159 and miR166, respectively. Both of the target genes are essential for fungal virulence [Arabidopsis cells by secreting exosome-like extracellular vesicles can also transfer small RNAs into fungal pathogen Botrytis cinerea. At the infection sites, these sRNA-containing vesicles accumulate and then are taken up by the fungal cells. Delivered host small RNAs induce the silence of fungal genes that is critical for pathogenicity. TAS1c-siR483 target two genes BC1G_10728 and BC1G_10508 from B. cinerea, and TAS2-siR453 targets BC1T_08464. All of the three genes involving in vesicle trafficking pathways are critical for pathogenicity [BC1G_10728 encodes a vacuolar protein sorting 51 and plays a crucial role in Candida albicans virulence [Arabidopsis has adapted exosome-mediated CKRI mechanism as part of its immune responses during the evolutionary arms race with the pathogens [Conversely, hosts also can transfer naturally occurring small RNAs into pests or pathogens to attenuate their virulence  infection by diminishes the repressive roles of OsMADS23, OsMADS27a, and OsMADS57 and concomitant by the up-regulation of OsRDR1 expression. Thus, miR444 can indirectly activate the OsRDR1-dependent antiviral RNA-silencing pathway. Over-expression of osa-miR171b conferred less susceptibility to rice stripe virus infection by regulating the target OsSCL6. OsSCL6-IIa/b/c was down-regulated or up-regulated in plants, where osa-miR171b was over-expressed or interfered [During pathogen infection, plant small RNAs play key roles in gene regulation level. According to the targets of miRNAs that how to respond to the pathogen infection, miRNAs were divided into active and repressed regulation in basal resistance . In the ors TIR1 . Repressresponse . Overexpterfered .NFYA in immunity against the infection of bacterial wilt Ralstonia solanacearum [Magnaporthe oryzae in Arabidopsis and rice, respectively [MdWRKYN1 and MdWRKY26 expression, which decreases the expression of some pathogenesis-related genes, and results in susceptibility to Alternaria alternaria f. sp. mali. In Arabidopsis, miR396/GRF module mediates innate immunity against P. cucumerina infection without growth costs. Reduced activity of miR396 (MIM396 plants) was found to improve broad resistance to necrotrophic and hemibiotrophic fungal pathogens [TCP module involves in the rice blast disease. Increasing expression level of rice miR319 or decreasing expression level of its target TCP21, LIPOXYGENASE2 (LOX2) and LOX5 can facilitate rice ragged stunt virus (RRSV) infection [METHYLTRANSFERASE 2 increased resistance to hemibiotrophic  and necrotrophic (Plectosphaerrella cucumerina) fungal pathogens in Arabidopsis [GhMKK6 gene in cotton, ghr-miR5272a involved in the immune response. Over-expressing ghr-miR5272a increased sensitivity to Fusarium oxysporum by decreasing the expression of GhMKK6 and the followed disease-resistance genes, which lead a similar phenotype to GhMKK6-silenced cotton [Arabidopsis down-regulated the expression of NITROGEN LIMITATION ADAPTATION (NLA) gene. It suppressed the basal defense pathway by enhancing susceptibility to the cyst nematode Heterodera schachtii [In the negative regulation, overexpression their target genes could confer the resistance to pathogens in plants. miR169 suppresses the expression of nacearum  and the ectively . The traectively . In applathogens . MiR319/nfection , which cnfection . Inhibitd cotton . In addichachtii .OsLRR-RLK2 was induced, which is responded to Xanthomonas oryzae pv. Oryzae in rice [Except these miRNAs indirectly regulation the PTI pathway, a few of miRNAs were predicted to directly regulate the receptor-like genes. For example, when osa-miR159a.1 was repressed, the expression of  in rice . In futuMembrin 12 promote the secretion of antimicrobial PR proteins, which functions in ETI during infection of Pseudomonas syringae pv. Tomato in Arabidopsis [Pseudomonas syringae. During early infection, miR863-3p silences two negative regulators of plant defense, namely atypical receptor-like pseudokinase1 (ARLPK1) and ARLPK2, both of which trigger immunity through mRNA degradation. Later during infection, miR863-3p silences SERRATE, and positively regulates defense. And SERRATE is essential for miR863-3p accumulation by a negative feedback loop. Thus, miR863-3p targets both negative and positive regulators of immunity through two modes of action to fine-tune in the timing and amplitude of defense responses [In addition to the basal defense, miRNAs are also involved in ETI pathway to directly and indirectly regulate the disease resistance genes . MiR393*bidopsis . The miResponses .NBS-LRR defense genes is often lethal to plant cells, which is associated with the fitness costs. Thus, plants develop several mechanisms to regulate the transcript level of NBS-LRR genes. One of the key mechanism is the suppression of regulation network in microRNAs and NBS-LRRs, which may play a crucial role in plant-microbe interactions by sRNA silencing mechanism [NBS-LRR genes confer defense against the pathogen infections in gene dosage dynamic expression level by multiple duplications and diversification, while miRNAs minimized the cost of gene copies by inhibiting their expression [NBS-LRRs by targeting the similar motif sites [NBS-LRR genes. High expression of plant echanism . NBS-LRRpression . One miRif sites , which mCC-NB-LRR or TIR-NB-LRR gene classes was mostly characterized in eudicots. In most of the post-transcriptional regulation networks, the miRNA can trigger the 21-nt phased siRNA generation in NB-LRR transcripts, which were processed by RNA-dependent RNA polymerase 6 (RDR6) and DICER-LIKE 4 (DCL4) [Brassica miR1885 were validated to induce by Turnip Mosaic Virus (TuMV) infection, which cleaved TIR-NB-LRR class genes [TIR-NB-LRR immune receptors, both of nta-miR6020 and nta-miR6019 provide resistance to Tobacco mosaic virus (TMV) [Arabidopsis, miR472 modulated the disease resistance genes mediated by RDR6 silencing pathway [Medicago, miR2109, miR482/miR2118 and miR1507 were found to influence NB-LRR gene family [NB-LRR gene class with CC or TIR domains, which were proposed to function in the regulation of defense response or host specificity during rhizobium colonization [NBS-LRR gene class in Norway Spruce [Mla alleles [Mla alleles [The regulation between miRNAs and 4 (DCL4) . For exass genes . In Tobaus (TMV) ,41. In tus (TMV) . In Arab pathway . In Medie family . In legunization ,44. In ay Spruce . In mono alleles . In barlArabidopsis, 44 RLK subgroups were defined, and leucine-rich repeat receptor-like kinases (LRR-RLK) belong to the largest receptor-like kinase family and are focused by researchers [LRR-RLKs constitute 19 subfamilies, most of which were derived from the common ancestors in land plants. The proportions of LRR-RLK genes in Lycophytes and moss genome are 0.30% and 0.36%, respectively, while the proportions of LRR-RLK genes in angiosperms are 0.67–1.39% [LRR-RLK involved in the defense/resistance-related genes was less conserved than that involved in development. Defense-associated LRR-RLKs undergone many duplication events, and most of them were massively lineage-specific expansion mainly by tandem duplication [In land plants, RLKs expanded extensively and fulfilled these diverse roles including perceive growth hormones, environmental/danger signals derived from pathogens . In Arabearchers . Accordi67–1.39% , which ilication ,144. TheNBS-LRR genes as a class of ancient and conserved genes have been detected in gymnosperms, angiosperm plants and animals to ensure immunity [NBS-LRR genes have a great structural diversity in plants and animals. For example, TIR domains were established in the ancestor plants conifers and mosses, and also in animals shared functionality regarding innate immunity [NBS-LRR genes, tandem duplication in genome is the major expansion mechanism in plants. More than 60% of NBS-LRR genes organized in a general pattern of clusters in plant genomes . And in rice, maize, and sorghum, miR3117 also displayed lower expression levels.Although there are many miRNAs regulated c anther ,61,62. INBS-LRRs in plants. The highly duplicated NBS-LRRs were typically targeted by miRNAs (NBS-LRR genes were rarely regulated by miRNAs such as in Poaceae and Brassicaceae genomes. For example, some miRNAs also have a high duplication rate such as miR482/miR2118 in tandem duplication in genomes [Diverse miRNAs, as negative transcriptional regulators, inhibit y miRNAs B, while  genomes ,61,62, wNBS-LRRs from different gene families. And most of these new birth miRNAs target these NBS-LRR gene regions of conserved, encoded protein motif, which follow in the convergent evolution model (NBS-LRR genes in polyploid plants. These NBS-LRR associated miRNAs had a rapid diversity. The nucleotide diversity of the target site region in the wobble position of the codons drives the diversification of miRNAs. These characters of high duplication rate and rapid diversity were similar to their target genes. The co-evolutionary model between NBS-LRRs and miRNAs in plants makes the plants balance the costs and benefits of disease resistance [Newly emerged miRNAs were periodically derived from duplicated/redundant on model B. The misistance .TBF1 gene. It is called uORFsTBF1, which is a key immune regulator and its translation is transiently and rapidly induced upon pathogen challenge (uORFsTBF1-mediated translational control over the production of AtNPR1 in rice and an auto-activated immunity receptor snc1-1 in Arabidopsis did not reduce the plant fitness in the laboratory or