Increasing evidence suggests an unexpected potential for non-neutralizing antibodies to prevent HIV infection. Consequently, identification of functional linear B-cell epitopes for HIV are important for developing preventative and therapeutic strategies. We therefore explored the role of antigen-specific immune responses in controlling plasma viremia in SIV infected rhesus macaques.MAC251. Peripheral blood CD4+ T-cells were quantified. Plasma was examined for viremia, antigen specific IgG, IgA and IgM binding responses and neutralizing antibodies. Regions containing binding epitopes for antigen-specific IgG, IgM and IgA responses were determined, and the minimum size of linear Envelope epitope responsible for binding antibodies was identified.Thirteen rhesus macaques were inoculated either intravaginally or intrarectally with SIVThe presence of neutralizing antibodies did not correlate the outcome of the disease. In a few SIV-infected macaques, antigen-specific IgG and IgM responses in plasma correlated with decreased plasma viremia. Early induction and the breadth of antigen-specific IgG responses were found to be significantly correlated with the control of plasma viral load. Immunoglobulin classes share similar functional linear B-cell epitopes. SIV-specific linear envelope B-cell epitopes were found to be 12 amino-acids in length.Early induction of combination of peptide-specific IgG responses were found to be responsible for the control of plasma viral load and indicative of disease outcome in SIV-infected rhesus macaques and might be important for the development of therapeutic strategies for control or prevention of HIV/AIDS.The online version of this article (doi:10.1186/s12985-016-0652-x) contains supplementary material, which is available to authorized users. HIV-1 infection is associated with polyclonal B-cell activation, hypergammaglobulinemia, the presence of immature/transitional CD10+ or exhausted CD27 negative B-cells in blood , loss ofB-cell epitopes are described either as conformational  epitopes where multiple discontinuous amino-acids (aa) segments are folded to produce a unique conformational epitope complementary to the antibody, termed a contact epitope –14, or lMAC251 infected rhesus macaques (RMs). Regions containing binding epitopes for antigen-specific IgG, IgM and IgA responses during different stages of SIV infection were determined, and the minimum size of linear Env epitope responsible for binding Abs was identified. Our findings suggest that conformational IgG and IgM responses as well as breadth of different peptide-specific functional IgG responses are indicative of disease outcome. The presence of NAbs against neutralization-sensitive and resistant pseudovirus did not predict the outcome of the disease.The present study was designed to determine the importance of total immunoglobulin, antigen-specific immunoglobulin responses against whole viral lysate (WVL), peptides corresponding to Env, Gag, Nef, and Tat and neutralizing antibodies (NAbs) in controlling plasma viral load (pVL) in SIVAll animals in this study were housed at the Tulane National Primate Research Center (TNPRC) in accordance with the standards incorporated in the Guide for the Care and Use of Laboratory Animals . All of Macaca mulatta), initially negative for SIV, HIV-2, type D retrovirus and simian T-cell leukemia virus 1 infection were inoculated with 300–500 TCID50 of SIVMAC251-CX intravaginally (Ivag) or intrarectally (IR)  , 23 or bPB T-cell immunophenotyping was performed using anti-CD3-FITC (SP34-2), anti-CD4-APC (L200) and anti-CD8-PE (RPA-T8) monoclonal antibodies (MAbs) obtained from BD Biosciences as reported earlier , 26. At MAC251.6 and the neutralization-resistant SIVMAC251.30 pseudoviruses, using MLV-pseudotyped virus as a negative control for non-specific virus inhibition as described previously [Neutralizing antibody (NAb) activity was measured in 96-well culture plates by using Tat-regulated luciferase (Luc) reporter gene expression to quantify reductions in virus infection in TZM-bl cells against the neutralization-sensitive SIVeviously . TZM-bl MAC251 WVL was used as coating antigen  to quantify antibody responses against conformational/discontinuous epitopes. Total IgG, IgM and IgA concentrations were measured by ELISA as previously described [Antigen-specific IgG, IgA and IgM were detected in plasma using ELISA as previously described , 29, 30.escribed , where pSIV-Env , SIV-Gag , SIV-Nef , and SIV-Tat  15-mers with 11-aa overlap peptides (NIH AIDS Research and Reference Reagent Program) were used as coating antigen. Plates were coated with peptide pools  to determine antibody responses generated against linear/continuous epitopes by ELISA as described earlier , 30. Forpost hoc multiple comparison test for all means. Statistical differences between groups were tested using a two-tailed unpaired t-test. Pearson coefficient of determination analysis was performed to calculate correlation between pVLs and immunoglobulin responses and between pVLs and breadth of antigen-specific antibody responses using SAS software (version 9 in a windows environment). For all analyses, P values <0.05 were considered significant.Statistical significance for immunoglobulin quantitation data was determined using a one-way ANOVA. The Bonferroni method was used as a 10 6.3–7.8 RNA copies/ml of plasma) was detected between 14 and 21 day post infection  for SIV-Env, Gag and Nef antigens were used to identify functional linear B-cell epitopes using antigen specific ELISA protocol mentioned above Figs.  and 5. Ip > 0.05). V2-V3 of gp120 played an important role in inducing antigen-specific IgM responses.Overall, the majority of the three immunoglobulin classes bound the same regions of Env peptides  immunoglobulin responses were limited, and most Ab responses occurred in the p2, p1 and highly conserved p8 and p6 regions Fig. . PP11 haIn the IR inoculated SIV-infected RMs, Nef-specific IgG responses were limited to BG21 and appeared late during infection (95dpi). However, Nef-specific IgG responses were detected early at 55dpi only in GN91, a RM from Ivag inoculated SIV-infected group. Moreover, Ivag inoculated SIV-infected RMs that survived upto 257dpi exhibited Nef-specific IgG responses at late chronic phase of infection Fig. . SIV-NefTat-specific responses appeared as early as 73dpi in Ivag infected macaques compared to IR infected macaques where the responses were detected at 236dpi. GN91 was one of the several animals with strong responses to this antigen; however, it showed no differences in the early generation of these responses when compared to other animals.SIV-Envelope epitope mapping was performed with plasma collected from 4 chronically Ivag SIV-infected macaques  using the antigen-specific IgG ELISA protocol as described above. Five to 12-aa peptides from a 15mer SIV-Env peptide (sequence: KTVLPVTIMSGLVFH in V3 region) were synthesized commercially  and used as coating antigen at the concentration of 5 μg/ml to determine the exact length of a linear B-cell epitope. ELISA cutoff values were determined from SIV naïve macaques as described above. Positive IgG responses were detected when peptides with 12aa or greater in length were tested, which suggests that the minimum size of the functional linear epitope for eliciting antibodies was 12aa in length Table .Table 3DMAC251 and SIVMAC239 were shown to be relatively resistant to antibody-mediated neutralization by both autologous and MAbs treatment [MAC251 challenged macaques, none of the macaques were able to generate significant levels of neutralizing Abs against either pathogenic SIVMAC251 or laboratory-adapted SIVMAC251 [Identification of broadly neutralizing antibodies (bNAbs) directed at the Env protein provides a possible solution to generate antibody based vaccine strategies. However, all HIV vaccines assessed to date were unable to generate bNAbs. The contribution of bNAbs to the control of early HIV-1 infection remains uncertain, as bNAbs develop relatively late in infection. Approximately 10–25% of HIV-1 infected people develop bNAbs within 3-years post infection . Moreovereatment , 35. In 3 RNA copies/ml of plasma and had significantly upregulated WVL-specific IgG and IgM responses compared to CL87 suggesting that binding Ab responses might be playing an important role in clearing pVL. Our overall data implies that combination of different peptide-specific IgG responses are predictive of the control of plasma viremia compared to the peptide-specific IgM and IgA responses. Our findings corroborate prior studies showing that while gp41 and gp120 show high levels of binding to IgG, there appears to be no correlation with control of early viral load, and subsequent Env-specific IgG responses had little impact on disease progression [GN91 was able to control pVL to approximately 1 x 10gression –38 when gression , howevergression . While Ggression , indicatgression –48. In tMAC239 were shown to be strong targets for peptide-specific IgG, IgA and IgM responses in SIV infected macaques . Seventy percent of SIV-infected macaques generated antigen-specific IgG responses to this region, and several macaques also generated antigen-specific IgA responses to this region. Therefore, Env-specific immunoglobulins bind to several linear epitopes of recently identified bNAbs, with the exception of the CD4bs. In future studies, it will be important to define the plasma viral sequence data in SIV infected animals and determine if the presence of escape variant(s) have any correlation with the antigen-specific immunoglobulin responses.We also asked whether linear epitopes for antigen-specific immunoglobulins identified in this study corresponded to bNAb targets. The b12 bNAb recognizes almost exactly the same region as CD4, where these conserved regions of the CD4bs  fall witIn summary, quantitative and qualitative immunoglobulin responses were detected in SIV-infected macaques, where increased IgG responses were measured specifically against the gp41 region followed by variable regions V1-V3 of gp120, and NC and p6 region of Gag protein. Antigen-specific IgM and IgA responses were more limited but targeted towards similar regions of the Env and Gag proteins. Several regions in Env protein strongly bind to immunoglobulins and are important epitopes for bNAbs. Early induction and increased breadth of antigen-specific IgG responses might be crucial to the control of plasma viral load and predictive of disease progression in SIV/HIV infection.Our data strongly suggest that an early induction and increased breadth of peptide-specific IgG responses are indicative of disease progression. While Gag-specific responses may be argued to be an indirect indicator of HIV disease progression, recent identification of Abs against Nef and Tat proteins suggests that it may be possible to prevent and control HIV/AIDS by targeting the function of Tat and Nef proteins. Moreover, the presence of NAbs did not predict the outcome of the disease in our study where the generation of those antibodies might be too late to control the SIV infection. The experimental design does not address the role of T-cell responses and other innate immune responses in regulating plasma viral load in these SIV-infected animals.


