Single-cell RNA-sequencing methods are now robust and economically practical and are becoming a powerful tool for high-throughput, high-resolution transcriptomic analysis of cell states and dynamics. Single-cell approaches circumvent the averaging artifacts associated with traditional bulk population data, yielding new insights into the cellular diversity underlying superficially homogeneous populations. Thus far, single-cell RNA-sequencing has already shown great effectiveness in unraveling complex cell populations, reconstructing developmental trajectories, and modeling transcriptional dynamics. Ongoing technical improvements to single-cell RNA-sequencing throughput and sensitivity, the development of more sophisticated analytical frameworks for single-cell data, and an increasing array of complementary single-cell assays all promise to expand the usefulness and potential applications of single-cell transcriptomic profiling. Of these methods, RNA-sequencing (RNA-seq) enabled transcriptomic profiling at unprecedented sensitivity and breadth, leading to the discovery of new RNA species and deepening our understanding of transcriptome dynamics3. In recent years, low-input RNA-seq methods have been adapted to work in single cells4. These single-cell RNA-seq (scRNA-seq) technologies can quantify intra-population heterogeneity and enable study of cell states and transitions at very high resolution, potentially revealing cell subtypes or gene expression dynamics that are masked in bulk, population-averaged measurements6. In this review, we will discuss recent advancements and current limitations of scRNA-seq methodologies and highlight major applications of scRNA-seq in biological research.The advent of next-generation sequencing over a decade ago spurred the development of a host of sequencing-based technologies21. Currently published scRNA-seq protocols all follow the same general workflow: single cells are isolated; cells are lysed, and the RNA is captured for reverse transcription into cDNA; and the cDNA is pre-amplified and then used to prepare libraries for sequencing and downstream analysis. Kolodziejczyket al.22 provide a comprehensive review of individual scRNA-seq protocols and their relative strengths and weaknesses.Over the past six years, numerous scRNA-seq protocols have been developed23, amplification bias arising during pre-amplification limits the quantitative accuracy of scRNA-seq. Unique molecular identifiers (UMIs) can be used to barcode individual RNA molecules during the reverse transcription step, allowing direct transcript counting29, and many of the newer scRNA-seq protocols use UMIs to improve transcript quantitation19. Alternatively, exogenous RNA standards such as those from the External RNA Control Consortium (ERCC) can be “spiked in” with cellular RNA to map between relative and absolute transcript counts30. Stegleet al.31 provide a more detailed discussion of methods for scRNA-seq transcript quantitation and highlight some of the analytical challenges unique to single-cell data.Although cDNA pre-amplification is necessary because only minute amounts of RNA are captured from each cell17 and droplet-based19 cell-barcoding strategies have enabled the analysis of tens of thousands of cells in a single experiment. The high-throughput capacity of these new technologies will increase the resolution of single-cell experiments, improving their ability to detect rare cell subtypes or transitional states.scRNA-seq methods have also been improving in terms of throughput and scalability. Whereas most earlier methods have been limited to measuring hundreds or thousands of cells at a time, recent advancements in microwell9) is represented in the final sequencing libraries, scRNA-seq has limited sensitivity and is unable to reliably detect low-abundance transcripts33. The low amount of input material for scRNA-seq libraries also leads to high levels of technical noise, which complicates data analysis and can mask underlying biological variation37. Methods for modeling technical variation in scRNA-seq data have been proposed37; however, most approaches use the sample-to-sample variation in ERCC read counts to model and control for technical noise in the single-cell data and thus can be used only with experiments incorporating spike-in controls. Moreover, these approaches assume that the spike-in transcripts are treated the same as cellular RNA during library prep. However, naked spike-in RNA does not pass through cellular lysis and is not in complex with ribosomes or RNA-binding proteins. Thus, although spike-in procedures serve as useful indicators of transcript frequency and sensitivity in an experiment, there are many sources of variability that remain difficult to control in scRNA-seq.Current scRNA-seq technologies still face a number of challenges. Collectively, existing scRNA-seq methods have low capture efficiency. Because only a small fraction of each cell’s transcript complement  strategies (discussed further below), which detect transcripts through the binding of fluorescently labeled probes. However, althoughin situ sequencing methods preserve spatial information and can measure RNA expression patterns at subcellular resolution, these approaches are currently limited in throughput and require specialized tools which may not be widely accessible.For most single-cell isolation procedures, information about cells’ original spatial context and cellular environment is lost. Recently, computational methods have been developed to infer a cell’s original position in three-dimensional space from its transcriptional profile by using a reference gene expression map built from existing47, lncRNAs48, or circular RNAs50) or bacterial RNA21. Random hexamer priming has been suggested as a strategy to simultaneously capture both polyadenylated and non-polyadenylated transcripts in single cells21, and computationally selected “not-so-random” primers could potentially be used to capture poly(A)+ and poly(A)– species while depleting for ribosomal RNA51. Incorporating these alternative priming strategies into existing scRNA-seq technologies would enable the exploration of a wider spectrum of transcript types, broadening the scope and applicability of scRNA-seq.Finally, the bulk of scRNA-seq literature has focused solely on polyadenylated mRNAs; almost all published scRNA-seq protocols isolate cellular RNA by using poly-T priming, which captures only polyadenylated transcripts. Consequently, current methods are ill suited to investigate non-polyadenylated transcript classes, such as regulatory non-coding RNA  and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq)69 (measuring chromatin accessibility) have been adapted to work with single cells. These methods offer insight into the epigenetic heterogeneity within cell populations, and paired epigenomic and transcriptomic data could deepen our understanding of the mechanisms underlying gene expression regulation. Although direct comparison of a cell’s epigenomic and transcriptomic profiles is not currently possible, combining single-cell bisulfite sequencing or single-cell ATAC-seq with scRNA-seq from the same cell could enable such analyses in the future. Similarly, integrating scRNA-seq with single-cell proteomic methods71 would provide insight into post-transcriptional gene regulation and the degree to which mRNA expression is reflected at the protein level.Over the past few years, methods have also been developed to assay the epigenetic landscape of single cells: both bisulfite sequencing74, even within genetically homogenous cell populations75. Consequently, bulk measurements can mask important cellular heterogeneity76 and lead to averaging artifacts6. One major advantage of scRNA-seq is its ability to detect such cell-to-cell heterogeneity and capitalize upon it to uncover population structure and cell dynamics hidden at the group level.Recent studies have demonstrated high cell-to-cell transcriptomic variation77, spleen16, lung78, or brain83. Clustering methods77 or dimensionality reduction techniques78 can be used directly on single-cell expression data to group cells by transcriptomic similarity and to detect the underlying population structure in an unsupervised manner  in the expression data prior to applying trajectory analysis techniques94.scRNA-seq is also commonly used to study cellular transitions between different states and to map cell trajectories through processes like differentiation . Several95. Transitions between “on” and “off” states are governed by several stochastic processes97, and this phenomenon of “transcriptional bursting” is a major source of gene expression heterogeneity between cells. scRNA-seq can be used to explore transcriptional mechanics and to model the kinetics of stochastic gene transcription99  usually greatly exceeds the number of sample observations (cells measured), and this disparity poses challenges for parameter estimation108. Simplifying the model on the basis of prior knowledge or focusing on only a small subnetwork of key players may be necessary to make parameter estimation feasible110. Finally, experimentally validating inferred GRNs can be very difficult; whereas knocking out a single gene is relatively straightforward, disrupting interactions between two proteins or between a protein and its target sequence can be much harder, and very few hypothesized models have been rigorously tested thus far.The inherent gene expression variability between cells in scRNA-seq data can be used to infer gene regulatory networks (GRNs)ity103 . Network37. Pairing single-cell transcriptomic data with spatial information54 or orthogonal single-cell genomic assays68 also promises to provide new insights into transcriptional dynamics and the mechanisms underlying gene regulation.scRNA-seq technologies have advanced significantly since their inception, improving in terms of both transcript quantitation and experimental throughput. Whereas low capture efficiency and high levels of technical noise limit the sensitivity and accuracy of scRNA-seq, more sophisticated analytical frameworks are emerging to facilitate the interpretation of scRNA-seq data84. In the context of dynamic processes, cell trajectories reconstructed from single-cell transcriptomic data have provided insight into transient intermediate cell states and have helped to identify key regulator genes91. Finally, scRNA-seq also shows great potential for elucidating stochastic transcriptional kinetics and inferring gene regulatory networks. However, network inference from scRNA-seq data is computationally challenging and difficult to validate; inferred network models should thus be critically evaluated and experimentally tested where possible.scRNA-seq has been very effective at dissecting complex, heterogeneous cell populations, enabling unsupervised learning of population structure and the discovery of novel subtypes and rare cell species