in the field [Up to now, a number of genes were exemplified to be involved in plant immunity defense. By over-expressing such defense genes can dramatically enhance disease resistance in plants, while is often associated with significant penalties to fitness and make the resulting products undesirable. Thus, it is difficult in agricultural applications. Recently, it has been developed a strategy to utilize these disease defense genes from the angle of plant genes or their regulators . The strhallenge C, uORF. he field . This stP450 monooxygenase gene of cotton impaired larval tolerance of gossypol [Heterodera glycines Ichinohe. Double-stranded RNA complementary to cytochrome P450 lanosterol C14 alpha-demethylase-encoding genes of Fusarium in Arabidopsis and barley contributes to strong resistance to Fusarium species [MAPKK gene PsFUZ7 in wheat enhance stable resistance to wheat stripe rust [PsCPK1 in Puccinia striiformis f. sp. tritici conferred resistance of wheat to stripe rust [Transgene-derived artificial sRNAs in plants can induce the target gene silencing in certain interacting insects ,85, nemagossypol . HIGS of species . HIGS toipe rust . HIGS ofipe rust . By trancytochrome P450 lanosterol C-14alpha-demethylases of fungal gene can suppress fungal growth [CYP51-targeting dsRNA at a concentration range of 1–20 ng/mL, inhibited growth of Fusarium species [The pathogens and pests are capable to take up the double RNAs or small RNAs from the plants or the environments . Based ol growth . On barl species . It has  species . This SIPlants deployed PTI, ETI, and CKRI innate immune systems to arm race with different pathogen stresses. Pathogens developed more advanced effectors to defeat plant defense immunity. A number of genes have been exemplified to play important role between the host-pathogen interactions in plants. These signaling genes will be helpful to improve plant disease resistance against various pathogens. The sustainable and broaden spectrum resistance genes and their regulators such as miRNAs will be applied in developing crop varieties by introducing the molecular switch. From the cross-kingdom angle, the HIGS can also be used to crop breeding by transgenic approach, which can also confer the broaden spectrum resistance to hosts. The SIGS can also make plants yield the broaden spectrum resistance by spraying the designed dsRNAs/sRNA. Further function studies in plants will dissect more and more defense genes and hopefully unravel the intricate defense regulation network. More and more molecular technologies will be invented and adapted to help develop the eco-friendly disease-resistance cultivars.


MyHC-I, -IIa and -IIx genes were well expressed. In domestic pigs, the transformation was directed toward glycolytic myofibers in which the MyHC-IIb gene transcript was abundant. The myofiber succinate dehydrogenase (SDH) activity and lipid content reflected expression patterns of the MyHCs and not genes involved in lipid uptake and utilization . MyHC myofiber characteristics reflect the effects of selection pressure and rearing conditions on the growth and lean meat content and may be valuable for establishing a balanced breeding scheme focused on improving meat quality traits.This comparative study found that postnatal myofiber differentiation in the longissimus dorsi muscle of wild pigs was accelerated and directed toward oxidative myofibers in which the embryMyHC level decreased significantly faster in the WPs than in the DPs. The relative MyHC-I and -IIa expression was significantly higher in the WPs, and MyHC-IIb was substantially higher in the DPs. The differences in MyHC expression corroborated the number of SDH-positive myofibers and IMFLs. Expression of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) genes displayed only age-related variations. In summary, the evidence is provided for accelerated postnatal myofiber transformation directed towards oxidative myofibers in WPs. The SDH activity/staining intensity largely reflected the expression of MyHCs, and not genes involved in lipid uptake and utilization. This study aimed to compare age-dependent changes in the relative expression of genes encoding myosin heavy chain (MyHC) isoforms and selected lipid metabolism-related genes in the longissimus dorsi muscle of wild pigs (WPs) and domestic pigs (DPs). Muscles sampled from postnatal day one as well as three-week-old and two-year-old animals were used in quantitative polymerase chain reaction (qPCR) assays, histological evaluations of succinate dehydrogenase (SDH) activity, and intra-myofiber lipid (IMFL) assessment. Expression of the MyHC isoforms displayed the most extensive age- and breed-dependent changes within the first three postnatal weeks. The  MyHC-I) and three fast  MyHC isoforms ).low type . In vitrlow type . Additio quality ,13,17.MyHC expression profiles and lipid metabolism were studied in wild and modern highly selected pigs using two genetically distant European pig breeds reared in contrasted conditions. In our previous study, we performed enzyme-immunohistochemical analysis in WPs and DPs, and our results suggested distinct differences in the maturation of different myofiber types and their hypertrophic potential [To understand better the mechanisms behind meat quality deterioration related to domestication and selection pressure, age-related changes in otential . HoweverSus scrofa scrofa) and DPs (Large White) at three different ages as previously described [Muscle samples were collected from the longissimus dorsi muscle of European WPs , agarose gel electrophoresis and Agilent 2100 for the preliminary quantitation, testing of RNA degradation/potential contamination and RNA integrity check and quantitation, respectively. RNA samples that passed the quality control analysis were used in the subsequent steps. Five out of the 30 RNA samples, including four extracted from the WP muscle samples, were excluded due to RNA degradation  lower than 6, which was stringent considering that a RIN higher than 5 was suggested to represent good total RNA quality for downstream application [−ΔCt equation adopted from Livak and Schmittgen, 2001 [agex-Ctday1). No significant relationship was found between age/breed and 18S rRNA expression  according to the manufacturer’s protocol, including an on-column DNase digestion step. RNA sample quality control was performed by Novogene Co., Ltd.  using the following methods: (i) NanoDroplication ). One μglication . The euklication . The effpression ; therefoThe same muscle samples used in the qPCR analysis were also used to obtain transverse cryosections (10 μm), which were stained for succinate dehydrogenase (SDH) activity and the amount of lipids in the myofibers. The cryosections were cut using a cryostat Leica CM 1800 at −17 °C and mounted onto (3-aminopropyl) triethoxysilane (APES) coated slides. The oxidative capacity of the myofibers was determined by analyzing the mitochondrial SDH activity as previously described . The sec® Gold reagent . An optimized oil red O staining protocol developed by Koopman et al.  was usedA Nikon Microphot FXA microscope equipped with a DS-Fi1 camera and the NIS Elements D.32 imaging software  and a Leica multispectral confocal laser microscope  were used to examine the cryosections. Oil red O-stained lipids were detected under the confocal microscope using an excitation laser line at 543 nm (helium-neon). Optical sections (1.0 µm) were acquired, and images were produced using an 8-fold frame averaging a 1024 × 1024 pixel resolution. Representative images were presented using Adobe Creative Cloud . p-value was <0.05.Data were subjected to two-way analysis of variance. The latter included fixed effects of age , breed (WP vs. DP) and their interaction. The analyses were performed using the Sigma Plot 11.0 statistical software . Means were compared with Tukey’s multiple comparisons tests for unequal sample sizes and differences considered statistically significant when embry, -I, -IIa and -IIx isoform mRNAs were abundant in both breeds, the MyHC-I and -IIx levels were significantly lower in the DPs than in the WPs. The relative MyHC-IIb expression level was very low at this age in both the WP and DP piglets ,  are summarized in  0.056), . However 0.056), . We performed enzyme histochemistry assays for SDH activity and oil red O staining . The resMyHCs and selected lipid metabolism-related genes in the longissimus dorsi of WPs and DPs. The relative abundance of MyHC transcripts and the relationships among them have previously been studied in diverse pig breeds [MyHC expression [In this comparative study, we evaluated age-dependent changes in relative mRNA expression of g breeds ,25,26,27pression ,12,25,28pression ,20,26, apression ,11,29.MyHC mRNA expression profiles have been largely in agreement with enzyme-immunohistochemistry analyses of myofiber type compositions [MyHC mRNA and its corresponding protein in the longissimus dorsi muscle has also been demonstrated in growing commercial crossbred pigs [MyHC genes are transcriptionally regulated in porcine skeletal muscles [MyHC transcript levels in both breeds occurred during the first three postnatal weeks. From day one to three weeks postnatal, the order of MyHC transcript levels changed as follows: IIx > IIa > I > embry > IIb to IIx > IIb > IIa > I > embry. Although this pattern of postnatal transitions was the same in both breeds, the quantitative changes were substantially different between them. embryMyHC expression disappeared approximately 10-fold faster in the WPs than in the DPs, whereas MyHC-IIb increased