Chronic pain, including cancer-related pain, is a pain condition often caused by inflammation or dysfunctional nerves. Chronic pain treatment poses a significant health care challenge, where opioids especially morphine are widely used and patients often develop tolerance over time with aggravated pain. microRNA (miRNA) is known to play important roles in regulating gene expressions in the nervous system to affect neuronal network plasticity related to algogenesis and the developing of morphine tolerance. In this article, we reviewed studies conducted in rodent animal models investigating the mechanisms of miRNAs regulation in chronic pain with different phenotypes and morphine tolerance. In addition, the potential of targeting miRNAs for chronic pain and morphine tolerance treatment is also reviewed. Finally, we point out the directions of the future research in chronic pain and morphine tolerance. Chronic pain is usually caused by inflammation or damage to the tissues or nerves in the nervous system. It becomes a major challenge in clinic with worldwide incidence of 20–25%  is small non-coding functional RNA that negatively regulates multiple gene expressions. The sequences of miRNAs and their target sites in mRNAs are extensively conserved across species  Ago2 and TRBP proteins incorporates the guide strand into the RISC complex, and the passenger strand is degraded  RNA polymerase II transcripts miRNA genes to pri-miRNA  of specific-target mRNAs to degrade or destabilize the target mRNA to inhibit protein translation. Each miRNA might target several mRNAs and a single mRNA can also be targeted by several miRNAs , and neuropathic pain   into the rat masseter muscle. Bai et al. showed the downregulation of miR-10a, miR-29a, miR-98, miR-99a, miR-124, miR-134, and miR-183 in TG, of which miR-124 was widely described as a key miRNA in pain-processing. The expression of miR-124 was decreased in the early state after the model was created, but was increased in the late state  but not in a neuropathic pain model (transection of the sciatic nerve). It is suggested that miR-143 down-regulation in response to inflammatory cytokines could be responsible for the increase in versican V1/2 transcription and thereby contributed to the inflammation-dependent upregulation of versican in nociceptors  , miR-124 expression was decreased in putative nociceptive spinal neurons and non-nociceptive DRG neurons  can catalyze DNA 5-hydroxylmethylcytosine (5hmC) in neurons of mammals. TET-mediated hydroxy methylation of miR-365-3p regulated nociceptive behavior. TET1 and TET3, which catalyzed 5hmC in the miR-365-3p promoter were significantly increased in the spinal cord of formalin-induced nociceptive pain mice. In addition, potassium channel, voltage-gated eag-related subfamily H member 2 (Kcnh2) as a target of miR-365-3p, played a critical role in nociceptive pain , transcriptional regulators (MeCP2), and signaling molecules . Both intracellular and circulatory miRNAs were involved in nociceptive pain and the circulatory miRNA let-7b contributed to nociceptive pain as well as intracellular miRNAs. The miRNAs reported to be involved in nociceptive pain in rodent models are summarized in Table Lesions to the somatosensory nervous system frequently give rise to neuropathic pain, generally classified as either peripheral  or central  , which are consistently downregulated in pain conditions with distinct causes. The miR-182-96-183 cluster was downregulated in injured DRG and spinal cord neurons in neuropathic pain  differentially targeting the 3′-UTR sequences of voltage-gated potassium channels and modulatory subunits, was presented to target potassium channels. Overexpression of miR-17-92 cluster contributed to pain through down-regulating potassium channels expression. Conversely, blockade of miR-17-92 alleviates mechanical allodynia in SNL rats . The included literatures also reported two microRNA clusters, miR-182-96-183 and miR-17-92, which mainly targets mRNAs translated to voltage-gated sodium and potassium channels. In general, miRNAs modulate pain-related behaviors by targeting the voltage-gated channels, the signaling pathway inhibitors and the proinflammatory cytokines.Microglia modulated neurotransmission and neuroinflammation. And it has been increasingly recognized as major player in neuropathic pain  in primary spinal microglia. Interestingly, the analgesic effect could be impaired by the autophagy inhibitor, 3-methyladenine. Spinal nerve ligation decreased autophagic activity of primary spinal microglia, while miR-195 inhibitor increased autophagy  in SCI model. Conversely, overexpression of miR-128 significantly promoted the conversion between M1 phenotype and M2 phenotype. Furthermore, miR-128 overexpression obviously decreased the concentration of TNF-α, IL-1β, and IL-6. Although the target of miR-128 and the exact mechanism was unknown, miR-128 overexpression significantly downregulated the expression levels of P38 and P-P38 /signal transducer and activator of transcription 3 (STAT3) pathway, which was indirectly regulated by miR-218 in CCI rats. Overexpression of miR-218 down-regulated the expression of the suppressor of cytokine signaling 3 (SOCS3), leading to STAT3 activation. Moreover, downregulation of miR-218 suppressed microglia activation and STAT3 signaling activation in primary microglia. Thus, silencing of miR-218 may be a promising and novel treatment for neuropathic pain via inhibiting the activation of microglial cell STAT3 signaling and downstream proinflammatory genes of microglia  , which is the direct target of miR-186a-5p drove spinal astrocyte activation and neuropathic pain via CXCR5. CXCR5 expression induced by SNL was required for the SNL-induced activation of spinal astrocytes and microglia. The study revealed a neuronal/astrocytic interaction in the spinal cord by which neuronally produced CXCL13 activates astrocytes via CXCR5 to facilitate neuropathic pain  , the target gene of miR-23b was up-regulated in neuropathic pain. miR23b infusion significantly improved paw withdrawal thresholds. Moreover, miR23b expression also alleviated neuropathic pain through protecting GABAergic neurons against ROS/p38/JNK-mediated apoptotic death . Moreover, using mir-500 antagomir rescued the GABAergic synapses and attenuated the sensitized pain behavior. So miR-500 may be a novel therapeutic option for neuropathic pain  which played roles in releasing synaptic vesicles in hippocampal glutamatergic neurons and regulating the amount of neurotransmitter, was expressed inpeptidergic and non-peptidergic putative nociceptive neurons of DRG -dependent gene expression plays an important role in central sensitization  were targeted by miRNAs. And, it is important to emphasize that interruption to the positive feedback regulation between CREB/CRTC1 and miR-132 can effectively relieve bone cancer pain. The reported dysregulation of miRNAs involved in cancer pain is illustrated in Table in vitro and in vivo correlated with a downregulation of MOR. Injecting anti-let-7 into the brain attenuates opioid antinociceptive tolerance in mice  mRNA which is a transcript known to be intricately involved in dendritic spine density regulation with chronic morphine's consequences is a target of miR27a. miR27a was found to positively regulate Serpini1 mRNA in multiple neuronal cell lines (N2A and N1E-115 neuroblastoma cells). And, Serpini1 knockout mice developed analgesic tolerance at a slower rate than wild-type mice  and the mouse brain. miR-124 inhibited p65 and TRAF6-dependent TLR signaling, which causes immunosuppression of microglia. The study suggested that modulation of miRNAs is capable of preventing opioid tolerance-induced damage to microglia , has been identified to be a target of miR-219. In chronic morphine treatment mouse, miR-219 expression was downregulated with the increasing of CaMKIIγ and CaMKIIγ-dependent BDNF expression in DRG. In addition, morphine tolerance was markedly delayed by upregulating miR-219 expression or downregulating CaMKIIγ expression and was partially attenuated by BDNF via tyrosine receptor kinase B-Fc  was upregulated in mouse DRG following chronic morphine treatment. And upregulation of miR-375 level could significantly hinder morphine tolerance by inhibiting the JAK2/STAT3 pathway  to slightly modified the chemistry of the RNA oligonucleotides. In order to deliver miRNAs into the central nervous system, some studies use viral vectors such as adeno-associated virus (AAV), herpes simplex virus (HSV) and lentivirus (LV) to cross the blood brain barrier.Although only a few miRNA therapeutic clinical trials is currently conducted Bouchie,  and limiCauses of cancer pain are multifactorial and complex and are likely to vary with tumor-related and host-related factors and processes, and the pathophysiology is poorly understood  in mediating and maintaining nociceptive sensitivity, as well as neuropathic and nociceptive pain, has been ascertained in different studies  was dysregulated in microglia both in chronic pain and morphine tolerance. Since microglia is critical in aggravating chronic pain when morphine tolerance develops, miR-124 might be an interesting target for future studies. Although miR-93 and miR-338 were both dysregulated in chronic morphine-treatment bone cancer pain, but whether they were expressed in microglia is unknown. No doubt future studies are likely to provide better insights into miRNAs in microglia of morphine tolerance to reveal the function of miRNAs in the aggravation of chronic pain caused by morphine tolerance.Anti-miRNAs and miRNA-mimics has been frequently applied in animal models of chronic pain to accomplish diagnostic or therapeutic utilization. While the miRNA-therapy in human is warranted to be adequately studied. As