Next generation sequencing has shown the frequent occurrence of point mutations in the ubiquitin E3 ligase c-Cbl in myeloid malignancies. Mouse models revealed a causal contribution of c-Cbl for the onset of such neoplasms. The point mutations typically cluster in the linker region and RING finger domain and affect both alleles by acquired uniparental disomy. The fast progress in the detection of c-Cbl mutations is contrasted by our scarce knowledge on their functional consequences. The c-Cbl protein displays several enzymatic functions by promoting the attachment of differentially composed ubiquitin chains and of the ubiquitin-like protein NEDD8 to its target proteins. In addition, c-Cbl functions as an adapter protein and undergoes phosphorylation-dependent inducible conformation changes. Studies on the impact of c-Cbl mutations on its functions as a dynamic and versatile adapter protein, its interactomes and on its various enzymatic activities are now important to allow the identification of druggable targets within the c-Cbl signaling network. Myeloid malignancies comprise three broad categories: acute myelogenous leukemia (AML) characterized by accumulation of immature myeloid cells in the bone marrow, myelodypastic syndromes (MDS) associated with ineffective hematopoiesis and chronic myeloproliferative disorders (MPDs), usually associated with an increased production of terminally differentiated myeloid cells. Despite this heterogeneity, all myeloid malignancies originate from progenitors that normally give rise to terminally differentiated cells of the myeloid series. Genetic and epigenetic events occurring early in hematopoietic stem cell maturation can lead to myeloid malignancies. Mutations can disrupt the normal course of hematopoiesis, resulting in a wide range of defects affecting the myeloid lineages .Normal hematopoiesis involves a strict hierarchy of hematopoietic progenitor cells. The single primary source for the entire mammalian blood system is the hematopoietic stem cell (HSC), which is both multipotent and self-renewing in the absence of differentiation . HSCs inSome myeloid malignancies are characterized by prominent and characteristic disease-causing mutations. For example, the vast majority of Polycythemia vera patients carry gain-of function mutations in Janus kinase 2 (JAK2 V617F) , while aThe attachment of ubiquitin to its client proteins regulates many different biological processes and occurs by a complex enzymatic cascade . This prCbl proteins can exert multiple enzymatic functions. They can attach K48-branched polyubiquitin chains, resulting in proteasomal degradation of the decorated client proteins . Cbl E3 In addition to these enzymatic activities, the various domains in Cbl proteins enable its function as an adapter by allowing constitutive or phososphorylation-dependent association with a plethora of different proteins , 23. TheAlthough approximately 10% of myeloid neoplasms show c-Cbl mutations, their frequency varies considerably between the subtypes. For example, while de novo AML has a relatively low frequency of such mutations at around 1-3%, juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukaemia (CMML) have the highest frequencies of c-Cbl mutations of around 13-15% -35. MutaThe causal contribution of c-Cbl to the onset of myeloid malignancies was revealed in mouse models. c-Cbl-deficient mice are viable but show lymphoid hyperplasia, enhanced T-cell signaling and primary splenic extramedullary hemopoiesis . In contThe importance of c-Cbl to myeloid differentiation and cancer is likely due to its involvement in numerous signaling pathways and biological functions. Mutation of c-Cbl can lead to aberrant activation of the JAK-STAT and PI3K-AKT pathways to transmit mitogenic and/or survival signals -50. The in vitro and in vivo [+) in humanized CSF-1 newborn mice resulted in more efficient differentiation and enhanced frequencies of human monocytes/macrophages in the bone marrow, spleens, peripheral blood, lungs, liver and peritoneal cavity, pointing to its potential role in myeloid cell fate [Cbl also contributes to myeloid differentiation by regulating the activity of colony stimulating factor-1 (CSF-1) receptor (CSF-1R). In the hematopoietic system, CSF-1 is believed to act specifically on myeloid progenitors, starting from the CMP stage, and to favour the differentiation of CMPs into the monocyte/macrophage lineage . Indeed, in vivo . Furtherell fate . The magell fate -61. ThesAccumulating clinical evidence shows that progression mutations in c-Cbl and other key regulators occur during further clonal development of myeloid malignancies. Unlike the classical gain-of-function mutations exemplified by constitutive active JAK2 V617F) or BCR-ABL , mutant F or BCR-


Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. Our data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans.UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. Here, we show that UVRAG deficiency in  Drosophila Collection: Intestinal-stem-cell-specific loss of the Drosophila ortholog of the tumor suppressor UVRAG, which is implicated in colorectal cancer, leads to endocytic defects and dysplasia. UVRAG) encodes a subunit of the Vps34 kinase complex, which is important for the generation of phosphatidylinositol 3-phosphate (PI3P). UVRAG promotes the biogenesis of this phospholipid by binding to the autophagic tumor suppressor Beclin-1 within this complex  that produce enterocytes and enteroendocrine cells, similar to the functions of stem cells residing at the base of Lieberkühn's crypts in the mammalian intestine. Ingested pathogens and toxins damage the gut and trigger a regeneration response through increased proliferation of stem cells and differentiation of progeny, both in flies and mammals -mediated silencing of Uvrag in midgut ISCs of adult Drosophila using a standard temperature-sensitive gene expression system. This method allows genetic manipulation of escargot (esg)-positive ISCs and differentiating progenitors (EBs) in adult flies, as gene silencing (or overexpression) and GFP expression can be triggered by shifting animals to 29°C (Uvrag in esg-GFP-positive cells (where GFP is expressed under the esg promoter) strongly decreased the number of GFP-tagged FYVE dots, which mark PI3P-positive vesicles, indicating efficient gene silencing  in esg-GFP-positive cells resulted in a striking intracellular accumulation of the Notch ligand Delta . To confirm this finding, we generated mitotic clones in the gut that were homozygous mutants for previously described Uvrag-null alleles , in line with our RNAi data. Basal levels of the Wnt signaling ligand Wingless/Wg can be detected in ISCs and EBs , again indicating impaired endolysosomal degradation.Loss of UVRAG results in the accumulation of plasma membrane receptors and ligands in stalled endocytic compartments in cultured human cells, and also in cells of the developing pathways . During pathways . In line alleles . Both mu and EBs , and theFig. S1D).UVRAG- or Vps34-dependent production of the phospholipid PI3P is important for endosome maturation. We thus analyzed Rab7-positive endosomes in control and UVRAG loss-of-function stem cells. Indeed, quantification of Rab7-GFP vesicles revealed a clear increase in the percentage of cytoplasmic area occupied in UVRAG-deficient cells .Strikingly, the loss of UVRAG led to a remarkable expansion of both ISCs containing the endocytosed Notch ligand Delta and Delta-negative EBs B. The ovUvrag-mutant clones were induced by somatic recombination at the adult stage: GFP-positive ISCs that were homozygous mutant for either one of the two null alleles produced many more cells per clone than control cells during the same time window . Importantly, the expansion of Uvrag-mutant clones was suppressed by expression of wild-type Uvrag in these cells in both cases , confirming that the intestinal dysplasia is solely due to UVRAG deficiency. Uvrag overexpression on its own caused no obvious alteration of the esg-GFP compartment in the posterior midgut .Staining for the mitotic marker phosphorylated histone H3 revealed that ISCs proliferated more upon loss of UVRAG D. This wesg-Gal4-driven UAS-mCherry (esg-mCherry) expression and active Notch signaling even after becoming polyploid . The activity of target of rapamycin (Tor) kinase is relatively low in ISCs, which maintains their stem cell identity. Notch-mediated activation of this important positive regulator of cell growth is reported to promote the differentiation of EBs into enterocytes .ISCs have the ability to self renew and to generate EB daughter cells in which Notch signaling is activated by ISCs that are positive for its ligand Delta . In wildolyploid A. Moreoverocytes . In lineFig. S2C). As these cells represent another lineage that also differentiates from EBs , indicating increased retention of ingested food. As proper gut function is a crucial determinant of Drosophila lifespan  . In line with this, DSS killed flies undergoing UVRAG RNAi in esg-GFP-positive cells much faster than it killed control flies  . DSS treol flies C.Pseudomonas aeruginosa .We tested the activity of selected signaling pathways in UVRAG-deficient intestines that could play a crucial role in the proliferation of ISCs of the posterior midgut . Receptoupd1) and Unpaired3 (upd3)  B,C. Inte3 (upd3) B,C. ThesDrosophila) pathway, another positive regulator of ISC proliferation in Drosophila (puckered (puc), a gene that encodes a phosphatase that antagonizes JNK activity. We used a puc-lacZ reporter line as a readout for JNK activation in the context of UVRAG loss. This transcriptional reporter is only active in enterocytes in wild-type guts .We also looked at the Jun N-terminal kinase  in esg-GFP-positive cells undergoing UVRAG RNAi suppressed hyperproliferation and completely restored the number of ISCs and EBs to wild-type levels , indicating that the Wingless pathway is required for the maintenance of ISCs regardless of the genetic background.In contrast with these results, blocking Wingless signaling through the expression of a dominant-negative version of the downstream TCF transcription factor eliminated most esg-GFP-positive cells in UVRAG-deficient and also wild-type cells  or Basket (JNK) . In contrast, heterozygosity for Stat92E or Basket, but not Wingless, suppressed the hyperproliferation of UVRAG-deficient cells . These data indicate that STAT and JNK signaling are specifically required to sustain UVRAG-deficiency-induced intestinal dysplasia.We sought to test whether the above pathways are specifically involved in the dysplasia of UVRAG-deficient ISCs. We thus quantified the number of esg-GFP-positive cells using genetic backgrounds that were heterozygous for STAT, JNK and Wingless components. No statistically significant differences in the number of mitoses were observed in animals heterozygous for Uvrag animals because Notch RNAi rescues this mutant phenotype , instead of rescuing the dysplastic phenotype. Interestingly, overexpression of the active fragment of Notch could still induce the differentiation of UVRAG-deficient ISCs and EBs to enterocytes .Deregulation of Notch signaling has been found to cause a male genitalia rotation defect in hypomorphic mutant henotype . Notch ierocytes . We thusUVRAG is famous for its dual roles in autophagy as it has been implicated both in autophagosome formation as a Vps34 kinase complex subunit and in fusion with lysosomes because it binds to the HOPS tethering complex , 2008. WDrosophila (An established test for autophagic degradation (flux) is