to substantially higher levels in the DPs. embryMyHC is one of the developmental MyHCs that predominate during gestation. Developmental MyHCs are subsequently replaced by adult fast MyHCs  in the late gestation/early postnatal period [embryMyHC expression level reflects muscle maturity. embryMyHC was also reported in 6-week-old DPs [MyHC-IIx expression level observed in the 1-day-old wild piglets additionally confirmed that myofiber maturation and the substitution of developmental with adult MyHC was accelerated during the first post-partum hours in 1-day-old wild piglets.Analyses of ositions . A positred pigs . These r muscles ,9. We obl period . Thus, t-old DPs  and coulMyHC-I expression level was higher in 1-day-old wild piglets than in 1-day-old domestic piglets, perhaps as a consequence of an accelerated transformation from fast to slow MyHCs in the so-called hybrid I/II myofibers during the first hours post-partum. This effect could have been caused by (i) exposure to cold after birth and consequential shivering thermogenesis [PGC-1α expression level in the WPs than in the DPs, which is a well-documented cold- and exercise-induced coactivator [MyHC-I mRNA abundance subsequently increased in both breeds, but its expression was substantially higher in the WPs than in the DPs in all age groups. The results regarding the MyHC-I mRNA expression pattern observed in the present study appear to contradict those in another report, in which the MyHC-I mRNA levels were found to decrease with age in commercial pigs [MyHC-I may increase during the first postnatal week. Furthermore, long-lasting physical activity can also induce myofiber transformation as follows: IIb > IIx > IIa > I [IIa) myofibers. The substantially higher MyHC-IIb level in the DPs corroborates its abundance in the skeletal muscles of modern pig breeds selected for leanness and high growth performance [MyHC-IIb-positive myofibers of DPs have a larger cross-sectional area (CSA) than those of WPs [In addition to age, genes can be up- or down-regulated by a variety of triggering factors, such as nutrition, hormones, and physical activity. Different intrinsic and/or extrinsic insults during gestation and the early postnatal period may have an especially prolonged and substantial influence on MyHC expression and muscle characteristics . The MyHogenesis , (ii) a ctivator  and suppctivator , (iii) lctivator  and iv) ctivator . The MyHial pigs . Howeverial pigs , differe IIa > I . These fformance . At the e of WPs ; additioe of WPs . Therefoe of WPs ,35.MyHC mRNA expression patterns have been suggested to be good indicators of meat quality characteristics [MyHC expression patterns observed in the WPs and DPs were in agreement with the higher number of SDH-positive myofibers and the higher IMFL content (estimated using the oil red O staining intensity) observed in WPs than in DPs. These findings corroborate with previous studies reporting a positive correlation between the abundance of MyHC-I transcript and SDH activity [MyHC-IIb and the IMF levels [PGC-1α expression was higher in local Chinese pigs, together with higher MyHC-I, -IIa and -IIx transcript levels, higher IMF levels and superior meat quality than those of Landrace pigs [PGC-1α level was higher during the early postnatal period in the WPs than in the DPs but that the difference in expression between the breeds was not significant in the adult pigs. In both breeds, the relative PGC-1α expression level decreased during the first three weeks, during which time the myofibers underwent intensive contractile and metabolic specialization. The level stayed the same in the adult WPs, whereas the level increased after the first three weeks in the DPs. The latter result can be explained by the recent finding that PGC-1α plays a role in myoblast differentiation/maturation [PPARγ)—and fatty acid uptake —related genes were reported to be higher in local fatty pig breeds, such as the Rongchang breed in China, than in commercial Landrace pigs, whereas the expression level of the fatty acid oxidation-related gene CPT-1B was higher in Landrace pigs [Although the eristics , we haveactivity  or the Iactivity , and a nF levels . Based oF levels . Similarace pigs ,13. We sturation . AdditioThe present study confirms that WPs exhibit faster postnatal myofiber differentiation/maturation than DPs directed toward the development of oxidative myofibers, which are desirable for meat quality. The histochemical analysis of SDH activity and IMFL staining results corroborate with the MyHC expression pattern. However, the mRNA abundance of the studied genes involved in lipid uptake and utilization did not differ between the wild and modern domestic pig breeds, suggesting that post-transcriptional modifications regulate the metabolic activity of these enzymes.


Soon after they were first described in 1990, aptamers were largely recognized as a new class of biological ligands that can rival antibodies in various analytical, diagnostic, and therapeutic applications. Aptamers are short single-stranded RNA or DNA oligonucleotides capable of folding into complex 3D structures, enabling them to bind to a large variety of targets ranging from small ions to an entire organism. Their high binding specificity and affinity make them comparable to antibodies, but they are superior regarding a longer shelf life, simple production and chemical modification, in addition to low toxicity and immunogenicity. In the past three decades, aptamers have been used in a plethora of therapeutics and drug delivery systems that involve innovative delivery mechanisms and carrying various types of drug cargos. However, the successful translation of aptamer research from bench to bedside has been challenged by several limitations that slow down the realization of promising aptamer applications as therapeutics at the clinical level. The main limitations include the susceptibility to degradation by nucleases, fast renal clearance, low thermal stability, and the limited functional group diversity. The solution to overcome such limitations lies in the chemistry of aptamers. The current review will focus on the recent arts of aptamer chemistry that have been evolved to refine the pharmacological properties of aptamers. Moreover, this review will analyze the advantages and disadvantages of such chemical modifications and how they impact the pharmacological properties of aptamers. Finally, this review will summarize the conjugation strategies of aptamers to nanocarriers for developing targeted drug delivery systems. Aptamers are single-stranded RNA or DNA oligonucleotides that fold up into a distinctive 3D structure capable of binding with high affinity and specificity to small molecules up to entire organisms, with nanomolar range dissociation constants. Nucleic acid-based aptamers were first described in 1990, where the first aptamers were in vitro selected using a random library of single-stranded oligonucleotides sequences . NowadayAptamers need to be stabilized for in vivo use against nuclease degradation, and their small size makes them susceptible to renal filtration. Aptamers’ stabilization can be attained by chemically modifying them using different approaches. Moreover, introducing chemical modifications into nucleic acid libraries increases the interaction capabilities of aptamers and thereby their target spectrum . ModifieChemical modifications of aptamer oligonucleotides are needed mainly to enhance their resistance to nuclease degradation and lowering their renal filtration. Additionally, chemical modifications, in some cases, may increase the aptamer-binding affinity . Many apOther aptamer chemical modification strategies were also required for conjugating aptamers to different drug molecules  or for a13 to 1015 different folded oligonucleotide sequences with the target of choice. This library is composed of random sequences in the middle and constant regions at the 5′- and -3′ ends, which are used for primer annealing and amplification. After incubation, the bound sequences can be separated from the remaining unbound sequences, retained, and then amplified to be reintroduced in iterative selection cycles. Conditions can be changed through each cycle to achieve optimal aptamer target-binding affinity. Rounds of selection are repeated until optimal enrichment for the highest affinity and specificity aptamers are achieved. Approximately 20 rounds of selection would yield high target affinity aptamers . Th. Th57]. Moreover, the stability of selected non G-quadruplex aptamer (NG8) that was modified with 3′-biotin or 3′-inverted thymidine was increased. The 3′-biotin and 3′-inverted thymidine NG8 aptamer was strongly resistant to nuclease attack in serum compared to the unmodified NG8 aptamer. The 3′-inverted thymidine aptamer remained intact for up to 72 and 31 h in 5% and 10% FBS, respectively. The 3′-inverted thymidine modification showed higher stability than the 3′-biotin modification, but both modifications were significantly more stable than the unmodified aptamer .Riccardi et al. synthesized and characterized a dansyl-fluorescent thrombin-binding aptamer (TBA) analog, named tris-mTBA, functionalized with a 5′-biotin tag, for incorporation onto streptavidin-coated silica NPs. The results showed that tris-mTBAwas able to form an antiparallel G-quadruplex structure and retain the ability to form a duplex structure with its complementary strand (cTBA), which acts as an antidote to reverse the anticoagulation activity of TBA. Moreover, they proved that tris-mTBA inhibits the human thrombin activity 10-fold more efficiently than unmodified TBA and biotin-TBA and in a reversible manner. In addition, TBA analogs showed higher resistance to enzymatic degradation compared to the unmodified TBA due to a protective effect of the conjugating groups .Xenopuslaevis oocytes with a half-life of ~10 h, whereas control "normal" oligonucleotides are completely degraded in less than 30 min in both systems . Sy. Sy87].  