described above, miR-16 mimics has been performed in patients failing to respond to standard therapy. As a result, the highly specific miRNAs altered in different poor conditions with the same mechanisms are willing to be especially studied.In animal pain models such as rodent spinal nerve ligation, CFA-induced inflammation and bone cancer pain, wide-spread miRNA modulation is observed. This review summarized the mechanisms of miRNAs regulation in different pain conditions and morphine tolerance. The dysregulation of miRNAs in glial cells was highlighted due to the critical role of these cells in chronic pain. In addition, the use of anti-miRNAs and miRNA mimics as potential therapeutic agents were shown to be effective in reversing pain and morphine tolerance.The roles of miRNAs play in chronic pain are likely to vary depending on the exact causes of chronic pain. Further researches focusing on the understanding of key miRNAs in regulating chronic pain are needed to validate these miRNAs as potential therapeutic target. For examples, understanding the roles of miRNAs play in purinergic signaling network and the regulation of P2 receptors is of great importance. Since microglia is critical in aggravating chronic pain when morphine tolerance develops, miR-124 might be an interesting target for future studies as it was dysregulated in microglia both in chronic pain and morphine tolerance. For future chronic pain and morphine tolerance treatment, anti-miRNAs and miRNA-mimics may prove to be a valuable option once the link between specific miRNAs and chronic pain or morphine tolerance is well established.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vitis vinifera L.), network analyses have been mostly adopted to contribute to the understanding of the regulatory mechanisms that control berry composition. Whereas, some studies have used gene co-expression networks to find common pathways and putative targets for transcription factors related to development and metabolism, others have defined networks of primary and secondary metabolites for characterizing the main metabolic differences between cultivars throughout fruit ripening. Lately, proteomic-related networks and those integrating genome-wide analyses of promoter regulatory elements have also been generated. The integration of all these data in multilayered networks allows building complex maps of molecular regulation and interaction. This perspective article describes the currently available network data and related resources for grapevine. With the aim of illustrating data integration approaches into network construction and analysis in grapevine, we searched for berry-specific regulators of the phenylpropanoid pathway. We generated a composite network consisting of overlaying maps of co-expression between structural and transcription factor genes, integrated with the presence of promoter cis-binding elements, microRNAs, and long non-coding RNAs (lncRNA). This approach revealed new uncharacterized transcription factors together with several microRNAs potentially regulating different steps of the phenylpropanoid pathway, and one particular lncRNA compromising the expression of nine stilbene synthase (STS) genes located in chromosome 10. Application of network-based approaches into multi-omics data will continue providing supplementary resources to address important questions regarding grapevine fruit quality and composition.Representing large biological data as networks is becoming increasingly adopted for predicting gene function while elucidating the multifaceted organization of life processes. In grapevine ( Complex biological processes can be studied from a “multi-omics” perspective thanks to the recent improvements in genome-wide techniques and systems biology approaches. Each omics data type is particularly useful in elucidating the constituents and function of a particular cellular domain. Together, they constitute layers of biological complexity. Genomic data generated from genome sequencing projects are commonly used to ascribe molecular function and biological processes based on sequence similarity, while transcriptomics and metabolomics data typically provide a global “snapshot” of gene expression and metabolite dynamics in various biological contexts.For many omics data, interactions/associations between molecules can be represented as networks, where nodes  are connected by edges. These edges denote an association often inferred from correlational and informational theoretic measures such as Pearson correlation coefficient (PCC) and mutual information (MI), respectively. In the case of gene co-expression networks (GCNs), edges represent similar gene expression behaviors. Based on the “guilt by association” principle, genes involved in related processes share similar gene expression dynamics across a wide range of experiments  could act as important switches in the developmental regulation of metabolism during berry growth and ripening, given their high centrality (number of connections) or degree scores in their network. Dai et al.  showed tSTILBENE SYNTHASE regulator acting in a tissue- and/or stress-dynamic manner.The increased ease of transcriptome profiling, combined with availability of datasets shared by the grapevine research community in public repositories, has led to increased attention in the use of gene co-expression networks (GCNs) in the study of berry development and metabolism. GCNs can be classified into “condition-dependent” and “condition-independent” categories  in cv. “Corvina” grapes  -gene interactions. First, we re-analyzed a comprehensive berry ripening RNA-Seq transcriptome dataset , two anthocyanin-o-methyltransferases (AOMT1-2), the UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UFGT) and ANTHOCYANIN-3-O-GLUCOSIDE-6′′-O-ACYLTRANSFERASE (3AT). Cluster II consisted of genes encoding proanthocyanidin biosynthesis genes including three predicted galloyl glucosyltransferases, ANTHOCYANIDIN REDUCTASE (ANR), and LEUCOANTHOCYANIDIN REDUCTASE (LAR), as well as upstream flavonoid pathway genes such as CHALCONE SYNTHASE (CHS) and CHALCONE ISOMERASE (CHI). One predicted antisense lncRNA (VIT_203s0180n00020) collocated (within 50 kB) and positively correlated with one galloyl glucosyltransferase gene (VIT_03s0180g00200). This cluster also contained genes encoding one 4-coumarate-Co-A ligase (4CL), two flavonol synthases (FLS4-5), one flavanone-3-hydroxylase (F3H), and one caffeic acid 3-o-methyltransferase (COMT), all of which were negatively correlated with genes from cluster I. Furthermore, grapevine miRNAs miR169r/t and grape-m0534 were predicted to target 4CL and FLS4, respectively. Several genes belonging to cluster II and I shared negative correlations  were observed for Fla pathway genes sharing many positive correlations within each group Figure . Clusterases AOMT-2, the UPHENYLALANINE AMMONIA-LYASE (PAL) and STILBENE SYNTHASE (STS) gene families  also shared many strong positive correlations with PAL genes and one 4CL was positively correlated with many STS encoding genes. Gene expressions within this cluster were mainly late-ripening specific, with many of them peaking at harvest. Promoters from cluster IV were highly enriched for cis-regulatory elements including those for R2R3-MYB, AP2/ERF, WRKY, bHLH, and bZIP TF binding. In particular, the MYB binding site CCWACC was present in one CCoAMT, two C4H, 10 PAL, and 27 STS genes. The potential regulation of these genes by MYB transcription factors is supported by recent studies showing that several grapevine MYBs may have regulatory roles controlling the levels of small weight phenylpropanoids and stilbenes  was co-located and strongly co-regulated with all nine STS positioned on chromosome 10. Recent evidence from several functionally characterized lncRNAs in animals and plants suggest that lncRNAs could operate as decoys, guides, signals, and scaffolds, acting as single molecules or complexes regulating pre- and post-transcriptional processes  holding strong positive correlations within and between the two large s Figure . Two cinMulti-omics studies incorporating systems biology approaches in grapevine have facilitated the identification of new grape secondary metabolism regulators and have helped in the characterization of genome-wide responses to environmental factors. These studies have brought knowledge and new tools to understand how to modify and improve grape's quality. Additional efforts will still be needed to map protein-DNA and protein-protein landscapes at a large scale. Also, DNAse I hypersensitivity mapping could be useful to identify pioneering transcription factors controlling grape and wine quality traits.JTM conceived the article and planned its structure. DW and JTM searched and discussed the literature and wrote the manuscript. DW generated new network data. All authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nanophotonic technique has been attracting much attention in applications of nano-bio-chemical sensing and energy conversion of solar energy harvesting and enhanced energy transfer. One approach for nano-bio-chemical sensing is surface plasmon resonance (SPR) imaging, which can detect the material properties, such as density, ion concentration, temperature, and effective refractive index in high sensitivity, label-free, and real-time under ambient conditions. Recent study shows that SPR can successfully detect the concentration variation of nanofluids during evaporation-induced self-assembly process. Spoof surface plasmon resonance based on multilayer metallo-dielectric hyperbolic metamaterials demonstrate SPR dispersion control, which can be combined with SPR imaging, to characterize high refractive index materials because of its exotic optical properties. Furthermore, nano-biophotonics