to measure the level of the selective cargo p62, which is also known as Ref(2)P in osophila . No statosophila A,C. Simiosophila , did notosophila B,C.Fig.Atg5 or Atg14 suppressed the dysplasia induced by UVRAG loss . However, RNAi against Atg5 or Atg14 also led to loss of esg-GFP-positive cells and decreased the number of mitoses , suggesting that autophagy is required for ISC maintenance in general, instead of for the promotion of dysplasia specifically in the absence of UVRAG. Similarly, autophagy has recently been shown to maintain muscle stem cell function and survival in mice (Knocking down the core autophagy genes  in mice .DN), the Drosophila homolog of dynamin, which is a routinely used tool to block the endocytic downregulation of plasma membrane receptors  flies and involves the simultaneous activation of both JNK and STAT signaling , and gene and RNAi expression was induced in stem cells and EBs by shifting vials containing eclosing adult flies to 29°C for inhibition of thermo-sensitive Gal80 activity, until guts were examined . In all cases, the posterior midgut of female flies was examined. The stocks used in this study were: ts]esg-GFP , UAS-Atg5 RNAi [JF02703], UAS-Atg14 RNAi [108559KK]  , upd3-lacZ and upd1-lacZ , and the mutant alleles 1-12wg, 67c23wg, 1-8wg, 06346stat92e, HJstat92e, 1bsk and 2bsk .rpowitz) , UAS-mCh08559KK] , UAS-RabMS01357] , UAS-FYVFYVE-GFP , UAS-N R Jasper) , NRE-GFP for 1 h at room temperature. After extensive washes in PBST, samples were blocked in 3% BSA PBST for 2 h and incubated overnight with primary antibodies in 1% BSA PBST at 4°C. Samples were labeled with secondary antibodies in 1% BSA PBST at room temperature for 2 h. Samples were also washed after each antibody labeling step with PBST containing 4% NaCl to reduce non-specific background labeling. Fluorescent microscopy was performed as described previously . PrimaryPseudomonas was performed as described previously , Mann–Whitney U-test , log-rank Mantel–Cox  and ANOVA . Hochberg and Bonferroni post-hoc tests were performed for multiple comparisons to reduce type-II errors in ANOVA and Kruskal–Wallis analyses, respectively.All analyses were performed in SPSS Statistics 21 software (IBM). No data or animals were excluded from the analyses. After checking the normality of data obtained in each experiment, the appropriate statistical test was applied to calculate significance values between data sets, as described previously . The folntel–Cox B-D, Krus


Importantly, the cytotoxic activity profile of some compounds remained unaltered in epithelial-to-mesenchymal (EMT)-induced stable populations of cancer stem-like cells, which acquired resistance to the well-known ROS inducer doxorubicin. Compounds 1, 2 and 5 inhibited the clonogenicity of cancer cells and induced apoptotic cell death accompanied by caspase 3/7 activation. Flow cytometry analyses indicated that ligands were strong inducers of oxidative stress, leading to a 7-fold increase in intracellular ROS levels. ROS induction was associated with their ability to bind intracellular iron and generate active coordination complexes inside of cells. In contrast, extracellular complexation of iron inhibited the activity of the ligands. Iron complexes showed a high proficiency to cleave DNA through oxidative-dependent mechanisms, suggesting a likely mechanism of cytotoxicity. In summary, we report that, upon chelation of intracellular iron, the pro-oxidant activity of amine-pyrimidine-based iron complexes efficiently kills cancer and cancer stem-like cells, thus providing functional evidence for an efficient family of redox-directed anti-cancer metallodrugs.Differential redox homeostasis in normal and malignant cells suggests that pro-oxidant-induced upregulation of cellular reactive oxygen species (ROS) should selectively target cancer cells without compromising the viability of untransformed cells. Consequently, a pro-oxidant deviation well-tolerated by nonmalignant cells might rapidly reach a cell-death threshold in malignant cells already at a high setpoint of constitutive oxidative stress. To test this hypothesis, we took advantage of a selected number of amine-pyridine-based Fe(II) complexes that operate as efficient and robust oxidation catalysts of organic substrates upon reaction with peroxides. Five of these Fe(II)-complexes and the corresponding aminopyridine ligands were selected to evaluate their anticancer properties. We found that the iron complexes failed to display any relevant activity, while the corresponding ligands exhibited significant antiproliferative activity. Among the ligands, none of which were hemolytic, compounds  Cancer cells undergo metabolic adaptations to sustain their uncontrolled growth and proliferation. Diverse intrinsic and extrinsic molecular mechanisms contribute to this metabolic reprogramming to supply cancer cells with sufficient energy and biosynthetic capacity in the tumor environment ,2. AlterHighly reactive ROS are produced in cells by the incomplete reduction of molecular oxygen to water during aerobic metabolism. ROS are normally regulated by cellular defensive antioxidants ,10 and pIncreased ROS in cancer cells upregulates the antioxidant response, resulting in a new redox balance that enables these cells to maintain higher ROS levels than normal cells. Consequently, cancer cells exhibit persistent oxidative stress, which promotes cell proliferation but is insufficient to cause cellular death ,13. This2O3), which are potent ROS inductors, have been shown to promote CSC death . Ac. Ac1, 2 um azide , suggest1-Fe, 2-Fe, 3-Fe, 4-Fe and 5-Fe), which were expected to be potent ROS inductors together with the corresponding uncomplexed ligands, as potential antitumoral agents. Our results demonstrate that the iron complexes failed to display any relevant cytotoxic activity, whereas the iron-free organic counterparts were cytotoxic. In particular, compounds 1, 2 and 5 exhibited strong antiproliferative activity against a broad panel of molecularly diverse human cancer cells with IC50 values in the low micromolar range. Importantly, the cytotoxic activity profile of compounds 1 and 2 remained unaltered in EMT-induced stable populations of cancer stem-like cells, which characteristically exhibit resistance to the majority of commonly employed anti-cancer agents including the well-known ROS inducer doxorubicin , which cleaves DNA by hydrogen abstraction [1, 2 and 5 demonstrated DNA cleavage activity in cells, with kinetics that mirrored intracellular ROS accumulation. Analysis of the interaction of ligands with naked DNA revealed that only the Fe-complexed ligands displayed nuclease activity by inducing double strand breaks in the DNA in the presence of hydrogen peroxide. The DNA cleavage activity was quenched by different ROS scavengers, revealing that the nuclease activity of 1-Fe and 2-Fe involve different ROS, while the activity of 5-Fe is likely to be associated to singlet oxygen generation. Further, the nuclease activity took place without any DNA groove selectivity. Collectively, these results indicate that once bound to intracellular iron, the ligands induce a strong oxidative DNA damage through ROS, which results in double-stranded DNA breaks.Different anticancer compounds with metal binding properties can induce DNA strand breaks when binding redox active metals in the presence of oxygen . The bletraction . Similar1 and 2. Additional mechanisms may be involved in the anticancer activity of 5, which displays similar antiproliferative and proapoptotic activities but limited generation of ROS.The anti-cancer activity of amine-pyridine-based iron complexes relies on different inter-dependent processes, involving intracellular Fe(II) chelation, generation of ROS, DNA fragmentation through oxidative mechanisms, induction of cell cycle arrest and apoptosis . This moin vivo effects and the nature of interaction as cocktail partners either with current ROS-generating radio- and chemo-therapeutic regimens or with the newer therapies that do not directly generate ROS, our current findings illustrate that, upon chelation of intracellular iron, the pro-oxidant activity of amine-pyrimidine-based iron complexes efficiently kills cancer and ROS-refractory cancer stem-like cells. Thus, our study provides functional evidence for promising redox-directed anti-cancer metallodrugs.Cancer cells have increased steady-state ROS levels and are likely to be more vulnerable to damage by further ROS insults induced by exogenous agents ,72. Inde


MicroRNA is small non-coding RNA and can lead to translational repression or target degradation by base-pairing with complementary sequences of mRNA molecules. MicroRNA-155 (miR-155), one of the most studied microRNA, is the first one to be reported as oncogenic . We conc+Foxp3+ regulatory T cells (Treg) induction. On the other hand, miR-155 deficiency hampered the antitumor responses of both dendritic cells and T cells. Therefore, it appears that in our tumor models, miR-155 mediated a dominant immunosuppressive effect by MDSC, leading to the enhanced overall antitumor immunity in miR-155 deficient hosts.We demonstrated that host miR-155 deficiency promoted antitumor T cell immunity in multiple transplanted tumor models. Further analysis of immune cell compartments revealed that miR-155 was required for the accumulation and suppressive function of myeloid-derived suppressive cells (MDSC) in the tumor microenvironment. Apart from the direct modulation on MDSC, miR-155 was also required for the MDSC-mediated CD4Reduced colon inflammation and decreased colorectal carcinogenesis were also found in miR-155 deficient mice when azoxymethane (AOM) and dextran sodium sulphate (DSS) were combined to induce colon lesions. Furthermore, miR-155 was upregulated in MDSC either from tumor-bearing hosts or generated from bone marrow progenitors by GM-CSF and IL-6. These results support the notion that miR-155 is a prototypical microRNA bridging inflammation and cancer development . Althoug−/− MDSC has a lower level of arginase activity than WT counterparts, and inhibition of arginase-I with specific inhibitors completely abrogated the suppressive activity of WT MDSC and did not affect the miR-155−/− MDSC. Our data indicate that miR-155 may modulate arginase-dependent suppressive function of MDSC via targeting SOCS1.To identify the molecular mechanisms by which miR-155 regulates MDSC (Figure −/− MDSC, which would presumably limit the tumor angiogenesis. Given a contribution of miR-155 expression by cancer cells to tumor angiogenesis [More interestingly, we observed the decreased production of MMP-9 and VEGF from miR-155ogenesis , furtherin vivo.It is notable that our results on host miR155 deficiency and tumor growth differ from other recent studies , 8. DiffTaken together, our study highlights the essence of evaluating the intrinsic role of miR-155 carefully in distinct immune cell subsets, where miR-155 could be either protective or deleterious to antitumor immunity. In this regard, it would be safe and important to develop the anti-miR-155 cancer therapy in cell-specific manner. It is becoming evident that miR-155 functions as “OncomiR” in concert with “ImmunomiR” in orchestrating cancer growth and progression.