RNase A . MinakawO and 4′-C of the sugar ring produces an analog of ribonucleotide called locked nucleic acid (LNA) (A methylene linkage between 2′-id (LNA) . This moLNA/DNA chimera LNA5, forming a stable complex against HIV-1 trans-activating response (TAR) RNA, was synthesized from a shortened and stable version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicated that these modifications provide good protection towards nuclease digestion in bovine serum and keep the same binding affinity of the unmodified RNA aptamer . Shi et D value of 4.20 nM) . Th. Th12]. A thrombin-binding quadruplex aptamer with a UNA-modified thrombin-binding aptamer (UNA-modified TBA) was developed. It was found that UNA substitution in the loops of the quadruplex could increase the binding affinity and clotting time in blood samples. The UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties .Aptamers could be selected with LNA or UNA in their structure, which may give better results. Three different LNA-nucleoside triphosphates, LNA-TTP, LNA-ATP, and LNA-5-methyl-CTP, were tested as substrates for KOD DNA polymerase. The results showed that KOD DNA polymerase is good for the synthesis of DNA oligonucleotide duplexes containing LNA nucleotides .l-RNA series, as analogs to RE31-DNA aptamer for effective prolonging of the thrombin time, was evaluated by Kotkowiak et al. They showed that all modified residues can influence the thermal and biological stabilities of G-quadruplex in a position-dependent manner. The aptamers modified simultaneously with UNA and LNAs possess a two-fold higher anticoagulant effect. RE31 variants modified with nucleosides in UNA, LNA, or β-l-RNA series exhibited prolongation of aptamer stability in human serum .,52.14,52The phosphorothioate modification might influence the thermal stability of the quadruplex structure in different G-quadruplex-forming oligonucleotides, and the phosphorothioate backbone is considered to be responsible for strongly binding and inhibiting the gp120 envelope protein of the HIV . PartialPost-SELEX modifications by the addition of short phosphorothioate caps to the 3′- and 5′-ends of the 2′-amino-modified RNA and DNA aptamers against VPF/VEGF showed high binding affinity and increased nuclease resistance .D = 47 nM) without recognizing the other members of the selectin protein family. ESTA-1 aptamer bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovary, and skin without affecting normal organs -2′-deoxyuridine 5′-O-triphosphate was synthesized and used by Balintová et al. as substrate for KOD XL DNA polymerase in a primer extension (PEX) reaction to generate carborane-modified DNA or oligonucleotides. These carborane-modified hydrophobic aptamers may increase the potential interactions against hydrophobic proteins or analytes -2′-deoxyuridine (NapdU) replacing dT) that inhibit interleukin-6 (IL-6) signaling, a key component of inflammatory diseases, were found to be stable in serum and blocked the interaction of IL-6 with its receptor, IL-6Rα -1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNA molecule nor the details of its interaction with a hypoxanthine ligand, with a 3.7 -nM affinity of the riboswitch for guanine . S. S165]. Riccardi et al. designed a highly integrated and multifunctional nanosystem based on niosomal formulations. These niosomes were loaded with the nucleolipid Ru(III)-complex, HoThyRu, and then decorated with AS1411 aptamer via the post-insertion dispersion method. These niosome formulations showed increased antiproliferative activity when loaded with both the Ru(III)-complex and the AS1411 aptamer compared with all the tested controls. A valuable therapeutic window was found for the HeLa cancer cells for concentrations up to 3.5 μM. The final formulation provides several medical applications, including “on-demand” release, specific tissue/cell type targeting, in vivo imaging, and diagnosis .Aptamers can also be directly incorporated in the nanoparticle formulation. For example, gadolinium-doped luminescent and mesoporous strontium hydroxyapatite nanorods loaded with doxorubicin (Gd:SrHap-Dox) were coated with AS1411 aptamer. The coating was performed by fridge incubation of preformed drug-loaded nanorods with a G quadruplex structure of the AS1411 aptamer for 12 h, mainly through strong electrostatic forces. Aptamer-capped Gd:SrHapnanorods can be internalized into MCF-7 cells, resulting in pore opening and drug release .Post-insertion is mostly used to functionalize preformed liposomes with certain aptamers linked to a the lipid anchor, such as DSPE-PEG, to form micelles  49]..49].Willis et al. functionalized the membrane of lipid vesicles with nuclease-stable, anti-VEGF aptamer, using the diacylglycerol (DAG) lipid as an anchor. This conjugation did not influence the binding affinity of the aptamer, and the plasma residence time of the liposome-anchored aptamer was considerably improved compared with that of the free aptamer .Carbodiimides are frequently used in organic synthesis, bioconjugation, and drug delivery. For example, the water-soluble carbodiimide, 1-ethyl-3-(3-(dimethylaminopropyl)-carbodiimide (EDC), is a common reagent used for activating carboxylic acid residues to react with ligands containing amino groups, resulting in amide bond linkage  169]..169].2-PEG-lip) using 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC) and sulfo(N-hydroxysuccinimide) (sulfo-NHS). In Vitro targeting studies verified efficient and rapid uptake of the ESTA-conjugated liposomes (ESTA-lip). Moreover, the aptamer-liposomes were retained in a human breast tumor xenografted model without any decrease in the circulation half-life ..27].A DNA aptamer (sgc8) was successfully attached to the surface of liposomes using the thiol–maleimide chemistry . To prepA DNA aptamer was selected against mouse tumor endothelial cells (anti-mTEC aptamer) (AraHH001). This aptamer was functionalized to the surface of PEGylated liposomes using the thiol–maleimide crosslinking. This functionalization indicates the potential of the targeted delivery of anti-angiogenesis drugs into tumor endothelial cells  195]..195].Li et al. developed nucleolin-targeting liposomes guided by aptamer AS1411 to deliver anti-BRAF siRNA (siBraf) for the treatment of malignant melanomas. The AS1411 aptamer was covalently attached to siRNA-loaded lipoplexes via thiol–maleimide chemistry. This combination showed major silencing activity in A375 tumor xenograft mice and inhibited melanoma growth .An RNA aptamer (Apt1) against the CD44 receptor was selected  and conjDendrimers can also be guided by aptamers. Dendrimers are synthetic, highly branched (treelike) macromolecules with nanometric dimensions. Dendrimers have many functional groups on their surface, offering a high number of sites for the conjugation of targeting ligands . A gene Parallel chemistry to thiol–maleimide conjugation has been used by introducing a soft electrophile (as the Michael system) at the surface of the nanoparticle. This approach is most useful in dendrimer nanoparticles due to their versatile surface functional groups . Anti-nuAnother thiol–maleimide-related aptamer coupling was used in the conjugation of an EpCAM aptamer to carboxymethyl cellulose (CMC)-magnetic iron oxide nanoparticles (CMC-MNPs). CMC-MNPs interacted with 1,6-diaminohexane via carbodiimide coupling in order to introduce some amino groups to the CMC-MNPs’ surface. Next, thiolated EpCAM aptamer was covalently linked to amino-functionalized CMC-MNPs using the hetero-bifunctional crosslinker, 4-maleimidebutyric acid-NHS ester. This magnetic nanoparticle-aptamer probe was utilized for specific hepatocellular carcinoma imaging and treatment .3-(2-Pyridyldithio) propionyl hydrazide (PDPH) was an alternative aptamer bioconjugation cross-linker. PDPH is a heterofunctional crosslinker that possesses a carbonyl-reactive hydrazide group at one end and a sulfhydryl-reactive-pyridyl disulfide group on the other end .In a study by Pala et al., dextran-coated ferric oxide magnetic nanocarriers conjugated with the HER2 aptamers were developed to induce hyperthermia in SK-BR-3 and U-87 MG cells. The hydrazide group firstly reacted with the dextrin moiety to form a hydrazone bond, followed by conjugation with 5′-thiolate HER2 aptamer to the pyridyl end to form a disulfide bond. The aptamer-tagged nanoparticles were highly specific towards the HER2-expressing cells and a 90-fold lower dose was required to kill 50% of the targeted cells compared to the aptamer-free nanoparticle . HER2 apThis approach controls the number of aptamers on the surface of nanoparticles and offers more versatility to the functionalization method. This technique is based on a DNA strand conjugated on the surface of the nanoparticle that is a complementary fit to the intended aptamer . Baek etA DNA-based nanostructure with self-assembly and a pyramid cage composed of four oligonucleotides was introduced by Charoenphol and Bermudez . DNA nanAnother interesting DNA icosahedra nanostructure, loaded with doxorubicin for targeting MCF-7 breast cancer cells, was functionalized with anti-mucin1 (anti-MUC1) aptamers through cDNA strand conjugation. Aptamer-conjugated