could enable innovative energy conversion such as the increase of absorption and emission efficiency and the perfect absorption. Localized SPR using metal nanoparticles shows highly enhanced absorption in solar energy harvesting. Three-dimensional hyperbolic metamaterial cavity nanostructure shows enhanced spontaneous emission. Recently ultrathin film perfect absorber is demonstrated with the film thickness as low as ~1/50th of the operating wavelength using epsilon-near-zero (ENZ) phenomena at the wavelength close to SPR. It is expected to provide a breakthrough in sensing and energy conversion applications using the exotic optical properties based on the nanophotonic technique. Recent sensing and energy conversion applications are reviewed using surface plasmon resonance and nanophotnic techniques. Nanophotonic techniques have been actively studied and applied in various fields from sensing, subwavelength imaging, to energy conversion in the last decade ,12,13,14Energy conversion is other main applications using nanophotonics. Nanaoparticle-based localized SPR plays a key role in hybrid nano-bio structures to enhance absorption efficiency of light-acceptor materials, such as photosystem I molecules, in energy harvesting by amplifying the surrounding electric field of metal nanoparticles when lights are illuminated . This en−5 in refractive index unit [p-polarized light at an incident angle exceeding the critical angle for total internal reflection, the evanescent wave vector  of evaporating nanofluids. The dry-out pattern shows the traditional coffee-ring pattern due to self-pinning along the edge of droplets. The SPR imaging technique shows that it can detect the optical property variation of effective refractive index in a label-free and quantitative manner. The effective refractive index of nanofluids was successfully measured through total internal reflection (TIR) and verified using SPR reflectance imaging, as shown in  imaging . It presin situ detection.Very recently, SPR holography technique is demonstrated to record the SPR reflectance imaging and phase at the same time as in 2 dielectric) with a very low metal filling factor [2 with Au 5 nm and SiO2 100 nm thickness) at an incidence angle of 45 degree. The signatures and dispersion control of surface plasmon resonance from 1 to 1.8 μm is experimentally demonstrated using a periodic multilayer metallo-dielectric structure. The dispersion control of SPR, by simple variation of filling factor between metal and dielectric materials, provides extensive convenience to select the correct resonance wavelength of the test medium in nano-bio-chemical sensing applications. In addition, it is expected to suggest new sensing techniques using the exotic optical properties of HMM.Currently metallo-dielctric hyperbolic metamaterials (HMM) have attracted growing interest in a vast range of applications, including sub-wavelength imaging, spontaneous emission enhancement, and near-field energy transfer, because of their unique optical properties and simple fabrication. Spoof surface plasmon resonance, based on HMM, demonstrates effective SPR dispersion or wavelength control and exotic optical properties, which can be combined with SPR imaging for the selection of the SPR wavelength, depending on samples, providing higher sensitivity and enhanced spatial resolution. g factor . HMM is Furthermore, nano-biophotonics could enable innovative energy conversion such as the increase of absorption and emission efficiencies and the perfect absorption. Localized SPR using metal nanoparticles shows highly enhanced absorption in solar energy harvesting. et al. [2) layers  layers a with a 2O3 /10 wt % SnO2 sputtering target at room temperature under a base vacuum pressure of 10−7 torr, followed by a 10-minute anneal in Ar gas at 700 °C. Four samples were prepared with differing ITO thicknesses of d = 24, 53, 88, and 137 nm. Subsequently, a 200 nm layer of Ag was deposited on the ITO films using electron beam evaporation. The absorption spectra obtained at the incidence angles for which the maximum absorption was obtained are shown in The other energy conversion application is perfect absorption with low-loss and ultra-thin materials using the epsilon-near-zero (ENZ) phenomena near SPR wavelength region, as in Sensing application demonstrates that SPR imaging can detect the effective optical properties of nanofluids during evaporation-induced self-assembly process in label-free, real-time, and full-field manner. With the development of hyperbolic metamaterials (HMM), it is also shown that the one of the main parameters controlling SPR imaging, SPR wavelength, can be selectively chosen depending on the sample by simply adjusting the filling factor between the metal and dielectric films in HMM. The unique optical property of HMM is expected to provide a new sensing scheme with higher sensitivity. Furthermore, energy conversion applications, such as solar energy harvesting and perfect absorption, are presented to increase energy transfer rate with metal nanoparticles using localized SPR, three-dimensional hyperbolic metamaterial cavity, and ultra-thin film based on epsilon-near-zero (ENZ) phenomena.


There is evidence that eukaryotic miRNAs  play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host–pathogen interaction. To date, different classes of regulatory small-RNAs have been identified which differ in their biogenesis, length, and tissue distribution . Among tMature miRNAs are loaded into Argonaute proteins (mainly AGO2) to form the miRNA-induced silencing complex (miRISC) . One strMicroRNAs were shown to play a role in the replication and propagation of viruses, including cellular antiviral responses and/or in the promotion of viral infections through complex regulatory pathways. It was also shown that miRNAs encoded by viral genomes can be expressed in host cells and participate in the lifecycle and in the cellular consequences of infection. This article review focuses on recent advances in the understanding of miRNA expression and function during viral infections, aiming at providing insights into their role in the pathogenesis of these infections. The article also discusses future directions for research and clinical use of miRNAs as antiviral agents.In early phases of viral infections, innate sensors of host cells detect viral products and initiate signal cascades involved in the antiviral responses. Antiviral miRNAs are components of this response. Some viruses have the capacity of manipulating host miRNAs into escaping an antiviral response or to promote viral infection.To initiate and maintain an antiviral innate response while the more specific adaptive response takes place, cells have developed a microbial pathogen recognition system based on pathogen recognition receptors (PRRs). PRRs, which include Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors and Nod-like receptors, recognize a broad spectrum of motifs common to viral pathogens and this recognition activates a downstream antiviral cascade that includes miRNAs . For insFigure 1), by a mechanism involving the Activator protein 1 (AP1) transcription factor and DNA hypomethylation , miR-155 and miR-223 expression is induced in macrophages through a RIG-I/JNK/NF-κB-dependent pathway. MiR-155 expression is also induced in an infection with Epstein–Barr virus (EBV) in B-cell lymphomas   and FOXOvia AGO effectors , which target several components of the host silencing machinery including small RNA processing, stability and activity ffectors . The 2b ffectors , while tffectors . These mFlaviviruses, which are single-stranded (ss) positive-RNA viruses, interfere with the miRNA processing machinery through an accumulation of the non-coding (nc) subgenomic flavivirus RNA (sfRNAs) that induces sequestration of the dsRNA binding proteins Dicer and AGO2 . This viPoxviridae family with a linear double-stranded DNA genome. VACV abrogates the expression of Dicer independently of VACV decapping enzymes via a mechanism that remains to be clarified .An indirect regulation of miRNA biogenesis has been observed in HeLa cells infected by vaccinia virus (VACV), a member of the larified   expresses a small U-rich ncRNA, HSUR 1, which contains a sequence complementarity to miR-27 . BindingFigure 1).Human cytomegalovirus (HCMV) expresses an nc-transcript, called miRNA decay element (miRDE), that contains several binding sites for miR-17 family members, the binding of which causes their degradation. This proviral function leads to an upregulation of viral DNA synthesis and viral production during lytic infection  -infected human rhabdomyosarcoma (RD) and SK-N-Sh cells. MiR-296-5p targets both capsid protein VP1 and VP3 coding regions in the viral genome as a response to viral infection . Other mPicornaviridae family that provokes cardiomyopathies. In HeLa cells, miR-342-5p targets the 2C-coding region of the viral RNA, which results in its degradation (Coxsackievirus B3 (CVB3) is an RNA virus belonging to the radation .Herpes simplex virus type 1 (HSV-1) replicates in epithelial cells and persists in a latent form in sensory neurons. Lytic replication and reactivation from latency depend on the expression of viral Infected Cell Protein 0 (ICP0), which is controlled by the cell-specific miR-138 in neurons .Another example is provided by miR-548g-3p that targets the Stem Loop A promoter element of DENV 5′-UTR, inhibiting the recruitment of the viral RNA-dependent RNA polymerase (NS5) to the viral genome and resulting in a blockade of viral replication .via downregulation of ERK/MAPK, oxidative stress and PI3K/AKT pathway activation (KUN-induced miR-532-5p downregulates SESTD1 (SEC14 and spectrin domains 1) and TAB3 (TGF-beta activated kinase 1/MAP3K7 binding protein 3) mRNAs to block WNV replication , thereby inhibiting viral replication . MiR-199tivation . The WNVlication .In fine, the stability and translational efficiency of cellular mRNA targets (“targetome”) is increased in infected cells . The liver-specific miR-122 is an example of such a strategy adopted by HCV  . In infed by HCV . MiR-122d by HCV  while itd by HCV . In addid by HCV . Similard by HCV . Finallyinvolved .Figure 3). EV71 infection in RD cells also led to elevated levels of miR-146a. In this model, AP1 is the key transcription factor involved and miR-146a suppresses IRAK1 and TRAF6 expression leading to the inhibition of IFN production, and viral evasion to host immune attacks .In DENV and VSV-infected monocytes/macrophages, and in JEV (strain JaOArS982) -infected human microglial brain cells (CHME3), the induction of miR-146a expression suppresses type I IFN production by targeting IL-1 receptor-associated kinase 1 (IRAK1), IRAK2, and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby enhancing viral replication or allowing viral escape to cellular immune response  .Besides host miRNAs and the various roles they play in the virus–host interaction and resulting infection, certain viruses have been shown to generate their own miRNAs, which play a role in the viral life-cycles and may induce metabolic perturbations in the infected cells , forming chimeric HSV pri-snRNA/v-miRNAs. These chimeric transcripts are then processed by the Integrator host complex into pre-miRNAs from the 3′-end and Herpesvirus saimiri U snRNAs (HSURs) from the 5′-end extremities . These vAnother class of DROSHA-independent miRNAs includes the Murine Gammaherpesvirus 68 (MHV68) v-miRNAs which are essential for transition to latency in the key virus reservoir of memory B-cells and for pathogenesis . These vAlthough this is debated, it appears that some cytoplasmic RNA viruses could generate v-miRNAs . The WNVThe discovery that cytoplasmic RNA viruses could be engineered to produce functional miRNAs emphasizes the existence of cytoplasmic, non-canonical, miRNA biogenesis pathways. For instance, infection of BHK cells by cytoplasmic recombinant Sindbis viruses (rSINVs) allows the expression of virus-derived cytoplasmic pri-miRNAs (c-pri-miRNAs). The viral-induced translocation of DROSHA into the cytoplasm initiates the processing of these c-pri-miRNAs and the biogenesis of miRNAs .in silico and in vivo  3′-UTR, thereby sustaining the latency phase . LikewisViral-miRNAs can also regulate the expression of host mRNAs. v-miRNAs from KSHV target different host mRNAs, including the IκBα mRNA, activating NF-κB signaling and preventing viral lytic replication . SimilarSeveral v-miRNAs enhance viral survival by targeting genes involved in the antiviral immune response. For example, the KSHV-expressed miR-K12-10a directly reduces the expression of TWEAKR (TNF-like weak inducer of apoptosis receptor) , whereasTable 1). Thus, specific v-miRNAs could represent an interesting target for antiviral interventions.These observations indicate that viruses, including DNA viruses, cytoplasmic RNA viruses and retroviruses, produce v-miRNAs through or independently of the DROSHA-dependent maturation pathways. These v-miRNAs generally play a proviral role by enhancing the viral life-cycles, impairing the expression of genes involved in antiviral responses or regulating cell metabolism (in vitro and in vivo proof-of-concept studies. A specific inhibitor (antagomir) of miR-122  entered into human clinical trials  during viral infections and the antiviral response, in order to improve our antiviral All authors contributed to the planning, writing, and revision of the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EGFR wild-type (WT) patients in the ARCHER 1009 trial. Similarly, it failed to demonstrate any survival benefits as compared to placebo in EGFR WT subsets progressing on chemotherapy and at least one previous first-generation TKI (erlotinib or gefitinib) in the BR.26 trial. In the case of EGFR-mutant NSCLCs, a pooled analysis of the ARCHER 1009 and ARCHER 1028 trials comparing the efficacy of dacomitinib vs. erlotinib in chemotherapy-pretreated, EGFR TKI-naïve patients showed a trend to a longer progression-free survival (PFS) and overall survival in favor of dacomitinib that did not reach statistical significance, with a higher rate of treatment related adverse events . On the other hand, the clinical activity in patients with EGFR-mutant NSCLCs with acquired TKI resistance that were included in phase II/III trials was equally poor . Therefore, with the results of the ARCHER 1050 trial (NCT01774721) still pending, the current clinical development of dacomitinib is largely focused on EGFR-mutant, TKI-naïve patients. Here, we review the most relevant clinical data of dacomitinib in advanced NSCLC. We discuss the potential role of dacomitinib in pretreated EGFR WT and EGFR-mutant (TKI-naïve and TKI-resistant) patients. Finally, we briefly comment the available clinical data of dacomitinib in HER2-mutant NSCLC patients.Dacomitinib is a second-generation, irreversible, covalent pan-HER tyrosine-kinase inhibitor (TKI). It showed potent EGFR signaling inhibition in experimental models, including first-generation TKI-resistant non-small cell lung cancer (NSCLC) cell lines. This preclinical efficacy did not translate into clinically meaningful treatment benefits for advanced, pretreated, molecularly unselected NSCLC patients enrolled in two parallel phase III trials. Dacomitinib and erlotinib showed overlapping efficacy data in chemotherapy-pretreated  Second-line treatment options for advanced non-small cell lung cancer (NSCLC) patients have substantially expanded in the past few years. Docetaxel- or pemetrexed-based chemotherapy and erlotinib were the only three drugs approved in our setting until year 2014, achieving an approximate 8–10% of response rates (RRs), median 4 months of progression-free survival (PFS) and 8–10 months of overall survival (OS) , and bevIn the absence of significant differences in terms of efficacy, the choice between pemetrexed- or docetaxel-based second-line chemotherapy is largely driven by three factors: histology, as pemetrexed is restricted to non-squamous tumors, type of platinum doublet used during first-line treatment, with pemetrexed being increasingly incorporated into the first-line or maintenance treatments, and differences in toxicity profiles. On the other hand, when deciding between chemotherapy and erlotinib, apart from clinical factors, EGFR mutation status is the main biomarker that determines treatment selection.EGFR-mutant tumors (EGFR-sensitizing mutations). As the clinical activity of EGFR TKIs in TKI-naïve, EGFR-mutant tumors is comparable between treatment-naïve or platinum-pretreated patients  cancers, RRs and survival were significantly lower with gefitinib- compared to platinum-based chemotherapy in the IPASS study  in treatment-naïve patients was restricted to those with patients , first- SS study . HoweverSS study –13. Moreificance –16.EGFR activating mutations . Moreover, it has inhibitory activity against gefitinib-resistant exon 20 insertions and acquired resistance exon 20 T790M mutations in preclinical lung cancer models. Unlike gefitinib or other first-generation TKIs, dacomitinib, as a pan-ERBB inhibitor, also inhibits the activity of both WT and mutant HER2 kinase , PFS (8 weeks), and OS (26 weeks) to those of the overall study population (p = 0.01). However, no differences in OS were noted   and EGFR WT/KRAS WT NSCLCs . Dacomitinib did not improve OS compared to erlotinib in patients with EGFR WT cancers  and PFS  compared to placebo in patients progressing on chemotherapy and EGFR TKIs, it failed to demonstrate improved OS  , and OS  compared to erlotinib. Results were almost identical for patients with either KRAS or EGFR WT NSCLCs  and OS  outcomes that somehow favored dacomitinib , dacomitinib showed only modest efficacy, with an objective RR of 12%, 3 months of median PFS, and 9 months of median OS. No responses were seen in patients with tumors harboring the most common HER2 activating mutation (c. 2324_2325ins12) (HER2-amplified cancers (n = 4) (HER2 amplification) and what specific HER2 genotypes are true predictive targets for the benefit of dacomitinib.In the largest prospective phase II study conducted to date in patients with 25ins12) . Intrigu (n = 4) . More stEGFR-sensitizing mutations and does not prolong OS compared to placebo in heavily pretreated patients either. Also, dacomitinib does not overcome EGFR T790M-mediated acquired resistance in EGFR-mutant NSCLCs at tolerable doses in humans. In non-T790M-mediated resistance, in which functional activation of HER pathway or acquired HER2 activating mutations have been described in some cases  with potent activity against the T790M mutation , median PFS , and median time to treatment failure  over gefinitib. However, there were no OS differences among treatment arms in this phase IIb trial (n = 319). Pre-specified subgroup analysis according to mutation type (exon 19 deletions vs. L858R mutations) did no show significant differences in OS either. Overall, treatment-related adverse events (mainly skin rash and diarrhea) and serious adverse events were more common with afatinib (EGFR-mutant NSCLCs.In this regard, whether second-generation EGFR TKIs in TKI-naïve patients are superior to first-generation TKIs in EGFR-mutant NSCLCs is not fully answered to date. In the LUX-Lung 7 trial, afatinib significantly increased RRs (70 vs. 56%; afatinib . TherefoAll authors contributed equally to this work.LP-A has received advisory honoraria from Roche, Lilly, MSD, Novartis, BMS, Clovis, Amgen, Boehringer Ing., Astra Zeneca, and Pfizer, not related to this work. The other 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.