Neonatal encephalopathy following birth asphyxia is a major predictor of long-term neurological impairment. Therapeutic hypothermia is currently the standard of care to prevent brain injury in asphyxiated newborns but is not protective in all cases. More robust and versatile treatment options are needed. Angiogenesis is a demonstrated therapeutic target in adult stroke. However, no systematic study examines the expression of angiogenesis-related markers following birth asphyxia in human newborns.This study aimed to evaluate the expression of angiogenesis-related protein markers in asphyxiated newborns developing and not developing brain injury compared to healthy control newborns.Twelve asphyxiated newborns treated with hypothermia were prospectively enrolled; six developed eventual brain injury and six did not. Four healthy control newborns were also included. We used Rules-Based Medicine multi-analyte profiling and protein array technologies to study the plasma concentration of 49 angiogenesis-related proteins. Mean protein concentrations were compared between each group of newborns.Compared to healthy newborns, asphyxiated newborns not developing brain injury showed up-regulation of pro-angiogenic proteins, including fatty acid binding protein-4, glucose-6-phosphate isomerase, neuropilin-1, and receptor tyrosine-protein kinase erbB-3; this up-regulation was not evident in asphyxiated newborns eventually developing brain injury. Also, asphyxiated newborns developing brain injury showed a decreased expression of anti-angiogenic proteins, including insulin-growth factor binding proteins -1, -4, and -6, compared to healthy newborns.These findings suggest that angiogenesis pathways are dysregulated following birth asphyxia and are putatively involved in brain injury pathology and recovery. In angiogenesis, a coordinated orchestra of proteins causes new blood vessels to sprout and mature from existing vessels. This process begins with the degradation of the local extracellular matrix and the weakening of tight junctions and interactions between endothelial cells and pericytes. Vascular destabilization permits endothelial cell proliferation and migration to a site of nascent tube formation. Angiogenesis is completed as intercellular interactions and the extracellular matrix are re-established to stabilize the new blood vessels . Recent Neonatal encephalopathy is a major predictor of neonatal death and long-term neurological deficits, including cerebral palsy, intellectual disability, and epilepsy . CurrentWe hypothesized that angiogenesis is activated following birth asphyxia, and that this activation may differ between asphyxiated newborns developing and not developing brain injury. Thus, the objective of this study was to evaluate the expression of angiogenesis-related protein markers in asphyxiated newborns treated with hypothermia developing and not developing brain injury compared to healthy control newborns.We conducted a cohort study of term asphyxiated newborns admitted to our neonatal intensive care unit who met the criteria for induced hypothermia –18: (1) Clinical data, including gestational age, birth weight, sex, Apgar score at 10 minutes, use of intubation and chest compression at birth, arterial cord pH, and initial infant blood gas pH were collected prospectively for each asphyxiated newborn. The hour of life at which hypothermia was initiated was also calculated and recorded.Healthy term newborns with normal brain MRI findings were included as healthy controls. The research protocol was approved by the research ethics board from the Montreal Children’s Hospital, McGill University Health Centre, and informed written consent was obtained from the parents on behalf of their newborns in all cases.The presence or absence of brain injury in asphyxiated and healthy control newborns was defined by magnetic resonance imaging obtained around day 10 of life (range: day 9–13 of life), as they have previously been reported to define precisely the extent of the brain injury in these newborns –21. NeurNurses collected blood samples in tubes containing potassium EDTA at 24 hours of life for healthy control newborns and at 6, 24, 48, 72, and 96 hours of life for asphyxiated newborns treated with hypothermia. Blood samples were immediately centrifuged at 3600 rpm for 6 minutes at room temperature. Following centrifugation, plasma was removed, aliquotted, and stored at -80°C until tested. Plasma samples were coded, and protein markers were measured blindly with respect to patient identity and disease.http://www.rules-basedmedicine.com). These mixtures of sample and capture microspheres were thoroughly mixed and incubated at room temperature for 1 hour. Multiplexed cocktails of biotinylated, reporter antibodies for each multiplex were then added robotically and, after thorough mixing, were incubated for an additional hour at room temperature. Multiplexes were developed using an excess of streptavidin-phycoerythrin solution, which was thoroughly mixed into each multiplex and incubated for 1 hour at room temperature. The volume of each multiplexed reaction was reduced by vacuum filtration and the volume increased by dilution into matrix buffer for analysis. Analysis was performed in a Luminex 100 instrument and the resulting data stream was interpreted using proprietary data analysis software developed at Rules-Based Medicine (Myriad RBM). For each multiplex, both calibrators and controls were included on each microtiter plate. 8-point calibrators were run in the first and last column of each plate and 3-level controls were included in duplicate. Testing results were determined first for the high, medium and low controls for each multiplex to ensure proper assay performance. Unknown values for each of the analytes localized in a specific multiplex were determined using 4- and 5- parameter weighted and non-weighted curve fitting algorithms included in the data analysis package. Expression of 49 angiogenesis-related proteins was thus analyzed. Analyzed proteins, listed in “The samples were thawed at room temperature, vortexed, spun at 13,000 x g for 5 minutes for clarification and 40 mcL were transferred to a master microtiter plate for multi-analyte profiling (MAP) antigen analysis. Using automated pipetting, an aliquot of each sample was introduced into one of the capture microsphere multiplexes of the Rules-Based Medicine (Myriad RBM) Custom Human multi-analyte profile (MAP)  . For graphical representation, ratios of group means were calculated and log transformed to obtain fold change data for each protein marker. In addition, to improve data interpretation, a DAVID functional clustering of Gene Ontology Terms was performed [Protein expression data were represented as mean concentration ± standard deviation for each group of newborns. To test differences in protein expression between groups, we used Mann-Whitney U tests. A erformed –24.Twelve asphyxiated newborns treated with hypothermia and four healthy control newborns were included in this study. Six of the asphyxiated newborns developed brain injury; four developed a basal ganglia injury pattern “” and twoMean protein concentration values were compared between asphyxiated newborns developing and not developing brain injury and healthy newborns at 24 hours of life “”.Asphyxiated newborns treated with hypothermia showed altered expression in 10 of the 49 angiogenesis-related proteins compared to healthy newborns “”. Matrix). Two of these proteins, angiopoietin-2 (Ang-2) and hepsin destabilize the vasculature to permit new sprouting. Expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is important for endothelial cell survival, proliferation, and migration, was decreased. Neuropilin-1 (NP-1), receptor tyrosine-protein kinase erbB-3 (Erb-B3), and YKL-40, which promote similar endothelial cell effects, also showed diminished expression. Expression of the angiogenic inhibitors IGF-BP-1, IGF-BP-4, and IGF-BP-5 and the apoptotic protein sortilin was also decreased. When performing a DAVID functional clustering of Gene Ontology Terms using the 49 angiogenesis-related proteins as background and the abovementioned 10 dysregulated proteins as the Gene List, one cluster of proteins stood out all related to extracellular signaling activities ; one protein could not be grouped.When comparing asphyxiated newborns developing and not developing brain injury at 24 hours of life, the expression of 10 angiogenesis-related proteins was decreased in the prior group compared to the latter “”. Two ofComparisons in protein expression between asphyxiated newborns not developing brain injury and healthy newborns, as well as between asphyxiated newborns developing brain injury and healthy newborns were included in “Mean protein concentration values were also compared between asphyxiated newborns developing and not developing brain injury throughout the first days of life, i.e., at 6, 24, 48, 72, and 96 hours of life “”. Among This is the first systematic study of the expression of angiogenesis-related protein markers following birth asphyxia in human newborns. The expression of angiogenesis-related proteins was clearly dysregulated in asphyxiated newborns treated with hypothermia compared to healthy newborns, suggesting that angiogenesis plays an important but under-recognized role following birth asphyxia.Interestingly, asphyxiated newborns who did not develop brain injury showed increased expression of four proteins involved in endothelial cell survival, proliferation, and migration  compared to healthy newborns, while only one such protein  was up-regulated in asphyxiated newborns developing brain injury. In comparison to asphyxiated newborns not developing brain injury, those who did develop an injury showed decreased expression of HB-EGF, NP-1, Erb-B3 and YKL-40, four proteins that promote endothelial cell survival, proliferation, and migration. This suggests that up-regulation of pro-angiogenic proteins may be neuroprotective or neurorestorative in asphyxiated newborns. Previous studies examining the individual roles of these proteins support their isolated effects on neuroprotection and neurorestoration. For example, animal models of adult stroke show that NP-1 facilitates VEGF signaling to promote neurorestoration . HB-EGF-Intriguingly, asphyxiated newborns developing brain injury showed decreased expression of four anti-angiogenic proteins  compared to healthy newborns, while only two of these proteins  were down-regulated in asphyxiated newborns not developing brain injury compared to healthy newborns. In addition, IGF-BP-1, -4, and -5 were less expressed in asphyxiated newborns developing brain injury compared to those not developing brain injury. Evidence suggests that IGF-BP-1, -4, -5 and -6, which bind IGF-1, inhibit angiogenesis –33. WhilIn this study, protein expression was also altered in other steps of the angiogenesis pathway, beginning with vascular destabilization. MMP-9 expression was decreased among asphyxiated newborns not developing brain injury. Previous studies have found that MMP-9, which degrades the extracellular matrix to facilitate vascular sprouting , is incrVEGF-C expression was decreased in all asphyxiated newborns, which may represent a mechanism for protecting against vascular permeability-related edema –48. ThisThis study also demonstrated decreased BDNF expression in all asphyxiated newborns. Previously, in studies of asphyxiated human newborns, increased BDNF levels have been found in cord blood at birth , blood sAmong the proteins important for vascular stabilization, collagen IV expression was decreased in asphyxiated newborns developing brain injury compared to healthy newborns. No previous studies of collagen IV expression following birth asphyxia exist; however, this protein is an essential component of the extracellular matrix, and its breakdown is necessary for new vessel sprouting. Therefore, decreased collagen IV expression may again indicate some compensatory mechanism for a failure to adequately up-regulate pro-angiogenic proteins in asphyxiated newborns developing brain injury.Among apoptotic proteins, FasR was up-regulated in all asphyxiated newborns while FasL was down-regulated in asphyxiated newborns developing brain injury compared to healthy newborns. Additionally, sortilin expression was decreased in asphyxiated newborns developing brain injury compared to those not developing brain injury. No previous studies of serum concentrations of these proteins in asphyxiated newborns exist; however, Fas signaling has been shown to contribute to brain injury following neonatal hypoxia-ischemia .This study was intended to highlight whether angiogenesis pathways are preferentially activated following birth asphyxia. As per the DAVID functional clustering of Gene Ontology Terms, the dysregulated angiogenesis-related proteins were mostly