DNA icosahedra nanostructures showed an efficient and specific internalization for killing epithelial cancer cells .ALK gene expression, leading to growth arrest and apoptosis ..227].Escherichia coli, that are arranged in a hollow spherical nanostructure ..32].N3-AS1411 aptamer with a ω-alkyne-functionalized polyether by click chemistry using tricarboxylate ligand (BimC4A) to stabilize Cu (I) during the cycloaddition. APH molecules, being loaded with doxorubicin, are actively internalized via endocytosis into MCF-7 cells and selectively kill nucleolin-expressing target cells (Aptamer-polymer hybrids (APHs) were synthesized based on the coupling of the 3′-et cells  230]..N3-AS141Although aptamers’ low toxicity and immunogenicity have been demonstrated by the majority of studies published in the literature, some reported cases indicated that administrated doses of aptamers were associated with a degree of toxicity and immunogenicity. As an example, macugen, an anti-VEGF aptamer, showed minimal toxicity and no serious drug-related side effects when injected as single or multiple doses into humans and animals ,280,281.Aptamers are specific nucleic acid-based binding ligands that have refined properties, making them good candidates for various biomedical applications at the diagnostic, therapeutic, and targeted drug delivery levels. Since the discovery of aptamers, several chemical modifications have been introduced to enhance their resistance against nucleases and improve their stability in vivo. As for targeting ligands, aptamers have been successfully conjugated to nanocarriers, drug molecules, and nucleic acids, and they have been successfully implicated in the selective targeting and delivery of therapeutic payloads into targeted cells. Currently, there are several aptamers in various stages of clinical trials, with many already showing promising preclinical data. The field of aptamers is relatively new, but there is a growing body of evidence that it will contribute significantly to the future advancement of the new generation of targeted therapeutics.Aptamers stand for a remarkable new family of medicinal agents placed between conventional organic molecules and biological drugs. Aptamers have size advantages, can be easily chemically synthesized, are largely non-toxic and non-immunogenic, and have high affinities and specificities that are comparable to antibodies. Aptamers can be synthesized and selected to a range of targets more efficiently.Despite all these advantages, aptamers suffer from decreased in vivo stability and high renal excretion. Chemical modifications of aptamers can enhance their pharmacokinetic properties and improve their bioavailability. Moreover, aptamer chemical or physical conjugation to different types of drugs or even to target nanoparticles can expand the range of their medical applications.


Dear Editor,Polycystic ovary syndrome (PCOS) is a common female reproductive endocrinopathy that afflicts up to 10%–15% of women in reproductive age worldwide Nestler, . Women wTo establish a cell model for investigating PCOS disease, we first generated iPSCs from the skin tissues of three PCOS- and three non-PCOS patients. The primary fibroblasts of the patients were transduced with lenti-viral vectors expressing OCT4, SOX2, C-MYC and KLF4 according to a direct reprogramming protocol  were collected between PCOS- and non-PCOS-derived iPSCs. Among these DEGs, we were interested in genes related to neuroendocrine and metabolic processes in PCOS. The enriched top 40 up- and down-regulated genes were shown in heatmaps  were verified by RT-qPCR, according to functional annotation and their fold change  involved in glycolysis process were expressed abnormally, indicating deregulation of glucose metabolism (glycolysis and gluconeogenesis) in PCOS . Filtering by COS Fig. . In ordeFinally, for better understanding the biological effects of deregulated neuroendocrine genes in PCOS, the neural stem cells (NSCs) were differentiated from PCOS- and non-PCOS-derived iPSCs according to a retinoic acid (RA) induction method , which promotes steroidogenesis and follicular development in females Below is the link to the electronic supplementary material.


Dendritic cells (DCs) are the bridge between innate and T cell-dependent adaptive immunity and are promising therapeutic targets for cancer and immune-mediated disorders. Upon stimulation by pathogen or danger-sensing receptors, DCs become activated and poised to induce T cell priming. Recent studies have identified critical roles of metabolic pathways, including glycolysis, oxidative phosphorylation, and fatty acid metabolism, in orchestrating DC function. In this review, we discuss the shared and distinct metabolic programs shaping the functional specification of different DC subsets, including conventional DCs, bone marrow-derived DCs, and plasmacytoid DCs. We also briefly discuss the signaling networks that tune metabolic programs in DC subsets. Dendritic cells (DCs) are an innate immune cell population with the capacity to process and present antigenic peptides on major histocompatibility complex (MHC) molecules to antigen-specific T cells. Thus, the hallmark function of DCs is to induce T cell-mediated immunity to foreign antigens and tolerance to self-antigens . DCs arein vitro from GM-CSF (GM-CSF-BMDCs) or FLT3L (FLT3L-BMDCs), have been essential for advancing our understanding of DC biology. It is important to note that BMDCs are heterogeneous, but certain subpopulations have reported similarities between moDCs, pDCs, and cDCs , plasmacytoid DCs (pDCs), and monocyte-derived DCs moDCs; . cDCs arand cDCs .Metabolism is the process by which cells acquire and process nutrients to fulfill energy and biosynthetic requirements for biological functions . RecentlCompared to resting DCs, activated DCs have increased bioenergetic and biosynthetic demands that are required for protein and membrane synthesis to promote DC maturation. These requirements are met by rapid rewiring of glucose metabolism. Glucose uptake increases upon TLR stimulation, where it can be shuttled into the glycolytic pathway and serve as a precursor for adenosine triphosphate (ATP) production generated by conversion of pyruvate into lactate, even in the presence of oxygen. These are classical features of aerobic glycolysis or Warburg metabolism .Both GM-CSF-BMDCs and cDCs rapidly increase their glycolytic rate within minutes after TLR stimulation, which is maintained for several hours and returns to prestimulation levels when inducible nitric oxide synthase (iNOS) is not expressed . Moreovevice versa after activation  is favored over glycolysis under resting conditions and tivation , a more  T cells . Thus, mWhat regulates the balance between glycolysis and OXPHOS during DC activation? GM-CSF-BMDCs and cDCs have different ways to regulate these pathways. GM-CSF-BMDCs upregulate the expression of iNOS following TLR stimulation . iNOS gede novo fatty acid synthesis that is essential for DC activation.As mentioned above, activated DCs also have increased biosynthetic demands that are required for protein and membrane synthesis. Intermediates produced from glycolysis and mitochondrial metabolism are also important biosynthesis intermediates. Among these intermediates, acetyl-CoA is an important precursor used to generate fatty acids through the enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase FASN; . Upon DCin vivo (+ cDCs) or GM-CSF-BMDCs have normal functional capacity and activation, as revealed by normal expression of CD86 and MHC II, secretion of IL-12/23p40 and TNFα, and T cell priming ability following a bacterial infection  or secreted . These processes are regulated by the fatty acid synthesis-dependent expansion of the endoplasmic reticulum (ER) and Golgi networks in GM-CSF-BMDCs . Consistin vivo . A thirdnfection . Thus, Aper se, dictates DC function. In support of this view, activation-induced fatty acid synthesis results in increased lipid storage in lipid droplets in GM-CSF-BMDCs and cDCs  or blocking the function of Cpt1a (using the drug etomoxir or short-hairpin RNA) decreases TLR-induced production of IFNα, TNFα, and IL-6 by pDCs  on pDCs . The transport of fatty acids into the mitochondria is mediated by Cpt1a . DCs can by pDCs . Qiu et  on pDCs . Mitocho on pDCs . The comBOX 1Does the function or differentiation of different DC subsets rely on distinct metabolic programs?What are the precise roles of different metabolites in orchestrating the function or differentiation of DC subsets?Is there any cross-talk between metabolites and other signaling pathways, such as epigenetic regulation, for specifying the function or differentiation of DC subsets?Could reprogramming of DC metabolism be used as an efficient method for DC-based immunotherapy?Mechanistically, autocrine or paracrine type I IFNs likely regulate this futile cycle of fatty acid synthesis/FAO in pDCs , an ideaThe phosphatidylinositol-3-OH kinase (PI3K)-Akt is a key regulator of metabolic reprogramming . Indeed,in vitro , the serine/threonine kinases whose activity are necessary to limit organ size and suppress tumorigenesis . We recet OXPHOS . ConsequEmerging evidence provides new insight into the metabolic regulation in DCs. However, we are just starting to understand DC metabolism, and many interesting questions remain to be answered Box . InsightXD wrote the manuscript and organized the review. NC wrote part of the manuscript. HC wrote and edited the manuscript and provided overall instructions.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.