Prostate cancer recurrences are common, even with twenty-first-century primary prostate cancer treatment modalities. The most common salvage treatment is (delayed) hormonal therapy, which is often associated with serious side-effects. Due to the risk of significant toxicity, whole-gland targeted salvage treatments remain unpopular. Consequently, developments in focal therapies have arisen. Magnetic resonance imaging (MRI)-guided focal salvage high-dose-rate brachytherapy (HDR-BT) is a novel treatment aiming for minimal toxicity in recurrent prostate cancer patients. Repeating focal treatment could, therefore, be possible in case of post-salvage recurrence. We report the case of a 77-year-old man who underwent repeat focal HDR-BT. Despite improvements in primary prostate cancer care, 15%-55% of patients undergoing radiotherapy develop biochemical recurrence after a 10 years' follow-up . In the In an effort to reduce toxicity in the salvage setting, research has shifted towards organ-preserving approaches. Although prostate cancer is usually multifocal, the “index lesion” hypothesis states there is one clinically important tumor focus in the prostate (the index lesion), which harbors the metastatic precursor cell . After wThe Radiation Oncology department of the University Medical Center Utrecht (UMCU) is equipped with a 1.5T MRI treatment facility, enabling brachytherapy treatment under MRI guidance. The convergence of these technologies allows for an optimal implantation procedure, supporting focal treatment. In 2013, the UMCU introduced MRI-guided, single-fraction (19Gy) focal high-dose-rate brachytherapy (HDR-BT) with iridium-192 as the salvage treatment for local radio-recurrent prostate cancer. With this treatment, the radiation dose to the tumor is escalated while exposure to the surrounding organs at risk (OAR) is limited. Results with regard to toxicity are promising and, therefore, the question arises whether re-treatment with focal HDR-BT is possible for future post-salvage recurrences. This could prevent or further delay the initiation of ADT, thereby avoiding hormone-induced toxicity. Furthermore, postponing castration resistance could potentially increase prostate cancer-specific survival. We present a novel case of second MRI-guided focal salvage HDR-BT.A 77-year-old male visited the radiation oncology department for a follow-up consultation nine years after initial prostate cancer treatment with whole-gland Iodine-125 brachytherapy (145Gy). His further medical history consisted of an asymptomatic thoracic aortic aneurysm and his medication included antihypertensive drugs, a cholesterol-lowering statin, and an anticoagulant. The initial cT1ciT2aNxMx prostate tumor (staged on MRI) was located in the left peripheral zone  7.9 ng/ml). Treatment-related toxicity involved increased urinary frequency and transient obstructive complaints for which he received tamsulosin 0.4 mg once daily for approximately 18 months post-implantation. His erectile function declined but was sufficient for penetrative intercourse without the need for supporting medication. PSA levels dropped to a nadir of 0.2 ng/ml one year after treatment and remained stable during the first three years of follow-up. Later, a steady upward trend was seen with a PSA doubling time (PSADT) of 12 months up to the level of 2.5 ng/ml seven years post-treatment. This was considered a biochemical recurrence according to the Phoenix definition (PSA nadir+2 ng/ml) and radiographic evaluation for recurrent disease was performed using 3T multiparametric-MRI (mp-MRI) and F18-Choline PET-CT. However, there were no signs of local recurrence or distant metastases, nor on repeat imaging one year later.When the patient returned to our department, the PSA level had further increased to 6.7 ng/ml (prostate-specific antigen doubling time (PSADT) 18 months) and a 68Ga-PSMA PET-CT followed. The scan revealed local high uptake in the right dorsal peripheral zone next to the prostate midline and in the right seminal vesicle Figure  and MRI-Focal salvage treatmentPrior to treatment, the gross tumor volume (GTV), clinical target volume (CTV), defined as GTV with a five-millimeter margin, and the organ at risk (OAR)  were delineated based on a 3T mp-MRI and 68Ga-PSMA PET-CT scan. For the CTV to planning target volume (PTV), the margin was 0 millimeter as the source and dose distribution along the tumor in brachytherapy. With the patient in the lithotomy position and under spinal anesthesia, seven MR-compatible catheters were placed in and around the right peripheral zone and seminal vesicle via the perineum . Once again, disease status evaluation was performed with 68Ga-PSMA PET-CT and 3T mp-MRI. As compared to the first diagnostic PET-CT scan, less PSMA uptake was visible in the right peripheral zone. A new suspect lesion of approximately 10 millimeters was suggested in the left dorsal peripheral zone, in close relation to the seminal vesicle . The same recurrence location was revealed on the 68Ga-PSMA PET-CT and confirmed by the mp-MRI Figure  and MRI-Re-salvage treatmentEleven years after the initial prostate cancer treatment and two years after receiving the first MRI-guided focal salvage HDR-BT, the patient was re-treated. A total of eight catheters were placed in and around the recurrence lesion Figure . D95 forWith local salvage treatments, toxicity is a major issue that must be weighed against the benefits of delaying the onset of metastatic disease or, in particular cases, providing a cure. Focal treatment aimed at the tumor lesion instead of the whole prostate gland is a novel development in the span of salvage treatments. Powered by technical advances in diagnostic modalities and the increasing possibilities of MRI guidance before and during treatment, targeted therapy aims to reduce toxicity. With limited toxicity, repetitive salvage treatment could be possible for future local recurrences and ADT (and its side effects) could be postponed or even prevented.This report describes the first case of repeat MRI-guided focal salvage HDR-BT for prostate cancer recurrence. After both focal treatments, the patient experienced minor toxicity (maximum grade 2), which was limited to urinary retention and frequency symptoms, transient flatulence complaints, and a temporary decrease in erectile function. Because of the relatively short follow-up time after the second treatment, there is no information on long-term toxicity yet. However, results with regard to acute toxicity are promising and major complications in the future are not expected due to the high level of dose control with respect to the OAR. The combination of MRI guidance and the steep dose fall-off in brachytherapy allows for high precision in administering the radiation dose to the tumor. In the described case, dose constraints for the OAR were not exceeded during both focal HDR-BT treatments.Within the literature, there are few papers covering repeat salvage therapy. One case report by Claren et al. on second salvage treatment using whole-gland HDR-BT (5x7Gy) showed limited toxicity (grade 2 urinary incontinence). After 24 months, a PSA nadir of 0.03 ng/ml was reached . More reChoosing the appropriate salvage treatment strategy for recurrent prostate cancer is a complex matter and patient selection for focal treatment depends on many factors. To estimate the risk of toxicity, it is essential to establish any pre-existing urinary or bowel symptoms. Time from treatment to biochemical relapse and PSA kinetics, such as PSADT, have prognostic relevance with respect to salvage oncologic outcomes . ApproprMRI-guided focal salvage HDR-BT is a novel modality within the range of local treatment options for recurrent prostate cancer. This case report highlights the potential of this therapy with regard to re-treating locally recurrent prostate cancer after previous salvage treatment. Re-salvage could further delay or even avoid the need for ADT, thereby minimizing the risk of exposure to hormone-related toxicity. The joint use of MRI guidance and HDR-BT allows for targeted therapy with minimal risk of toxicity. Therefore, focal re-treatment seems possible. Further experience in treating patients with re-recurrent local prostate cancer will yield more knowledge on long-term outcomes.


In prostate cancer (PCa), similar to many other cancers, distant organ metastasis symbolizes the beginning of the end disease, which eventually leads to cancer death. Many mechanisms have been identified in this process that can be rationalized into targeted therapy. Among them, epithelial-to-mesenchymal transition (EMT) is originally characterized as a critical step for cell trans-differentiation during embryo development and now recognized in promoting cancer cells invasiveness because of high mobility and migratory abilities of mesenchymal cells once converted from carcinoma cells. Nevertheless, the underlying pathways leading to EMT appear to be very diverse in different cancer types, which certainly represent a challenge for developing effective intervention. In this article, we have carefully reviewed the key factors involved in EMT of PCa with clinical correlation in hope to facilitate the development of new therapeutic strategy that is expected to reduce the disease mortality. N-cadherin, as well as several E-cadherin transcriptional repressors including zinc finger proteins Snail/SNAI1 and Slug/SNAI2, twist-related protein 1 (Twist 1) and zinc finger E-box-binding homeobox 1 and 2 (ZEB1 and ZEB2) are highly elevated during EMT  mainly governs the phenotypic change of polarized ectodermal epithelial cells into migratory mesenchymal cells that ultimately constitute the mesodermal layer of the embryo . EMT occring EMT , leadingN-cadherin can increase prostate tumor spheroid formation by elevating expression of stemness markers such as c-Myc, Klf4, Sox2 and Oct4 via ErbB signaling pathway [N-cadherin and Vimentin are highly upregulated in CD44-positive populations, compared to CD44-negative ones [N-cadherin [Accumulating studies have demonstrated that activation of EMT transcription factors induces acquisition of stem cell properties in epithelial cells and contributes to the emergence of tumor-initiating cell population in several cancer types such as breast and pancreatic cancer ,12,13. I pathway . In addiive ones . Using tive ones . Howevercadherin , suggestEMT-related transcription factors such as Snail, Slug and Twist are shown to confer chemo-resistance in ovarian, breast and nasopharyngeal carcinoma ,19,20. IThe tumor surrounding microenvironment has been shown to play an important role in eliciting EMT of carcinoma cell through paracrine/endocrine fashion. Many extracellular signals are responsible for cell–cell communication that alter PCa cell behavior through a receptor-dependent manner. Several peptide hormones such as TGF-β, IL-6, FGF and Wnt are detected in prostatic stromal cells, which are associated with cancer progression ,25,26,27IL-6 is also demonstrated to be an EMT inducer leading to PCa invasiveness  and its N-cadherin, vimentin and decreases epithelial marker, E-cadherin, leading to cell invasion [Accumulating evidence has demonstrated that fibroblast growth factor (FGF) family is associated with EMT in PCa. An in vitro study using PCa cell lines demonstrated that FGF2 increases mesenchymal markers, invasion . Moreoveinvasion .N-cadherin and vimentin become elevated after ADT [PCa is a typical androgen-dependent disease and ADT is a standard treatment for patients with metastatic disease. It is known that androgen receptor (AR) can induce the expression of several proteases such as MMP2/9 and TMPRSS2 