involved in extracellular signaling activities, but also in neurogenesis and programmed cell death. Given that this study demonstrates a significant role for angiogenesis after birth asphyxia, further investigation with larger sample sizes and targeted analysis of angiogenesis-related protein expression is definitively warranted. An important next step also includes validation of the dysregulated proteins using a distinct technique (e.g. Western blot). Such investigation may lead to the discovery of potential novel therapeutic targets. It would have also been ideal to study a group of asphyxiated newborns not treated with hypothermia so to distinguish how the hypothermia treatment rather than the asphyxia influenced some of the observed changes. However, as cooling is now the standard of care, it is no longer ethically possible to not receive hypothermia to assess the evolution over time of these angiogenesis-related proteins.In this study, systemic plasma samples were analyzed rather than brain-specific samples. While studying cerebrospinal fluid would have provided a more direct view of the brain’s angiogenic response, ethical and practical concerns make it difficult to collect serial cerebrospinal fluid samples in sick newborns. Birth asphyxia has global effects on the newborn body, so altered plasma protein expression reflects peripheral as well as brain injuries . NonetheIn conclusion, this study highlights that angiogenesis pathways are dysregulated following birth asphyxia and are putatively involved in brain injury pathology and recovery. Our results suggest that a failure to up-regulate pro-angiogenic proteins may leave these newborns more susceptible to eventual brain injury. Furthermore, those newborns who fail to up-regulate pro-angiogenic proteins may attempt to compensate by down-regulating anti-angiogenic proteins. Overall, it seems that angiogenesis enhancement may be an important determinant of injury outcome following birth asphyxia. These findings may have future therapeutic applications to optimize eventual recovery following asphyxia and should be further investigated.S1 FigExpression of 49 angiogenesis-related proteins, which were selected based on angiogenic involvement and assay availability, was analyzed.(TIF)Click here for additional data file.S2 Figp values were calculated to highlight significant protein expression differences between the groups (* p < 0.05). For graphical representation, ratios between group mean concentrations were calculated and log transformed to obtain fold change data for each marker. (A) Fold changes in protein expression between asphyxiated newborns not developing brain injury and healthy newborns. Eleven angiogenesis-related proteins showed altered expression. MMP-9, KLK-5, VEGF-C and BDNF levels remained reduced; however no change was evident in the levels of Gal-3. FABP-4 expression remained elevated. In addition, a greater number of proteins enhancing endothelial cell survival, proliferation and migration were up-regulated among asphyxiated newborns not developing brain injury. Expression of neuropilin-1 (NP-1), which is a VEGF receptor-2 (VEGFR-2) co-receptor, was increased. The expression of other proteins important for endothelial cell survival, proliferation, and migration, including glucose-6-phosphate isomerase (G6PI) and receptor tyrosine-protein kinase erbB-3 (Erb-B3), was also increased. The anti-angiogenic proteins Fib-1C and IGF-BP-6 remained decreased. Among the apoptotic proteins, no change was apparent in FasL expression; however FasR expression remained increased. When performing a DAVID functional clustering of Gene Ontology Terms using the 49 angiogenesis-related proteins as background and the abovementioned 11 dysregulated proteins as the Gene List, two clusters of proteins stood out, one related to neurogenesis  and one related to extracellular signaling activities ; three proteins could not be grouped. (B) Fold changes in protein expression between asphyxiated newborns developing brain injury and healthy newborns. Thirteen angiogenesis-related proteins showed altered levels. While MMP-9 expression was not different between the two groups, KLK-5, VEGF-C and BDNF still showed reduced expression. Among the proteins enhancing endothelial cell survival, proliferation and migration, the expression of Gal3 and PlGF was still increased; but the expression of NP-1, G6PI, Erb-B3 and FABP-4 was not different, and the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was decreased. Collagen-IV, which is important for vascular stabilization, showed reduced expression. Among the anti-angiogenic proteins, Fib-1C and IGF-BP-6 expression remained decreased. Additionally, IGF-BP-1 and IGF-BP-4 were decreased, reflecting decreased expression of a greater number of anti-angiogenic proteins in these newborns compared to newborns not developing brain injury. Finally, the apoptotic proteins sortilin and FasL remained decreased, while FasR remained increased. When performing a DAVID functional clustering of Gene Ontology Terms using the 49 angiogenesis-related proteins as background and the abovementioned 13 dysregulated proteins as the Gene List, two clusters of proteins stood out, one related to programmed cell death  and one related to extracellular signaling activities ; four proteins could not be grouped.Mean concentrations in each group were compared. (TIF)Click here for additional data file.


Obesity-induced chronic inflammation plays a fundamental role in the pathogenesis of metabolic syndrome (MS). Recently, a growing body of evidence supports that miRNAs are largely dysregulated in obesity and that specific miRNAs regulate obesity-associated inflammation. We applied an approach aiming to identify active miRNA-TF-gene regulatory pathways in obesity. Firstly, we detected differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) from mRNA and miRNA expression profiles, respectively. Secondly, by mapping the DEGs and DEmiRs to the curated miRNA-TF-gene regulatory network as active seed nodes and connect them with their immediate neighbors, we obtained the potential active miRNA-TF-gene regulatory subnetwork in obesity. Thirdly, using a Breadth-First-Search (BFS) algorithm, we identified potential active miRNA-TF-gene regulatory pathways in obesity. Finally, through the hypergeometric test, we identified the active miRNA-TF-gene regulatory pathways that were significantly related to obesity.The potential active pathways with FDR < 0.0005 were considered to be the active miRNA-TF regulatory pathways in obesity. The union of the active pathways is visualized and identical nodes of the active pathways were merged.We identified 23 active miRNA-TF-gene regulatory pathways that were significantly related to obesity-related inflammation.The online version of this article (doi:10.1186/s12859-015-0512-5) contains supplementary material, which is available to authorized users. The prevalence of obesity has increased drastically worldwide. Overweight represents major health burdens and is associated with a cluster of metabolic disorders, such as insulin resistence and cardiovascular disease . ObesityAdipocytes and infiltrating inflammatory cells located within the adipose tissue (AT) secrete a number of bioactive molecules, such as tumor necrosis fator-α (TNF-α), interleukin (IL)-6 and adiponectin collectively called adipokines. They are labeled as anti-inflammatory or pre-inflammatory adipokines ,5. ObesiIn recent years, a lot of evidence has highlighted the modulating roles of miRNAs in inflammatory system ,9. MiRNAIn this study, by using hypergeometric test we found 23 active post-transcriptional regulatory pathways in obesity based on obesity-related mRNA and miRNA expression profiles.We applied a novel approach to identify active miRNA-TF regulatory pathways in obesity. Firstly, we found differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) from mRNA and miRNA expression profiles which were derived from the Gene Expression Omnibus (GEO) database . SecondlThe mRNA and miRNA expression profiles (GSE2508 and GSE18470) of human subcutaneous adipose were downloaded from GEO database  approach , and falFor miRNA expression profile, we filtered out samples with Type 2 Diabetes Mellitus. Then 6 lean and 13 obese samples were kept for the further analysis. The differentially expressed miRNAs were detected using SAM.We generated TF and miRNA regulatory network by merging miRTarBase,miRecords, TransmiR,TRANSFAC, and TarBase -16. HereThe known obesity-associated genes ,19 and mIt was reported that some disease-related key genes could hide among non-DE genes ,22. So wBased on the potential active miRNA-TF-gene regulatory subnetwork, all directed acyclic paths from 0-indegree nodes to 0-outdegree nodes were found. The gene/miRNA with 0-indegree is located upstream of the regulatory pathway. The gene/miRNA with a 0-outdegree is located downstream of the regulatory pathway. And the upstream genes/miRNAs’ activation could cause a cascade effect which results in the regulation of downstream gene/miRNA expression and leads to obesity disease. In this study, we defined potential active regulatory pathways as the directed acyclic paths with more than 2 nodes as, which contained at least one DE node and no more than one non-DE node or nodes without expression values between the two DE nodes .CR) of known obesity-associated genes and miRNAs in the potential active pathway is used to measure the strength of the relationships between the potential active pathway and obesity. CR was calculated according toCoverage rate  of the known obesity-associated genes and miRNAs of the potential active pathway was used to measure the strength of the association between the potential active pathway and obesity. Next, we identified the significantly active pathways using a hypergeometric test. The potential active pathways with FDR < 0.0005 were considered to be the active miRNA-TF regulatory pathways in obesity. As a result, we identified 23 active pathways  [2, and hsa-miR-145, has- miR-29b through these 2 TFs make regulation on CCL2. VEGFA stimulates angiogenesis in adipose tissue,SERPINE4 belongs to pro-inflammatory adipokines, and RBP4 is associated with insulin resistance and visceral fat distribution [In these 23 pathways, 6 adipokines including IL-1β, CCLe tissue . Obesity obesity . CCL2 haammation -27. The ribution . They arribution -31. By uribution . MiRNAs ribution ,32,33. HTaken together, our results suggest that specific miRNAs may be important regulators of inflammation in human circulation through their effects on adipokines levels. This may be mediated by indirect effects on TF circuits. miRNAs could therefore constitute novel potential targets for treating obesity comorbidities.We identified 23 active miRNA-TF-gene regulatory pathways that were significantly related to obesity-related inflammation.


Similarity among food allergens is a great problem affecting the specificity of diagnosis and treatment of allergic patients. Rice is often considered as a hypoallergenic foodstuff, but there are several reports documenting rice-induced allergy. We have observed that 80% of patients with food (including wheat) and pollen allergies have increased IgE antibodies against rice proteins.The extracts from wheat flour (Sulamit cultivar) or long-grain rice (Oryza sativa L.) were prepared from raw and boiled forms. The PBS-soluble and SDS-soluble proteins were analyzed by 1- or 2-DE and IgE binding components were characterized by immunoblot, basophil activation test, skin prick test and identified by MALDI-TOF mass spectrometry techniques.We have demonstrated that boiling decrease solubility and IgE reactivity of PBS-extracted rice and wheat proteins, but in SDS-extracts this reactivity was only slightly affected. The sera of patients highly positive on the IgE immunoblot, positive in basophil activation and skin prick test with boiled rice components were used for characterizing the IgE-binding proteins separated by 1-D or 2-D electrophoresis. Employing mass spectrometry, we identified 22 rice SDS soluble proteins. Six of them were new potential rice allergens: glutelin C precursor, granule-bound starch synthase 1 protein, disulfide isomerase-like 1-1 protein, hypothetical protein OsI_13867, putative acid phosphatase precursor 1, and a protein encoded by locus Os02g0453600. These proteins retaining IgE-binding capacity also after thermal processing potentially represent important allergens in rice-containing foodstuffs. All of the identified rice proteins differed from known wheat allergens, except proteins belonging to the á amylase/trypsin-inhibitor family.We could suggest that the patients with high IgE antibodies specific to rice, positive on immunoblot, should be further tested by skin prick test with boiled rice proteins before rice recommended as a suitable diet alternative.