Disease association Z scores in DISH were highly correlated with those from imputed HLA genotypes in null model datasets . We applied DISH to two previous GWAS datasets in Asian systemic lupus erythematosus and European rheumatoid arthritis populations. There was a high correlation between Z scores in the DISH and HLA genotype imputations, showing the same disease-susceptible and protective alleles. This study illustrated the usefulness of the DISH method in understanding and identifying disease-associated HLA variants in human diseases while maintaining individual-level data security.Strong genetic associations in the region containing human leukocyte antigen (HLA) genes have been well-documented in various human immune disorders. Imputation methods to infer HLA variants from single nucleotide polymorphism (SNP) genotypes are currently used to understand HLA associations with a trait of interest. However, it is challenging for some researchers to obtain individual-level SNP genotype data or reference haplotype data. In this study, we developed and evaluated a new method, DISH ( HLA genes are highly polymorphic. According to the IPD-IMGT/HLA database2 (Release 3.34.0 on Oct-18 2018), 8 major HLA genes  have >16,000 HLA alleles, encoding for more than 13,000 protein variants.Human leukocyte antigens (HLAs) present short peptides of self or foreign antigens on the cell surface to T lymphocytes4. To further understand the genetic architecture and functional variants in HLA disease association, HLA imputation has been widely used to infer individual HLA classical alleles and amino acid residues from single nucleotide polymorphism (SNP) genotypes using a hidden Markov model (HMM)-based imputation method6. However, individual-level SNP data to impute HLA alleles are typically limited or difficult for other researchers to access because of potential ethical concerns regarding sharing individual data and data security. Moreover, reference panels to provide long-range haplotypes constructed from HLA and SNP variants are not always available publicly. For example, a large European HLA reference panel (T1DGC panel) was initially provided with the SNP2HLA program5, but is no longer available in order to protect personal genetic information.Genetic associations within the major histocompatibility complex (MHC) region, which contains HLA genes, has been well characterized in various human inflammatory disorders, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)P values for SNPs, has become more prevalent in recent years due to voluntary sharing and journal policies. Herein, we introduce a computational tool to impute HLA summary association statistics from SNP summary association statistics  using linkage disequilibrium (LD) information between SNP and HLA variants in European and Asian populations. This method uses existing statistical techniques to impute the associations of untyped SNPs with a trait of interest based on the summary association statistics of typed SNPs. We illustrate that imputation of HLA association statistics can help understand causal HLA alleles hidden in nearby SNP association signals.In contrast to the individual-level GWAS data, sharing summary association statistics, including effect sizes, standard errors, Z-scores, and N where the variance V is variance-covariance/correlation matrix and is equal to the LD correlation (r) matrix. Based on the conditional expectation of MVN variates, several studies have provided programs to impute Z scores of untyped SNP from Z scores of typed SNPs and reference LD information10. In this study, we applied the same mathematical approach to impute HLA association statistics. Z scores in a given locus are partitioned into Z scores of typed SNPs (ZSNP) and untyped HLA variants (ZHLA). The conditional expectation of ZHLA given ZSNP is estimated byHLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1 and -DRB1) and 399 polymorphic amino acid positions into 10,450 haplotypes in 5,225 unrelated Europeans5. The Asian reference dataset was phased by the same method to include 4,758 SNPs, 86 one-field and 163 two-field HLA alleles  and 1,528 polymorphic amino acid position into 1,708 haplotypes in 854 unrelated Asians. All the two-field HLA alleles were obtain by high-resolution sequence-based HLA typing12. The reference datasets were used as reference panels in imputing individual-level genotypes of HLA variants. The more detailed results of the reference datasets are described elsewhere13. Our computational strategy, DISH (direct imputing summary association statistics of HLA variants) is implemented in R and is publicly available at https://github.com/Ben-JWL/DiSH. We note that individual-level data of both the European and Asian references are not publicly available at this time. Instead, we precomputed and provided covariance matrices to protect individual genetic information and to make imputations run faster. The imputation reliability at each HLA variant was assessed by r2pred, the variance of the conditional variable 10.Local allelic correlations of genetic variants cause correlations of disease association statistics including Z scores of the same variants. Z scores can be approximated by multivariate normal (MVN) distribution entclass1pt{minimaWe generated two null-model datasets from pre-existing European and Asian genome-wide association studies (GWAS) data, respectively. European GWAS data was obtained from the Wellcome Trust Case-Control Consortium (WTCCC) (dataset accession ID: EGAD00000000021 and EGAD00000000022). A total of 2,962 WTCCC2 controls from the 1958 British Birth Cohort were genotyped by both Illumina 1.2 M array and Affymetrix_6.0 array. Data were merged and processed by general quality control (QC) procedures. The Asian SLE GWAS data was obtained from our previous study. In brief, a total of 5,342 unrelated QC-passed Korean subjects, including 849 SLE cases and 4,493 controls, were genotyped by Illumina Omni1 arrays and Human610/660W-Quad arrays. The 10,000 null-model datasets were generated by randomly assigning phenotypes (1000 cases and 1000 controls in each dataset) to samples from the original GWAS data. The null-model datasets were used to calculate Z scores of each SNP and to impute Z cores of untyped variants by DISH using an ethnicity-matched reference LD matrix. The accuracy of imputed Z scores in the DISH method was evaluated by comparing with Z scores calculated from imputed individual genotypes that were generated by HMM-based genotype imputation using SNP2HLA.14 and systemic lupus erythematosus in Asians13, respectively. For the European dataset, only association statistics for SNP2HLA-imputed variants was publicly available. From the statistics summary, we arbitrarily created a DISH input consisting of only the biallelic SNPs with official SNP names. For the Asian dataset, we calculated actual Z scores from genotyped data in order to impute DISH Z scores of untyped HLA variants using DISH-based methods.In addition, we applied our method to previously reported GWAS summary association statistic data and individual-level data from rheumatoid arthritis in EuropeansZ scores for genetic associations of untyped SNP and HLA variants from typed SNPs in a 5-Mb window of the MHC region based on a single variance-covariance matrix of the total variants listed in the reference dataset (see Details in Methods). To evaluate the performance of HLA statistic imputation, we generated 10,000 null model datasets from previously reported Asian13 or European (WTCCC2) GWAS data by randomly selecting 1,000 disease-affected cases and 1,000 controls and obtained Z scores of untyped variants by HMM-based and DISH-based imputation. The number of overlapping SNPs between the GWAS data and the ethnicity-matched reference variance-covariance matrix was 3,073 SNPs in Europeans and 2,582 SNPs in Asians. The HMM-based Z scores were obtained by testing for disease associations with SNP2HLA-imputed dosages of untyped variants. The DISH-based Z scores of untyped variants for disease association were calculated from Z scores of typed variants. The correlation coefficient (r) between the two independently generated sets of Z scores with an r2pred value ≥ 0.5 was 0.934 in the Asian dataset and 0.960 in the European dataset  compared to HMM-based Z scores. However, this deflation is critical to control the type I error in SNPs with strong P values . The two-field alleles of HLA-DRB1 showed similar association significance levels using DISH-based and HMM-based strategies. The Z score ranking of HLA-DRB1 alleles was also highly consistent , while HLA-DRB*04:01 showed the most significant protective association . Similarly, RA association of HLA-DRB1 alleles displayed good correlation between both methods (Spearman’s rank correlation coefficient r = 0.974) with the riskiest allele HLA-DRB*04:01  and the most protective allele HLA-DRB*13:01  for European RA and Asian SLE populations, we showed that MVN-based DISH-imputed Z scores calculated from individual-level genotypes of typed variants were highly correlated with HMM-based Z scores calculated from individual-level imputed genotypes by SNP2HLA. Moreover, the most protective, susceptible alleles