underlying cell invasion ,41,42. Ifter ADT . Furtherfter ADT ,48. For fter ADT ,50,51.N-cadherin and vimentin expression but no change in E-cadherin expression [Clinically, there is a positive correlation between AR and β-catenin in high-grade PCa . It certpression .Accumulating evidence has demonstrated that interplay between signal transduction pathways in response to external stimuli is a critical mechanism to drive the development of metastatic CRPC. In particular, signaling pathways involved in the initiation of EMT often lead to suppression of E-cadherin, resulting in enhanced cell proliferation and metastasis. Particularly, the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway integrates external growth factor stimulations with internal cellular processes. TGF-β can initiate EMT by dissociation of E-cadherin/catenin complexes from the actin cytoskeleton via PI3K/Akt signaling . In thisN-cadherin and Snail [N-cadherin and vimentin [Moreover, another critical downstream effector of Akt during prostate tumorigenesis is the mammalian target of rapamycin (mTOR) kinase. Hyper-activation of mTOR is observed in nearly 100% of advanced PCa ,61. In and Snail . Overallnd Snail . Meanwhind Snail . Functiond Snail ,66,67. Ind Snail ,69,70. Ovimentin . Moreovevimentin ; this isE-cadherin is an essential cell–cell adhesion molecule involved in maintaining the epithelial integrity of the carcinoma cell. Hence, loss of E-cadherin becomes a critical step for EMT initiation and is mainly regulated by several transcriptional repressors such as Snail , Slug 7, Twist ,75 and ZSnail is a zinc-finger protein binding to E-box sequences of the E-cadherin promoter . AberranN-cadherin and vimentin, and increasing E-cadherin expression then diminish tumor cell migration and invasion motility [Slug is a dominant regulator of EMT in many cancers including PCa. In addition to acting as an E-cadherin transcriptional repressor, Slug also regulates other factors leading to EMT in PCa. An in vitro study showed that Slug up-regulates both CXCL12 and CXCR4 and impacts on CXCL12/CXCR4 signaling downstream target gene, MMP9, leading to highly invasiveness of PCa , implyinmotility . Clinicamotility . Taken tN-cadherin expression [N-cadherin transcriptional activation via binding of Twist to the E-box regulatory element within the N-cadherin gene, suggesting that Twist acts as pivotal transcription factor in the metastatic progression of PCa.Twist is a basic helix-loop-helix protein that plays critical roles during development and tumorigenesis. Many studies have demonstrated that Twist can activate EMT, and that it enhances cell migration via binding to the promoter of the E-cadherin gene. In the past, the mechanistic association between Twist and transcriptional repression of E-cadherin has been shown in many malignancies including esophageal squamous cell carcinoma , bladderpression . This stZEB1 is a zinc finger homeodomain transcriptional repressor that regulates skeletal patterning during development and suppresses E-cadherin transcriptional activity in multiple malignancies.Clinically, ZEB1 is elevated in high-grade prostatic tumors, compared to benign or lower grade PCa specimens . A recenN-cadherin, fibronectin, ZEB1 and vimentin in highly metastatic PC3 cells [Forkhead box (FOX) proteins constitute a large family of 19 subgroups of transcriptional regulators that contain an evolutionary conserved DNA binding domain (Forkhead or winged-helix). Among them, FOXA1 is known as a pioneering transcription factor for AR ,98,99. HC3 cells . OverallEpigenetic regulation is considered as a key initial step in mammalian development. Since EMT occurs during embryogenesis, it is conceivable that epigenetics also plays a critical role in pathologic EMT. Accumulating evidence has demonstrated that both hyper- and hypomethylation of DNA are involved in the deregulation of several genes contributing to PCa progression ,107,108.N-cadherin, leading to subsequent up-regulation of E-cadherin [Moreover, histone modification of critical genes has similar effect on EMT induction during PCa metastasis. The histone methyltransferase, MMSET/WHSC1 (Multiple Myeloma SET domain), is capable of facilitating EMT in PCa cells via induction of Twist1, which in turns suppresses E-cadherin expression . In addicadherin . OverallN-cadherin, Twist and Snail are regulated by miR-29b, which is also down-regulated significantly in PCa cell lines and PCa patient specimens when compared to normal prostate epithelial cells and adjacent benign tissue, respectively. Ectopic expression of miR-29b in PCa cells is capable of suppressing PCa invasiveness in vitro, and diminishing secondary colonization at the lungs and liver following intravenous injection in vivo, suggesting miR-29b acts as an anti-metastatic miRNA that is down-regulated during PCa progression [N-cadherin and Snail, and induction of E-cadherin in LNCaP and C4-2B cell lines, overall suggesting that miR-34a-LEF1 regulation plays an important role in the metastatic progression of PCa [MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression via post-transcriptional silencing of target genes. miRNA regulation is highly associated with multiple biological processes such as differentiation, proliferation, migration, survival and invasion. Several miRNAs are known to target transcription factors contributing to the mesenchymal phenotype in PCa . For incgression . Meanwhigression . Similarn of PCa . In addin of PCa . FindingN-cadherin level, and causing morphological change to the epithelial phenotype.In contrast to tumor suppressor miRNAs, aberrant expression of oncogenic miRNAs is observed in highly aggressive PCa associated with EMT. A study using intra-cardiac inoculation of PCa cells in mice demonstrated the oncogenic role of miR-409 in PCa bone metastasis . InhibitIn addition, several studies demonstrated a negative feedback loop between miRNA and EMT transcription factors. For example, ZEB2 known as a direct target of miR-145 can also suppress miR-145 at transcription level. This double negative feedback loop between ZEB2 and miR-145 determines the invasiveness and stemness properties of PCa and contributes to the bone metastasis . MoreoveN-cadherin expression through modulating TGF-β1 signaling, and hence increase PCa cell migration and invasion motility [Long non-coding RNAs (lncRNAs), such as the prostate specific prostate cancer antigen 3 (PCA3/DD3), also plays a critical role in PCa EMT. Silencing of PCA3 in LNCaP cells modulates the expression pattern of several cancer-related genes coding EMT markers such as MTA2 and PLAUR. Meanwhile, PCA3 is shown to facilitate PRKD3-mediated invasion and migration via competitive sponging of miR-1261 . In addimotility .The initiation of EMT is considered the initial step leading to cancer metastasis that is expected to contribute to the poor prognosis of cancer patient. Thus, targeting EMT is likely to improve the overall survival of a patient. EMT is a highly regulated process that can be engaged by the reciprocal interaction between tumor surrounding microenvironment and cancer cells. Through extensive survey in PCa, several key inducers associated with the specific signaling pathways and their regulations have been reported. With respect to the role of EMT in cancer metastasis, stemness, and chemo-resistance, apparently, these key regulators can be druggable targets to be a new generation of cancer medicine as a targeted therapeutic strategy. In this case, small molecule inhibitor such as EZH2 inhibitor or certain unique miRNA such as miR-200  and miR-


In favor of their outgrowth, cancer cells must resist immune surveillance and edit the immune response. Cancer immunoediting is characterized by fundamental changes in the cellular composition and the inflammatory cytokine profiles in the microenvironment of the primary tumor and metastatic niches, with an ever increasing complexity of interactions between tumor cells and the immune system. Recent data suggest that genetic instability and immunoediting are not necessarily disparate processes. Increasing mutational load may be associated with multiple neoepitopes expressed by the tumor cells and thus increased chances for the immune system to recognize and combat these cells. At the same time the immune system is more and more suppressed and exhausted by this process. Consequently, immune checkpoint modulation may have the potential to be most successful in genetically highly altered and usually extremely unfavorable types of cancer. Moreover, the fact that epitopes recognized by the immune system are preferentially encoded by passenger gene mutations opens windows of synergy in targeting cancer-specific signaling pathways by small molecules simultaneously with antibodies modifying T-cell activation or exhaustion.This review covers some aspects of the current understanding of the immunological basis necessary to understand the rapidly developing therapeutic endeavours in cancer treatment, the clinical achievements made, and raises some burning questions for translational research in this field. Tumor immunotherapy has a long-standing history. Starting with the work of William Coley in the 1890s , 3. In seg brentuximab vedotin [Substantial progress has later been made with the introduction of monoclonal antibodies (MAb) inducing apoptosis and/or eliciting antibody- or complement-dependent cytotoxicity after binding to tumor antigens. Just to name a few, anti-CD20- , anti-He vedotin , gemtuzu vedotin , trastuz vedotin , rovalpi vedotin , denileu vedotin ) have aleg programmed death 1 (PD1)), and alterations of other key characteristics . In iews see  and furtiews see ) in ordeiews see , 25.via CD28/cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (eg ipilimumab), or interfering with T-cell exhaustion via the PD1/PD ligand 1 (PDL1) axis  display impressive therapeutic efficacy in melanoma [The generality of these mechanisms across most -if not all- cancer types implies a yet unexploited applicability of drugs targeting immune suppression in a wide range of tumor entities. In fact, antibodies counteracting suppression of the T-cell receptor (TCR) signaling melanoma –32, squamelanoma  and non-melanoma , squamoumelanoma , renal [melanoma , urothelmelanoma  and Hodgmelanoma , 39. Antmelanoma  or CTLA-melanoma  resultedmelanoma .There is need to systematically clarify the potential exploitation of targeting individual receptors expressed by T-cells, with the aim of circumventing the immunosuppressive effects cleverly used by cancer cells to evade host anti-tumor immune responses. In brief, T-cells exhibit various activating and inhibitory ‘checkpoint’ receptors or molecules Fig. .Fig. 1Cheg in [Activating costimulatory immune checkpoint molecules expressed by T-cells include (i) the B7-CD28 superfamily, which encompasses CD28 (the receptor for CD80 and CD86), CD278 (inducible T-cell costimulator (ICOS) and TREML-2/TLT-2 (Trem-like transcript 2 protein), and (ii) members of the tumor necrosis factor receptor (TNFR) superfamily such as CD27, CD134 (OX40), CD137 (induced by lymphocyte activation (ILA)), CD270 (herpesvirus entry mediator (HVEM)) and CD357 (glucocorticoid-induced TNFR family related gene (GITR)) (reviewed eg in ).Inhibitory checkpoint molecules found on T-cells include CD152 (CTLA-4), CD223 (lymphocyte activation gene 3 (LAG3)), CD272 (B- and T-lymphocyte attenuator (BTLA)), CD279 (PD1) and CD366 (T-cell immunoglobulin and mucin protein 3 (TIM3)), V-domain Ig suppressor of T-cell activation (VISTA), as well as the newly discovered T-cell immunoreceptor with Ig and ITIM domains (TIGIT).eg by expression or