Since 2001 we have utilized a novel surgical approach for Pancoast tumors in which lobectomy and mediastinal lymph node dissection are performed directly though the chest wall defect. The defect is then patched at the completion of the procedure ("cut-in patch-out") thereby avoiding a separate thoracotomy with rib spreading. We undertook a study to compare outcomes of this novel "cut-in patch-out" technique with traditional thoracotomy for patients with Pancoast tumors.We retrospectively identified 41 patients undergoing surgical resection of Pancoast tumors requiring en-bloc removal of at least 3 ribs at our institution from 1999 to 2012. Surgery was accomplished by either a "cut-in patch-out" technique (n = 25) or traditional posterolateral thoracotomy and separate chest wall resection (n = 16). Multiple variables including patient demographics, neoadjuvant therapy, extent of resection, and pathology were analyzed with respect to outcomes from morbidity, narcotic use, and oncologic perspectives.Baseline demographics, neoadjuvant therapy, and perioperative factors including extent of surgery, complete resections (R0), nodal status and lymph node number, morbidity, and mortality were similar between the two groups. The mean duration of out-patient narcotic use was significantly lower in the "cut-in patch-out" group compared to the thoracotomy group . Using multivariate regression analysis, the traditional thoracotomy technique  was independently associated with prolonged oral narcotic requirements (>100 days). Additionally, five year survival for the "cut-in patch-out" group was 48% versus the traditional group at 12.5% (p = 0.04).Compared with a traditional thoracotomy and separate chest wall resection approach for P-NSCLC, a "cut-in patch-out" technique offers an alternative approach that appears to have at least oncologic equivalence while decreasing pain. We have more recently adapted this technique to select patients with pulmonary neoplasms involving chest wall invasion and believe further investigation is warranted.The online version of this article (doi:10.1186/s13019-014-0163-z) contains supplementary material, which is available to authorized users. Although initially characterized by a radiologist, Henry Pancoast, in 1924, surgical resection of a superior sulcus or Pancoast tumor was not attempted until the 1950s . CurrentSince 2001 we have utilized a novel surgical approach for P-NSCLC requiring en-bloc removal of at least 3 ribs where lobectomy and mediastinal lymph node dissection are performed though the defect after chest wall resection. The defect is patched at the completion of the procedure ("cut-in patch-out") thereby avoiding a separate thoracotomy with rib spreading. The aim of this study was to compare short and long term outcomes between P-NSCLC patients undergoing this novel technique and a traditional posterolateral thoracotomy approach.Under the Indiana University School of Medicine Institutional Review Board approval, a query of an institutional database was undertaken. Forty-one patients undergoing surgical resection of a P-NSCLC requiring en-bloc removal of at least 3 ribs from 1999 to 2012 were identified. Since 2001, 25 patients were identified who underwent a novel "cut-in patch-out" approach. Sixteen patients underwent resection of a P-NSCLC by a traditional posterolateral thoracotomy and separate chest wall resection who served as controls. Patients with anterior based apical tumors requiring a partial sternotomy approach (n = 6) and P-NSCLC who required en bloc removal of only 2 or fewer ribs (n = 13) were excluded from this study.Patient demographic data including age, gender, neoadjuvant chemotherapy/radiation therapy, and co-morbidities were collected. Operative and pathologic variables including tumor size, histology, stage, number of ribs resected, estimated blood loss, type of surgical resection , and number of mediastinal lymph nodes harvested were collected. Short term post-operative data including duration of intravenous narcotics, length of hospital stay, morbidity (pulmonary and non-pulmonary morbidity) and mortality were recorded. Survival data was verified with the Social Security Death Index. Long-term postoperative data including duration of daily narcotic use following discharge were collected and verified by a statewide narcotic tracking system. This system, under the auspices of the state government, provides an online database of all prescribed controlled substances, including narcotics. Finally, status at time of last follow up was obtained.th interspace thoracotomy with rib spreading for hilar dissection and lobectomy along with complete peribronchial and mediastinal lymph node dissection. Apical en bloc chest wall resection was typically accomplished after hilar dissection in these cases. After 2001, a majority of patients with P-NSCLC underwent resection using a "cut-in patch-out" technique which did not involve a separate thoracotomy. With the "cut-in patch-out" technique, an extended posterolateral incision was also utilized. In these cases however, the pleural space was initially entered in the interspace estimated to be 3 to 5 cm anterior and inferior to the tumor location, as determined by preoperative CT scan. The tumor was palpated and, if clear of the interspace entry site, the interspace was opened posteriorly to the costovertebral angle. The anterior aspect of the chest resection was then performed 3 to 5 cm anterior to the tumor location, again confirmed visually or by palpation. A one cm segment of rib was excised anteriorly to improve chest wall mobility which was facilitated by upward scapular retraction  version 20.0 for Windows. Values are presented as means with standard deviations unless otherwise specified. Continuous variables were compared using student t-test while categorical variables were compared with the chi squared. Survival and recurrence analysis was performed using the Kaplan-Meier method. A multivariate regression analysis for prolonged duration of oral narcotics (>100 days) was adjusted for narcotic use preoperatively, the number of ribs resected, estimated blood loss, length of hospital stay, and surgical technique. All tests were two-sided, with 0.05 serving as the level of significance.In total, 25 patients underwent the "cut-in patch-out" approach while 16 patients underwent a traditional posterolateral thoracotomy and separate chest wall resection. Patient characteristics are presented in Table Perioperative factors were also similar between both groups Table . All patPostoperative outcomes are presented in Table Daily outpatient narcotic use was significantly lower in the "cut-in patch-out" group  Figure . With reResults of multivariate regression analysis to identify risk factors for prolonged oral narcotic use (>100 days) is given in Table The results of this study demonstrate that a "cut-in patch-out" approach to P-NSCLC can offer a reduction in postoperative pain when compared with a traditional approach. Although not seen in the immediate post-operative course with intravenous narcotic use, the benefit became apparent after discharge analyzing the duration of oral narcotic requirement by both univariate and in a multivariate model. From an oncologic standpoint, the number of mediastinal lymph nodes retrieved and surgical margin status were similar between the two groups. More importantly, five-year recurrence rates and survival were both improved in patients undergoing the "cut-in patch-out" technique suggesting this approach offers at least an equivalent oncologic outcome.A "cut-in patch-out" type of surgical approach to P-NSCLC, was initially reported by Kent et al. from Memorial Sloan-Kettering Cancer Center using a polytetraflouroethylene patch for chest wall reconstruction in 2004 . We haveThere have been many options described for chest wall reconstruction after P-NSCLC resection including polypropylene and polytetrafluoroethylene prosthetic mesh ,4],13],[13]4],4],. We haveAlthough previous work has investigated the prevalence of post-thoracotomy pain, there is little information on pain after a chest wall resection. One retrospective analysis of thoracotomy patients by Keller et al. determined that 50% of those undergoing chest wall resections with thoracotomy developed significant post-thoracotomy pain compared with 11% of those undergoing thoracotomy without chest wall resection . A more The factors that have been shown to be associated with the best outcomes for P-NSCLC resections include R0 resections, absence of positive nodes, and anatomic lobar versus wedge pulmonary resections . AlthougThere are several limitations to this study. First, this is a small retrospective series at a single institution. Although reduction in oral narcotic requirements were apparent in the "cut-in patch-out" group, larger and more uniform patient subsets are needed to determine differences from long-term oncologic perspectives including the ability to achieve tumor free margins. Another limitation is that although the majority of patients appeared to be taking similar narcotic doses, we were only able to determine the use of narcotics per se and could not obtain accurate dosage information. Additionally, although it appeared that non-narcotic pain medications such as non-steroidal anti-inflammatory drugs or gamma-aminobutyric acid analogs were used sparingly in our series, we were unable to conclusively make this determination for accurate analysis.In conclusion, this study demonstrates that compared with a traditional approach involving separate thoracotomy and chest wall resection for P-NSCLC tumors, a "cut-in patch-out" technique may reduce outpatient narcotic requirements and appears to result in equivalent oncologic outcomes including survival rates. We therefore believe this approach warrants further investigation. Furthermore, based on this favorable experience we have more recently adapted a variation of this "cut-in patch-out" technique using non-absorbable prosthetic mesh for select patients with non P-NSCLC and chest wall invasion.DW, IO, and KK participated in study design. DW, IO, TB, KR, DC, and KK drafted the manuscript. All authors read and approved the final manuscript.