were identical in both methods, illustrating the usefulness of the DISH method in looking for the most significant alleles in HLA fine-mapping studies. The observed associations of HLA-DRB1 alleles with RA or SLE in DISH-based and HMM-based approaches were supported by previous studies16 that tested for the association for sequenced HLA-DRB1 alleles.In most cases, it is difficult to interpret the biological meaning of SNP associations in the MHC region because of few GWAS variants in the HLA coding variants and numerous trait-associated SNPs due to high LD in the MHC region. In this study, the DISH method was developed to impute association statistics of untyped HLA variants directly from the statistics of typed SNPs. This method works based on the LD information in covariance matrices from European or Asian reference samples without individual-level GWAS genotype and reference data. Several methods to infer Z scores of untyped variants have been reported in recent yearsZ scores, r2pred, and P values of HLA and SNP variants listed only in the reference data. The HLA association summary statistics are generated for one-field and two-field alleles and all polymorphic amino acid residues in HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1 and -DRB1. Thus, the trait-associated classical HLA alleles of the major HLA genes could be identified and the important amino-acid residues or positions could be further narrowed down from DISH outputs. However, we note that it is also possible not to detect a true primary association of HLA genes in DISH outputs especially when minor HLA genes  or synonymous/intronic/UTR variants in the tested HLA genes are the actual primary causes. Furthermore, extensive and high LD patterns in the MHC region may interfere with detecting the true association signals especially for underpowered GWAS data before and after DISH-based imputation.This method provided 8. Third, although the order of significant SNPs sorted by P value was highly correlated between DISH and SNP2HLA methods, actual P values could be substantially different. Small differences in Z scores may show as large differences in the logarithmically scaled P values.There are three major limitations to the DISH approach due to no individual-level data, similar to other association statistic-imputing programs. First, HLA genes are highly polymorphic at the amino acid or gene levels. However, significance levels for disease association can be calculated only for bi-allelic variants . The association statistics for multi-allelic variants  are not able to be imputed from SNP association results and LD information. Second, it is not possible to correct for confounding factors  and perform a conditional analysis that adjusts for the most significant trait-associated variant to identify another independently associated variant. There is a different statistic-imputing program (DISSCO) correcting for confounding covariates but requiring individual-level covariatesIn summary, we introduced the publicly available DISH method to impute HLA associations directly from summary association statistics of nearby SNP associations. This method is useful for understanding and identifying disease-associated HLA variants in human disease while maintaining individual-level data security.Supplementary Information


Immunotherapy is a currently popular treatment strategy for cancer patients. Although recent developments in cancer immunotherapy have had significant clinical impact, only a subset of patients exhibits clinical response. Therefore, understanding the molecular mechanisms of immunotherapy resistance is necessary. The mechanisms of immune escape appear to consist of two distinct tumor characteristics: a decrease in effective immunocyte infiltration and function and the accumulation of immunosuppressive cells in the tumor microenvironment. Several host-derived factors may also contribute to immune escape. Moreover, inter-patient heterogeneity predominantly results from differences in somatic mutations between cancers, which has led to the hypothesis that differential activation of specific tumor-intrinsic pathways may explain the phenomenon of immune exclusion in a subset of cancers. Increasing evidence has also shown that tumor-intrinsic signaling plays a key role in regulating the immunosuppressive tumor microenvironment and tumor immune escape. Therefore, understanding the mechanisms underlying immune avoidance mediated by tumor-intrinsic signaling may help identify new therapeutic targets for expanding the efficacy of cancer immunotherapies. The recent developments in cancer immunotherapy show significant clinical impact. Particularly, monoclonal antibodies targeting the immune checkpoints cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have shown dramatic efficacy and have been approved by the FDA for cancer treatment , 6. TherThe tumor microenvironment consists of the non-cancerous cells present in the tumor, which includes immune cells, fibroblasts, and cells that comprise the blood vessels , 8. It hSeveral host-derived factors may also contribute to immune escape. Inter-patient heterogeneity predominantly results from differences in somatic mutations between cancers , which hTumor-intrinsic signaling pathways are considered to be oncogenic pathways. Increasing evidence has shown that tumor-intrinsic signaling plays a key role in regulating the immunosuppressive tumor microenvironment and tumor immune escape , 18. SucThe innate and adaptive immune cells in the tumor microenvironment harbor both tumor-promoting and tumor-suppressing activities, which may predict clinical outcome , 20. It + dendritic cells (DCs), thereby preventing cross-priming of antitumor T cells .V600-mutant metastatic melanoma, supporting the long-term first-line use of this combination therapy [V600-mutant unresectable or metastatic melanoma patients treated with a combination of dabrafenib plus trametinib show a clear benefit over patients receiving vemurafenib monotherapy, such as survival advantage as well as avoidance of disease-associated and adverse-event-associated symptoms, which supports this combination therapy as a standard of care for this population [V600-mutation melanoma compared with that of vemurafenib monotherapy [V600-mutation melanoma and was not associated with new toxic effects (NCT01682083) [V600-mutant non-small cell lung cancer [NCT02130466), combination therapy with dabrafenib, trametinib, and pembrolizumab conferred longer progression-free survival and duration of response with a higher rate of grade 3/4 adverse events compared with that of dabrafenib and trametinib doublet therapy [Dabrafenib is another selective inhibitor of mutated forms of BRAF kinase, and trametinib is another inhibitor of MEK 1/2. It has been shown that prolonged survival of more than 3 years is achievable with dabrafenib plus trametinib in patients with BRAF therapy . BRAFV60pulation . Anotherotherapy . Adjuvan1682083) . Moreoveg cancer , 101. In therapy .Moreover, Uppaluri et al.  performeOver the past decade, tryptophan catabolism has been considered a mechanism of innate and adaptive immune tolerance. Tryptophan catabolism is a central signaling pathway that maintains homeostasis by inhibiting the immunity that would result from uncontrolled immune responses. It is driven by the key enzymes IDO1 and tryptophan-2,3-dioxygenase 2 (TDO), which result in local depletion of tryptophan and accumulation of tryptophan catabolites, including kynurenine and its derivatives. This regulation of metabolism leads to a local immunosuppressive microenvironment resulting from several mechanisms whose respective roles remain incompletely understood.Drugs targeting this signaling pathway and specifically IDO1 have already underwent clinical trials with the aim to revert immunosuppression induced by cancers . RecentlTargeting tryptophan catabolism combined with other therapeutic strategies may improve the efficacy of cancer immunotherapy. This combination strategy has potential as an alternative for patients whose tumors do not respond to standard therapy . Other tThe spindle assembly checkpoint kinase TTK (Mps1), a key regulator of chromosome segregation, is a novel therapeutic target of small-molecule inhibitors. Treatment of a xenograft model of a CTNNB1-mutant cell line with the TTK inhibitor NTRC 0066-0 resulted in complete inhibition of tumor growth .Small-molecule inhibitors or siRNA for targeting STAT3 signaling have also met with success in mice tumor models , 114. StDeregulation of the PI3K/Akt signaling pathway that leads to enhanced Akt activity is one of the most frequent changes in tumors. In a phase I trial (NCT01476657), duvelisib  demonstrated promising clinical activity and an acceptable safety profile in relapsed/refractory T cell lymphoma , 118. ThWhen compared to parental cells, the cell invasion and migration abilities of OXA-R cells as well as the expression of metastasis-related markers decreased after treatment with the NF-κB inhibitor QNZ . MoreoveFinally, rapamycin was used for targeting TLR4, which triggered immune escape of tumor cells and inhibited the TLR4-activated NF-κB signaling pathway, uncovering a novel mechanism behind the antitumor effects of rapamycin .In this review, we discussed the mechanisms of how oncogenic signaling mediates tumor immune escape, which includes decreased effective immunocyte infiltration and function and increased levels of immunosuppressive cells in the tumor microenvironment Fig. . Therefo