upregulation of inhibitory molecules by the cancer cells themselves, and/or by production of soluble factors by the cancer cells that result in downregulation or blockage of activating receptors, or in ligation and/or upregulation of inhibitory receptors on T-cells, respectively  or ≤ vs. >50% (for pembrolizumab) and ≤ vs. >1%, 5% or 10% (for atezolizumab) with many reasons suggested, but none being really convincing in explaining these differences , 44. Givie kill) tumor cells. In contrast to tumor-associated antigens (TAA), which are in essence massively overexpressed ‘normal antigens’ that also occur on normal, non-cancerous tissues of the body, TSA are true neoantigens that cannot be found on any non-malignant cell. TAA are much more common than TSA, and strategies targeting TAA molcules include eg the clinically widely successful targeting of CD20 in lymphomas and CD33 in AML, as briefly mentioned above. Targeting of TSA would in theory eliminate the bystander killing of normal cells, which also bear these molecules to a lesser extent .Notably, the mutational burden defined as the number of mutations per megabase, may correlate with -and thus predict the occurrence of- tumor-specific (neo)antigens (TSA) which are expressed on the tumor cell surface and presented to T-cells. T-cells exposed to TSA can learn to specifically target and eliminate  [The response and efficacy of checkpoint inhibitors seems to be highest in tumor types with the highest mutational burden  . This isIn NSCLC patients treated with the PD1-inhibitor pembrolizumab, progression-free survival massively differed according to mutational burden in an as yet unseen manner . This haMost patients with colon cancer usually do not respond to checkpoint inhibitors , howeverThe mutational burden varies substantially over a range of 3–4 logs in different tumor entities  and evenOn average, 2–4 oncogenic driver gene mutations are present in various tumors , 53. It eg in multiple myeloma), mostly via their involvement in generating translocations, which are often associated with adverse outcome [The AID (activation-induced cytidine deaminase)/APOBEC  gene family members are cytidine deaminases causing alterations in DNA and mRNA sequences by cytidine-to-uracil (C-U) transitions -with subsequent conversion of U to thymine (T) during DNA replication- a process called DNA/mRNA-editing that results in protection from parasitic viruses as well as protein and antibody diversity. Loss of cellular control of APOBEC activities results in DNA hypermutations, promiscuous RNA editing, and ultimately genetic instability and tumorigenesis . On outcome . In brea outcome . In line outcome .In non-invasive early urothelial cancers APOBEC-related mutational signatures were predominantly seen in high-risk tumors .In another small series of urothelial tumors, the expression of certain APOBEC family members  was associated with PDL1 positive mononuclear cells infiltrating the tumor and increased expression of the variants A3F_a and A3F_b correlated with upregulated expression of PDL1 on tumor cells , indicatIn high grade serous ovarian carcinomas APOBEC3 expression was significantly associated with T-cell infiltration and -seemingly paradoxically- with improved clinical outcome .Furthermore, breast cancer developed more commonly in women with germline APOBEC3B (A3B) deleting polymorphisms, but these women were not subject to unfavorable risk profiles or worse outcome , suggestOn the other hand, activated members of the APOBEC family might increase the number of neoantigens, cancer-specific T-cell clones and may induce a broader TCR repertoire. Thus, APOBEC family member expression, function, or mutational pattern could serve as a biomarker for the response to checkpoint inhibitors and other immunomodulatory drugs. Initial evidence supporting this hypothesis includes:All of the above data encourage deeper analyses of the correlation (and presumed interaction) between (i) APOBEC family member expression profiles, splice variants and/or polymorphisms and (ii) mutational burden, clonal evolution, and effects on expression profiles of immunomodulatory molecules and their function. This might lead to a better understanding and fine-tuning of immunotherapies in cancer.Often, driver gene mutations and associated atypical proteins remain immunologically silent. In fact, over 90% of cancer cell mutations recognized by CD4+ and CD8+ T-cells occur in passenger genes . This suIndeed, TCR diversity was associated with good clinical outcomes following treatment with the MAb ipilimumab targeting CTLA-4 in a small series of melanoma patients . This waMost analyses of biomarkers thought to predict response to checkpoint inhibitors are currently performed in primary samples of tumor tissue. However, tumor biopsies are sometimes difficult or even impossible to obtain and, depending on the location of the tumor, may be associated with relevant side effects such as an increased risk of bleeding, organ perforation and/or infection, as well as high medical costs for the procedure itself. In addition, tissue biopsies usually cannot be performed continuously during the course of the disease due to these potential risks. Therefore, the actual status of tumor clone evolution and expansion or reduction of T-cell clones capable of actively combating the tumor, remains obscure in patients treated with checkpoint inhibitors. The provision of a rationale for therapeutic decision making and the choice of the optimal immunomodulatory drug most suitable to fight malignant subclones, would ideally require the serial analysis of (i) representative tumor DNA from tissue biopsies, and (ii) various T-cell subsets from peripheral blood.Recently, the detection of circulating tumor DNA (ctDNA) has shown very high identification rates of mutations that were also found in primary tumors using a deep-coverage  next-generation sequencing test of 70 genes .ie type and frequency of mutations) and peripheral blood T-cell exhaustion profiles. This underlines the necessity to characterize peripheral blood T-cells within the frame of clinical trials that aim to aid the immune system to adapt to clonal tumor evolution via therapeutic immunoediting.Nevertheless, it has been shown that 9/10 gastrointestinal cancer patients had CD4+ and/or CD8+ tumor infiltrating lymphocytes that recognized 1–3 neo-epitopes from somatic mutations occurring in the respective cancers . Moreoveeg inhibition of PD1 and activation of CD137, or combined inhibition of inhibitory checkpoint molecules) has been demonstrated [. Early phase I data support the view that such combinations of repressive with activating MAbs are feasible . However, these therapeutic approaches must be viewed with caution and be closely monitored, given the overwhelming activation of autoimmunity which could arise.The approach of repressing multiple pathways, or of combining repressive with immunostimulatory antibodies seems particulary exciting and is currently investigated in numerous trials Table . In precnstrated –78. Earleg anti-PD1 or anti-PDL1 antibodies with anti-TIM3 antibodies is an approach that should be further evaluated in controlled clinical trials.Data in melanoma have shown that nivolumab outcompetes ipilimumab and that the combination of both is superior over single treatment strategies . In thisNotably, TIM3 is expressed on tumor-infiltrating Tregs , which suppress CD8+ cytotoxic T-cells (CTLs) . Blockin. In addition, drugs targeting the proteins of mutated driver genes might directly increase the re-activation of the specific immune system exerted by checkpoint inhibitors. In part, these effects may be caused by interference of some kinase inhibitors with signaling pathways essential for T-cell function, activation, survival and proliferation. Indeed, it has recently been shown that the mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor cobimetinib increased major histocomaptibility complex (MHC) class I molecule expression on cancer cells and induced a 17% response rate in colorectal cancer patients treated with the anti-PDL1 mAb atezolizumab [Kinase inhibitors might synergize with immunotherapy in combating cancer, even without direct interaction of the molecular targets. In fact, phase I clinical trials have shown a synergy between gefitinib, which targets EGFR with the PD1 checkpoint inhibitor durvalumab in EGFR mutated NSCLC patients with ~80% response rates . In addiolizumab . Midostaolizumab , yet doeolizumab . Its effThe Bruton’s tryosine kinase (BTK) inhibitor ibrutinib binds covalently to BTK, thus inhibiting B-cell-receptor-mediated proliferation, inducing apoptosis and migration of neoplastic B-cells out of the protective micromilieu of lymph nodes. The drug has shown impressive efficacy in CLL , 89, par. In addition, in murine B-Raf wild-type syngeneic transplantable tumors Raf-kinase inhibitors paradoxically induced hyperactivation of extracellular-signal regulated kinase (ERK) signaling and thus increased T-cell activation and signaling [In addition, PDL1-exposed lymphocytes cocultured with melanoma cell lines showed downregulation of MAPK signaling which could be reverted by the B-Raf inhibitor vemurafenib in vitro . In muriignaling . This maOther kinase inhibitors have been shown to increase tumor cell infiltration by T-cells, as detected in core biopsies of patients, which predicts a more favorable spontaneous clinical course and better response to neoadjuvant Her2-targeting agents in breast cancer , 97. The.Likewise, janus kinase 2 (Jak2) mRNA expression in breast cancer cells was associated with increased numbers of tumor infiltrating leukocytes and better prognosis . HoweverIt is clear that the current results achieved with checkpoint inhibitors in clinical practice are exciting, but far from being good enough. Therefore, various combinations with chemotherapy, radiotherapy or endocrine therapy are currently being tested in clinical trials. This approach was initially followed only reluctantly due to the widespread view that these chemotherapeutic drugs suppress the immune system . HoweverIn line with this hypothesis, it was demonstrated in a systemically progressive melanoma patient that local radioation therapy induced upregulation of the tumor antigen NY-ESO-1 and resulted in consecutive systemic resensitization towards ipilimumab . This obvia upregulation of MHC-I on the neoplastic myeloid-derived suppressor cells . In thiview see .eg in colorectal cancer . These iews see , 119). Aiews see ). In addiews see  and in views see . In addiiews see ), and aliews see .. For example, gut microbiota have been shown to be involved in the metabolization of cytotoxic drugs  and in iewed in –130.−/− mice have altered composition of the gut microbiota [Recently it was also reported that PD1crobiota , and thacrobiota . StudyinIn summary, despite the clinical benefit observed in a relevant proportion of patients by targeted immune checkpoint modulation, this field of research is still in its infancy. However, our increasing understanding of tumor immunology in general, and the immunoediting process exerted by cancer cells in particular, opens a wide window of opportunities to improve therapeutic immunomodulatory approaches against cancer, making translational science in this exciting field more important than ever.Which factors could serve as predictors of response to checkpoint mediators?ie peripheral blood or tissue biopsy) should the analysis of potential predictors/biomarkers be performed?At which time points and from which source(s) ?Which biological interactions with the tumor microenvironment might affect the response to checkpoint mediators?