Remarkable progress has been made in image recognition, primarily due to the availability of large-scale annotated datasets and deep convolutional neural networks (CNNs). CNNs enable learning data-driven, highly representative, hierarchical image features from sufficient training data. However, obtaining datasets as comprehensively annotated as ImageNet in the medical imaging domain remains a challenge. There are currently three major techniques that successfully employ CNNs to medical image classification: training the CNN from scratch, using off-the-shelf pre-trained CNN features, and conducting unsupervised CNN pre-training with supervised fine-tuning. Another effective method is transfer learning, i.e., fine-tuning CNN models pre-trained from natural image dataset to medical image tasks. In this paper, we exploit three important, but previously understudied factors of employing deep convolutional neural networks to computer-aided detection problems. We first explore and evaluate different CNN architectures. The studied models contain 5 thousand to 160 million parameters, and vary in numbers of layers. We then evaluate the influence of dataset scale and spatial image context on performance. Finally, we examine when and why transfer learning from pre-trained ImageNet (via fine-tuning) can be useful. We study two specific computer-aided detection (CADe) problems, namely thoraco-abdominal lymph node (LN) detection and interstitial lung disease (ILD) classification. We achieve the state-of-the-art performance on the mediastinal LN detection, and report the first five-fold cross-validation classification results on predicting axial CT slices with ILD categories. Our extensive empirical evaluation, CNN model analysis and valuable insights can be extended to the design of high performance CAD systems for other medical imaging tasks. I.Tremendous progress has been made in image recognition, primarily due to the availability of large-scale annotated datasets  training the “CNN from scratch” Previous studies have analyzed three-dimensional patch creation for LN detection Although natural images and medical images differ significantly, conventional image descriptors developed for object recognition in natural images, such as the scale-invariant feature transform (SIFT) In this paper, we exploit three important, but previously under-studied factors of employing deep convolutional neural networks to computer-aided detection problems. Particularly, we explore and evaluate different CNN architectures varying in width (ranging from 5 thousand to 160 million parameters) and depth (various numbers of layers), describe the effects of varying dataset scale and spatial image context on performance, and discuss when and why transfer learning from pre-trained ImageNet CNN models can be valuable. We further verify our hypothesis by inheriting and adapting rich hierarchical image features Two specific computer-aided detection (CADe) problems, namely thoraco-abdominal lymph node (LN) detection and interstitial lung disease (ILD) classification are studied in this work. On mediastinal LN detection, we surpass all currently reported results. We obtain 86% sensitivity on 3 false positives (FP) per patient, versus the prior state-of-art sensitivities of 78% Evaluation protocols and details are critical to deriving significant empirical findings II.We employ CNNs (with the characteristics defined above) to thoraco-abdominal lymph node (LN) detection  and interstitial lung disease (ILD) detection. For LN detection, we use randomly sampled 2.5D views in CT Until the detection aggregation approach Interstitial lung disease (ILD) comprises more than 150 lung diseases affecting the interstitium, which can severely impair the patient's ability to breathe. Gao et al. A.We use the publicly available dataset from Unlike the heart or the liver, lymph nodes have no pre-determined anatomic orientation. Hence, the purely random image resampling  and reformatting  is a natural choice, which also happens to yield high CNN performance. Although we integrate three channels of information from three orthogonal views for LN detection, the pixel-wise spatial correlations between or among channels are not necessary. The convolutional kernels in the lower level CNN architectures can learn the optimal weights to linearly combine the observations from the axial, coronal, and sagittal channels by computing their dot-products. Transforming axial, coronal, and sagittal representations to RGB also facilitates transfer learning from CNN models trained on ImageNet.This learning representation  is flexible, in that it naturally combines multiple sources or channels of information. In the recent literature In summary, the assumption that there are or must be pixel-wise spatial correlations among input channels does not apply to the CNN model representation. For other medical imaging problems, such as pulmonary embolism detection B.We utilize the publicly available dataset of In the publicly available ILD dataset, very few CT slices are labeled as normal or healthy. The remaining CT slices cannotTo leverage the CNN architectures designed for color images and to transfer CNN parameters pre-trained on ImageNet, we transform all gray-scale axial CT slice images via three CT window ranges: lung window range 1This can be achieved by segmenting the lung using simple label-fusion methods  There is no significant difference between two setups. Due to the high precision of CNN based image processing, highly accurate lung segmentation is not necessary. The localization of ILD regions within the lung is simultaneously learned through selectively weighted CNN reception fields in the deepest convolutional layers during the classification based CNN training As observed in III.In this study, we explore, evaluate and analyze the influence of various CNN Architectures, dataset characteristics  learning rate: 0.01, decreased by a factor of 10 at every 10 epochs. We use the Caffe framework AlexNet and GoogLeNet CNN models can be either learned from scratch or fine-tuned from pre-trained models. Girshick et al. Our hypothesis on CNN parameter transfer learning is the following: despite the disparity between natural images and natural images, CNNs comprehensively trained on the large scale well-annotated ImageNet may still be transferred to make medical image recognition tasks more effective. Collecting and annotating large numbers of medical images still poses significant challenges. On the other hand, the mainstream deep CNN architectures  contain tens of millions of free parameters to train, and thus require sufficiently large numbers of labeled medical images.For transfer learning, we follow the approach of We also perform experiments using “off-the-shelf” CNN features of AlexNet pre-trained on ImageNet and training only the final classifier layer to complete the new CADe classification tasks. Parameters in the convolutional and fully connected layers are fixed and are used as deep image extractors, as in Finally, transfer learning in CNN representation, as empirically verified in previous literature IV.In this section, we evaluate and compare the performances of nine CNN model configurations  on two important CADe problems using publicly available datasets A.We train and evaluate CNNs using three-fold cross-validation (folds are split into disjoint sets of patients), with the different CNN architectures described above. In testing, each LN candidate has multiple random 2.5D views tested by CNN classifiers to generate LN class probability scores. We follow the random view aggregation by averaging probabilities, as in We first sample the LN image patches at a Results for lymph node detection in the mediastinum and abdomen are reported in GoogLeNet-RI-H performs poorly, as it is susceptible to over-fitting. No sufficient data samples are available to train GoogLeNet-RI-H with random initialization. Indeed, due to GoogLeNet-RI-H's complexity and 22-layer depth, million-image datasets may be required to properly train this model. However, GoogLeNet-TL-H significantly improves upon GoogLeNet-RI-H . This indicates that transfer learning offers a much better initialization of CNN parameters than random initialization. Likewise, AlexNet-TL-H consistently outperforms AlexNet-RI-H, though by smaller margins . This is also consistent with the findings reported for ILD detection in GoogLeNet-TL-H yields results similar to AlexNet-TL-H's for the mediastinal LN detection, and slightly outperforms Alex-Net-H for abdominal LN detection. AlexNet-RI-H exhibits less severe over-fitting than GoogLeNet-RI-H. We also evaluate a simple ensemble by averaging the probability scores from five CNNs: AlexNet-RI-H, AlexNet-TL-H, AlexNet-RI-H, GoogLeNet-TL-H and GoogLeNet-RI-L. This combined ensemble outputs the classification accuracies matching or slightly exceeding the best performing individual CNN models on the mediastinal or abdominal LN detection tasks, respectively.Many of our CNN models achieve notably better (FROC-AUC and TPR/3FP) results than the previous state-of-the-art models The main difference between our dataset preparation protocol and that from B.The CNN models evaluated in this experiment are 1) AlexNet-RI ; 2) AlexNet-TL  are shown in To investigate the performance difference between five-fold cross-validation (CV) in CNN training is implemented with the Caffe D.Medical datasets are often “biased”, in that the number of healthy samples is much larger than the number of diseased instances, or that the numbers of images per class are uneven. In ILD dataset, the number of fibrosis samples is about 3.5 times greater than the number of emphysema samples. The number of non-LNs is Without loss of generality, after GoogLeNet is trained on the training sets under “Equal” or “Biased” priors, we compare its classification results on the balanced validation sets. Evaluating a classifier on a biased validation set will cause unfair assessment of its performance. For instance, a classifier that predicts every image patch as “non-LN” will still achieve a 70% accuracy rate on a biased set with 3.5 times as many non-LN samples as LN samples. The classification accuracy results of GoogLeNet trained under two configurations are shown in V.In this section, we determine and analyze, via CNN visualization, the reasons for which transfer learning is beneficial to achieve better performance on CAD applications.A.In B.We focus on analyzing visual CNN optimization traces and activations from the ILD dataset, as its slice-level setting is most similar to ImageNet's. Indeed, both datasets use full-size images. The traces of the training loss, validation loss and validation accuracy of AlexNet-RI and AlexNet-TL, are shown in The last pooling layer (pool-5) activation maps of the ImageNet pre-trained AlexNet VI.•Deep CNN architectures with 8, even 22 layers •The trade-off between using better learning models and using more training data •Limited datasets can be a bottleneck to further advancement of CADe. Building progressively growing , well annotated datasets is at least as crucial as developing new algorithms. This has been accomplished, for instance, in the field of computer vision. The well-known scene recognition problem has made tremendous progress, thanks to the steady and continuous development of Scene-15, MIT Indoor-67, SUN-397 and Place datasets •Transfer learning from the large scale annotated natural image datasets (ImageNet) to CADe problems has been consistently beneficial in our experiments. This sheds some light on cross-dataset CNN learning in the medical image domain, e.g., the union of the ILD •Finally, applications of off-the-shelf deep CNN image features to CADe problems can be improved by either exploring the performance-complementary properties of hand-crafted features We summarize our findings as follows.VII.In this paper, we exploit and extensively evaluate three important, previously under-studied factors on deep convolutional neural networks (CNN) architecture, dataset characteristics, and transfer learning. We evaluate CNN performance on two different computer-aided diagnosis applications: thoraco-abdominal lymph node detection and interstitial lung disease classification. The empirical evaluation, CNN model visualization, CNN performance analysis, and conclusive insights can be generalized to the design of high performance CAD systems for other medical imaging tasks.http://ieeexplore.ieee.org.Color versions of one or more of the figures in this paper are available online at


Four erlotinib-sensitive lung adenocarcinoma cell lines and their resistant derivatives were tested. Of the resistant cell lines, all but one showed a similar sensitivity to the tested drugs as their parental cells. HCC4006ER cells with epithelial mesenchymal transition features acquired resistance to the three microtubule-targeting agents, docetaxel, paclitaxel and vinorelbine, but not to cisplatin and gemcitabine. Gene expression array and immunoblotting demonstrated that ATP-binding cassette subfamily B, member 1 (ABCB1) was up-regulated in HCC4006ER cells. ABCB1 knockdown by siRNA partially restored sensitivity to the anti-microtubule agents but not to erlotinib. Moreover, the histone deacetylase inhibitor entinostat sensitized HCC4006ER cells to anti-microtubule agents through ABCB1 suppression. Our study indicates that sensitivity of tumor cells to cytotoxic agents in general does not change before and after failure of EGFR-TKIs. However, we describe that two different molecular alterations confer acquired resistance to EGFR-TKIs and cytotoxic agents, respectively. This phenomenon should be kept in mind in selection of subsequent therapy after failure of EGFR-TKIs.Various alterations underlying acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been described. Although treatment strategies specific for these mechanisms are under development, cytotoxic agents are currently employed to treat many patients following failure of EGFR-TKIs. However, the effect of TKI resistance on sensitivity to these cytotoxic agents is mostly unclear. This study investigated the sensitivity of erlotinib-resistant tumor cells to five cytotoxic agents using an  EGFR) is present in approximately 40% and 20% of these tumors in East-Asians and Caucasians, respectively . Ad. AdCDH1 egulated .ABCB1) was up-regulated by approximately 40-fold in HCC4006ER cells. On the other hand, no other ABC transporter family member was overexpressed to a similar extent in HCC4006ER cells  has also been reported to be a predictive marker for clinical outcome of taxane/vinorelbine-based chemotherapy . HoweverABCB1. siRNA-mediated knockdown of ABCB1 partially sensitized HCC4006ER cells to anti-microtubule agents  in HCC4006 cells did not affect sensitivity to any of the three anti-microtubule agents  inhibitor entinostat, which reverses EMT in HCC4006ER cells and restores sensitivity to erlotinib as described in our previous study . Here, eWe have previously shown that HCC4006ER cells are resistant to erlotinib through acquisition of EMT as characterized by the down-regulation of E-cadherin expression . In the Alternatively, there are several examples in which resistance to EGFR-TKI and chemotherapeutic agents shared the same molecular mechanism. PTEN deficiency renders PC9 cells resistant to cisplatin and also reduces their sensitivity to erlotinib . AXL oveWe also found that the class I HDAC inhibitor entinostat restored sensitivity to anti-microtubule drugs through down-regulation of ABCB1 protein. We have previously shown that entinostat restores sensitivity to erlotinib by promoting E-cadherin re-expression . These oMET amplification, loss of amplified EGFR and IGF1R hyperactivation) did not exhibit any detectable change in sensitivity to five cytotoxic agents. This is consistent with previous in vitro observations that demonstrated that EGFR-TKI resistant cells with T790M or MET amplification showed sensitivity to cytotoxic agents similar to that of their parental cells [In contrast to the above, five cell lines with acquired resistance to erlotinib, (two by T790M secondary mutations, one each by al cells ,29. Thesal cells .EGFR mutation conferred a “collateral” resistance to anti-microtubule agents via upregulation of ABCB1. However, this phenomenon appeared exceptional and chemosensitivity was not influenced by EGFR-TKI resistance in most cases.In conclusion, we have demonstrated that erlotinib resitstance in a cell line harboring


A specialized microenvironment in the bone marrow niche is composed of stromal cells including mesenchymal stem cells (MSCs) that might support leukemia progression. In this study, we investigated the reciprocal interaction between MSCs and Adult T-cell Leukemia (ATL) cells, as a model of hematological malignancies. We evaluated the role of paracrine interaction between MSCs and leukemic cells through soluble factors, exosomes, and direct interaction through adhesion (N-cadherin) and communication (Connexins).ATL cells used were either HTLV-I negative (Molt-4) or positive (C81 and HuT-102) cells. Indirect co-cultures between MSCs and ATL cells were assessed in Transwell chambers whereby MSCs were seeded in the bottom well and leukemic cells were in the upper insert. Direct co-cultures were performed at 1:1 ratio and cells were then sorted using CD73, MSCs’ cell surface marker. Leukemic cells-derived exosomes were isolated by ultracentrifugation and co-cultured with MSCs. Following co-cultures for 72 hours, we studied cell proliferation by Trypan Blue Exclusion assay and expression profile of metastatic and stemness markers by Real-time PCR and western blotting.After indirect co-culture, MSCs caused a moderate increase in ATL cells proliferation. In contrast, MSCs proliferation was only induced by HuT-102 cells. Although indirect co-culture did not cause major changes in leukemic cells expression profile of metastatic markers, direct interaction induced the expression of Cx43, SDF-1 and VEGF, especially in the adherent fraction, compared to the suspension fraction. Exosomes derived from C81 and HuT-102, HTLV-I positive cells, contained Tax oncoprotein and induced MSCs proliferation. This interaction led to an increase in MSCs cell number, a change in cellular morphology and an increase in the expression of VEGF and stemness markers, Oct-4 and Nanog.These findings demonstrate that indirect and direct interactions reciprocally affect MSCs and leukemic cells properties. Interaction through leukemia-derived exosomes modulates MSCs properties which might in turn contribute to leukemia progression.