Metal-on-metal (MoM) hip arthroplasties produce abundant implant-derived wear debris composed mainly of cobalt (Co) and chromium (Cr). Cobalt-chromium (Co-Cr) wear particles are difficult to identify histologically and need to be distinguished from other wear particle types and endogenous components  which may be present in MoM periprosthetic tissues. In this study we sought to determine whether histological stains that have an affinity for metals are useful in identifying Co-Cr wear debris in MoM periprosthetic tissues. Histological sections of periprosthetic tissue from 30 failed MoM hip arthroplasties were stained with haematoxylin-eosin (HE), Solochrome Cyanine (SC), Solochrome Azurine (SA) and Perls’ Prussian Blue (PB). Sections of periprosthetic tissue from 10 cases of non-MoM arthroplasties using other implant biomaterials, including titanium, ceramic, polymethylmethacrylate (PMMA) and ultra-high molecular weight polyethylene (UHMWP) were similarly analysed. Sections of 10 cases of haemosiderin-containing knee tenosynovial giant cell tumour (TSGCT) were also stained with HE, SC, SA and PB. In MoM periprosthetic tissues, SC stained metal debris in phagocytic macrophages and in the superficial necrotic zone which exhibited little or no trichrome staining for fibrin. In non-MoM periprosthetic tissues, UHMWP, PMMA, ceramic and titanium particles were not stained by SC. Prussian Blue, but not SC or SA, stained haemosiderin deposits in MoM periprosthetic tissues and TSGT. Our findings show that SC staining (most likely Cr-associated) is useful in distinguishing Co-Cr wear particles from other metal/non-metal wear particles types in histological preparations of periprosthetic tissue and that SC reliably distinguishes haemosiderin from Co-Cr wear debris. Metal-on-metal (MoM) implant components are composed of approximately 62% cobalt, 27–30% chromium, 5–7% molybdenum by weight with small amounts of nickel, iron, silicon, carbon and manganese –4. Resur12 to 2.5 × 1014 particles every year [Although MoM articulations generate less volumetric wear than non-MoM articulations, the number of wear particles produced is significantly greater as the particles are mainly nanometer in size –3. It haery year . Co-Cr pery year . In routery year , 16.Accurate identification of implant-derived wear particles in periprosthetic tissues is important in the histological assessment of implant failure –14. Co-CRepresentative sections of the hip capsule, femoral and acetabular membrane were obtained from 30 cases of failed (aseptic) MoM hip resurfacing /total hip arthroplasty. Specimens from 10 cases of failed non-MoM hip arthroplasties employing ultra-high molecular weight polyethylene (UHMWP), polymethylmethacrylate (PMMA), ceramic and titanium components were similarly examined. Representative sections from 10 cases of haemosiderin-containing diffuse tenosynovial giant cell tumour (TSGCT) were also examined.All specimens were fixed in 10% neutral buffered formalin, routinely processed and paraffin-embedded sections mounted on glass slides. The sections were cut at 4 µm. All sections were stained with Haematoxylin-eosin (HE), Perls’ Prussian blue (PB), Solochrome Cyanine (SC) and Solochrome Azurine (SA). Selected sections containing abundant exudate on the surface of MoM periprosthetic tissue were stained with Martius Scarlet Blue and Masson Goldners Trichrome, both trichrome stains for fibrin .Staining methods employed for HE, SC, SA, and PB were as described by Bancroft and Stevens . The metHaematoxylin-eosin (HE) - stained sections of periprosthetic tissue from failed MoM hip implants showed variably necrotic and degenerate connective tissue in which there was a foreign-body macrophage response to Co-Cr particles. Most macrophages and macrophage polykaryons (giant cells) had plump cytoplasm which stained either pale pink or slate grey, but some cells had pigmented cytoplasm with fine (sand-like), yellow/brown (Co-Cr) particles Fig. . Co-Cr pIn Solochrome Cyanine (SC) -stained MoM periprosthetic tissues, there was a discrete colour product (most commonly purple but ranging from magenta to blue) in the cytoplasm of macrophages and giant cells Fig. . On the Extracellular haemosiderin deposits were present in most specimens of MoM periprosthetic tissues and were highlighted by Prussian Blue (PB) staining. These deposits were SC-negative. Most macrophages were not PB-negative but cytoplasmic PB staining of some pigmented macrophages was noted; in these cells particulate Co-Cr metal wear debris was not stained by PB Fig. . PB staiIn MoM periprosthetic tissues stained with SA, macrophages and giant cells containing Co-Cr particles were unstained and there was no staining of identifiable (PB-positive) haemosiderin deposits.In HE stained sections of periprosthetic tissue from failed non-MoM hip implants, there was a foreign body response to specific implant-derived wear particles. Periprosthetic tissue containing UHMWP and titanium (+/− PMMA) particles elicited a heavy macrophage and giant cell response whereas a less pronounced macrophage response to ceramic wear particles was seen.In non-MoM periprosthetic tissue, there was no SC staining of phagocytic cells that contained PMMA, UHMWP Fig. , titaniuHaemosiderin deposition was noted on HE staining in extracellular connective tissue and phagocytic cells of the synovium in all 10 cases of diffuse TSGCT. There was strong PB staining of haemosiderin but no staining with either SC or SA, the deposits (as in HE-stained sections) appearing brown in colour Fig. .Fig. 6DiMetal-on-metal (MoM) hip implants are known to produce abundant Co-Cr-containing metal debris which may be in the form of wear particles, organometallic complexes, metal salts/oxides and/or free metal ions , 4, 11. 3+ phosphate [Solochrome Cyanine  is a polycyclic aromatic sulfonic acid which has numerous pseudonyms including eriochrome cyanine R, chromoxane cyanin R and chromoxane B . SC has hosphate –30; thishosphate , 31. MacCo-Cr metal wear particles have been subcategorised into larger conventional metallic particles generated by adhesion/abrasion and smaller (mainly nano-size) corrosion metal particles which are produced by tribocorrosion, the process of degradation of the surface of metal implants due to the combined action of mechanical loading and electrochemical corrosion , 4, 14. Histological identification of implant-derived wear particles is important in the diagnosis of implant failure. Individual Co-Cr particles, being nano in size, are too small to be seen under the light microscope and, even when they form visible aggregates, can be difficult to identify histologically. Our empiric observation that SC specifically stains macrophages containing metal debris in MoM periprosthetic tissue is thus likely to be of diagnostic utility in the identification of Co-Cr wear particles. It can be challenging even for experienced musculoskeletal histopathologists to identify morphologically a specific type of wear particle in periprosthetic tissue, particularly when implant components in an arthroplasty are of more than one biomaterial type; for example, in the periprosthetic tissues of MoM arthroplasties employing a titanium acetabular shell, both titanium and Co-Cr metal wear particles can be found in MoM periprosthetic tissues. We noted that titanium, as well as ceramic; PMMA and UHMWP particles were unstained by SC in non-MoM periprosthetic tissues.3+ is not a strong enough oxidising agent itself to oxidise the Fe2+ in ferrocyanide to Fe3+ and Yoshio et al. have shown that treatment of potassium ferrocyanide with Cr3+ ions produces a reddish orange colour [Metal-on-metal implant-derived metal wear needs to be distinguished not only from other exogenous wear particles but also endogenous cell and tissue products, such as fibrin and haemosiderin. Histologically, a necrotic superficial zone on the surface of MoM periprosthetic tissues is often seen in cases of adverse reaction to metal debris (ARMD), particularly in cases of pseudotumour. Metal wear debris in this zone is derived not only from the contiguous metal implant but also from necrotic and apoptotic macrophages that have released Co-Cr wear debris as a consequence of cytotoxicity , 32. We e colour .Most laboratory stains employed in histopathology are dye compounds that have been found empirically to distinguish one or more histological features of diagnostic significance. Solochrome Cyanine (SC) is likely to be useful in this regard in the histological assessment of MoM periprosthetic tissue. SC strongly and specifically stained metal wear debris in phagocytes and in the superficial necrotic zone, a feature consistent with the Cr component of metal wear being discretely stained. In addition, SC did not stain other implant-derived metal or polymer biomaterial wear particles and helpfully distinguished haemosiderin from corrosion metal wear debris.