Aedes aegypti or Aedes albopictus mosquitoes transmit the four dengue virus (DENV) serotypes to humans while probing for small blood vessels in the skin. DENV causes the most prevalent arthropod-borne viral disease in humans, yet no vaccine or specific therapeutic is currently licensed. Although primary DENV infection confers life-long protective immunity against re-infection with the same DENV serotype, secondary infection with a different DENV serotype can lead to increased disease severity via cross-reactive T-cells or enhancing antibodies. This review summarizes recent findings in humans and animal models about DENV infection of DCs, monocytes, and macrophages. We discuss the dual role of DCs as both targets of DENV replication and mediators of innate and adaptive immunity, and summarize immune evasion strategies whereby DENV impairs the function of infected DCs. We suggest that DCs play a key role in priming DENV-specific neutralizing or potentially harmful memory B- and T-cell responses, and that future DC-directed therapies may help induce protective memory responses and reduce dengue pathogenesis.Dendritic cells (DCs) are sentinels of the immune system and detect pathogens at sites of entry, such as the skin. In addition to the ability of DCs to control infections directly via their innate immune functions, DCs help to prime adaptive B- and T-cell responses by processing and presenting antigen in lymphoid tissues. Infected  Dendritic cells (DCs) reside and migrate into barrier tissues such as the skin and mucosal epithelium that are the sites of pathogen invasion. In the steady state, DCs display high levels of phagocytic activity, take up antigen, and probe for pathogens via pattern-recognition receptors. DCs express Toll-like receptors (TLRs) and C-type lectins as transmembrane proteins as well as intracellular sensors, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation factor 5 (MDA-5), and cyclic GMP-AMP synthase (cGAS) that recognize conserved microbial patterns , 2. Upon+ T-cells  reside in different tissues and fulfill distinct functions. Classical DCs (cDCs) display the characteristic DC dendrites, are present in LNs, spleen, and bone marrow as well as skin, lung, liver, and intestine, and have the greatest ability to stimulate naïve T-cells . All cDC T-cells . In addi T-cells , 7. In a T-cells . NeverthIn vitro-generated human moDCs are used widely to study DC biology  while feeding on blood vessels in the skin  can progress to a potentially life-threatening vascular leakage syndrome, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), the latter characterized by hypotension and circulatory failure . At presAedes mosquitoes that take a blood meal from a human with acute dengue viremia become infected and, after DENV spreads to the salivary glands, transmit the virus when feeding on a new individual. Mosquito saliva contains components that counteract the host hemostatic response and modulate immunity  and also -γ receptor (AG129) are susceptible to DENV infection and display a tropism similar to humans  mice generally do not sustain DENV replication or develop disease . In compo humans , 44–46. o humans , as wello humans , 49. Treo humans . Monocytin vitro support DENV infection  is a C-type lectin expressed on the surface of DCs and MΦs that recognizes mannose-type sugars on the surface of bacterial, fungal, and viral pathogens. Signals via DC-SIGN induce the phagocytosis of pathogens and contribute to host defense . HoweverAedes mosquitoes transmit DENV when probing for blood vessels in the skin, most studies have focused on DENV infection in tissues after the virus has spread via the blood. Few studies have examined DENV infection and the immune response in the skin. DENV infects epidermal Langerhans cells (LCs) in healthy human skin explants in vitro  of the FcRIIA gene, who have less capacity to opsonize IgG2 antibodies, showed reduced risk of developing DHF  and induce cell-intrinsic and cell-extrinsic innate immune responses. DENV infection stimulates responses via TLR7, TLR3, MDA5, and RIG-I –90 and iin vitro, and IFN-α production in pDCs appears independent of active viral replication within pDCs  , 100, whAntibody-enhanced DENV infection also is believed to contribute to the evasion of the innate immune response through a mechanism termed “intrinsic ADE” . A monoc+ T-cells or on MHC II to CD4+ T-cells; (b) expressing co-stimulatory molecules that activate T-cells; and (c) secreting chemokines and cytokines that attract T-cells and modulate the priming T-cell effector functions.Dendritic cells link innate and adaptive immune responses by integrating innate signals from PAMPs with pathogen-derived antigens to induce antigen-specific T-cell and B-cell responses. DCs achieve this by (a) taking up and processing antigen and presenting antigen-derived peptides on MHC I to CD8+ apoptotic cells in those co-staining for DENV E protein  are a functional read-out for DC–T-cell interactions, in which DCs from MHC mismatched donors activate allogeneic T-cells. DENV infection decreased the capacity of moDCs , 119 as s Figure B. Others or TNFα . The imp or TNFα . Althougce IFN-γ . More stce IFN-γ , 31.Also, activated T-cells can support the maturation of DCs. An initial study showed that co-culture of moDCs with a CD40L-transfected cell line restored the ability of DENV-infected moDCs to induce MLR responses  Figure C. Subseqin vitro-generated human moDCs have been used to study the role of DCs in inducing innate or adaptive immune responses during DENV infection. Nevertheless, diverse subsets of cDCs in lymphoid or non-lymphoid tissues execute specialized functions in presenting antigen and inducing CD4+ or CD8+ T-cell responses  that are essential for inducing and maintaining optimal innate and adaptive immune responses. This tropism of DENV appears to impair DC function, which may undermine the priming of DENV-specific memory responses. Is it possible to block DENV replication in DCs to reduce viral load and restore DC function, which could impact the generation of neutralizing or potentially harmful memory B- and T-cell responses? Can we as a field harness the knowledge gained about DC biology during DENV infection to prevent human disease? Can DC function be optimized in the context of live-attenuated DENV vaccines to stimulate protective immunity?Significant progress has been made on characterizing DENV infection and activation of DCs, as well as protective or enhancing T-cell and B-cell responses. Nevertheless, early events after DENV transmission in the skin require further study, including a greater understanding of early virus replication, local immune responses at the site of transmission , and immunomodulatory effects of mosquito saliva. Further effort should focus on how responses of DCs impact disease outcome in an acute infection and prime immunological memory responses that will affect dengue pathogenesis and disease severity in secondary DENV infections. A collaborative effort using a multi-disciplinary approach among experts in dengue virology, medicine, vector biology, and immunology is called for to reach these goals.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.


These purine cyclic nucleotides activate cAK and cGK, respectively. So far, there was no evidence of further cyclic nucleotides acting as second messengers. Meanwhile the existence of cCMP was described ,2. cCMP 5-24, suggesting a stimulatory function for PKA in cCMP-mediated MAPK phosphorylation. To elucidate the role of cGK in murine tissues in this process, we used cGKII knockout (cGKII KO) and cGKI knockout (cGKI KO) mice. We detected stimulation in the jejunum tissues from cGKI KO and cGKII KO mice. It is interesting to note that the phosphorylation in the jejunum cGKII KO tissue was significantly increased when compared with the WT and cGKI KO tissues, suggesting an inhibitory role for cGKII in cCMP-induced MAPK phosphorylation in the jejunum.To elucidate propable functions cCMP-binding and -activated proteins were identified using different methods. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins in murine tissue lysates, using 4-AH-cCMP agarose. cCMP (200 µM) was added (+) or omitted (-) during the affinity chromatography experiments to investigate the specificity of the binding. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. Interestingly, no specific interaction of MAPK with 8-AET-cGMP agarose was detected. Moreover, DB-cCMP (100 µM) was also able to stimulate the phosphorylation of p44/p42 MAPK. The phosphorylation of MAPK was inhibited by the addition of the PKA inhibitor ASThese results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum.