Arabidopsis thaliana uncovered homologous components and similar mechanisms in plants for monitoring protein folding and for retaining, repairing, and removing misfolded proteins. These studies also revealed critical roles of the plant ERQC/ERAD systems in regulating important biochemical/physiological processes, such as abiotic stress tolerance and plant defense. In this review, we discuss our current understanding about the molecular components and biochemical mechanisms of the plant ERQC/ERAD system in comparison to yeast and mammalian systems.A correct three-dimensional structure is crucial for the physiological functions of a protein, yet the folding of proteins to acquire native conformation is a fundamentally error-prone process. Eukaryotic organisms have evolved a highly conserved endoplasmic reticulum-mediated protein quality control (ERQC) mechanism to monitor folding processes of secretory and membrane proteins, allowing export of only correctly folded proteins to their physiological destinations, retaining incompletely/mis-folded ones in the ER for additional folding attempts, marking and removing terminally misfolded ones via a unique multiple-step degradation process known as ER-associated degradation (ERAD). Most of our current knowledge on ERQC and ERAD came from genetic and biochemical investigations in yeast and mammalian cells. Recent studies in the reference plant  Arabidopsis thaliana and other model plant species not only identified homologous ERQC/ERAD components but also revealed evolutionarily conserved features as well as unique aspects of the plant ERQC/ERAD mechanisms  is the cellular port of entry for secretory and membrane proteins to enter the secretory pathway and is a folding compartment for proteins to attain their native conformations through interactions with molecular chaperones, sugar-binding lectins, and folding enzymes . Misfoldchanisms , especia3Man9GlcNac2  from a membrane-anchored dolichylpyrophosphate (DolPP) carrier to the Asn residue  proteins that sequentially add a monosaccharide onto the DolPP linker or a DolPP-linked oligosaccharide precursor . The structure of a N-linked glycan plays an important role in the protein folding and quality control  and glucosidase II (GII), producing a monoglucosylated N-glycan, GlcMan9GlcNac2, which is recognized by the ER chaperone-like lectins, a membrane-anchored calnexin (CNX) and its ER luminal homolog calreticulin . The high-specificity high-affinity binding between GlcMan9GlcNac2 and CNX/CRT is crucial for folding a nascent polypeptide as CNX/CRT can recruit other ER-chaperones and folding enzymes, including protein disulfide isomerases (PDIs) essential for generating inter/intra-molecular disulfide bonds. The removal of the remaining Glc residue by GII releases the nascent glycoprotein from CNX/CRT, thus terminating its folding process . It is worthy to mention that the budding yeast (Saccharomyces cerevisiae), which is widely used for studying the ERAD process, lacks the CNX/CRT-UGGT system due to the presence of a catalytically inactive UGGT homolog  led to the discovery of Arabidopsis UGGT  and SDF2  are also involved in the biogenesis/folding control of EFR , which have a N-terminal ATP-binding domain and a C-terminal substrate-binding domain that recognizes and binds to exposed hydrophobic patches of incompletely/mis-folded proteins in an ATP-dependent manner . The Arastresses . In Arabeceptors . BiPs anl of EFR . BiPs wel of EFR . Anotherl of EFR , 2007. Tproteins , none ofproteins . FurtherN-glycan , catalyzed by homologous to mannosidase 1 (Htm1) in yeast and ER-degradation enhancing α-mannosidase-like proteins (EDEMs) in mammals, is required for generating the ERAD signal, an exposed α1,6 Man residue on an N-linked glycan . The Arabidopsis genome encodes at least two homologs of the yeast Htm1/mammalian EDEMs  or drouf Doa10A ] could uThe 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.


Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP.cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination.Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases.in vivo.Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy The online version of this article (doi:10.1186/s13046-015-0149-2) contains supplementary material, which is available to authorized users. Herpes simplex virus thymidine kinase (HSVtk) in combination with ganciclovir (GCV) and cytosine deaminase (CD) derived from bacteria or yeast combined with 5-fluorocytosine (5-FC) . Similarly to all other treatments, also this therapeutic system is limited. As we demonstrated previously, treatment efficacy can be influenced by expression level of enzymes involved in drug activation/degradation, intercellular communication or by expression of ABC transporters effluxing toxic metabolites out of cells. We have also demonstrated insufficient efficacy of the treatment by adipose tissue-derived MSC (AT-MSC) expressing CD::UPRT combined with 5-FC on adenocarcinoma-derived cell line MDA-MB-231/EGFP in vitro. On the other hand, this cell line was sensitive to treatment by HSVtk expressing AT-MSC and GCV ..19]].On the other hand, we demonstrated that various tumor cell lines differ in the response to CD::UPRT-MSC/5-FC or HSVtk-MSC/GCV treatment depending on the expression of enzymes of nucleotide metabolism and ABC transporters ,4. By “cIn various studies, the combination of CD/5-FC and HSVtk/GCV approaches has been described as synergic ,22 additE.coli CD/HSVtk fusion gene was demonstrated by simultaneous treatment of transgene-expressing cells with their specific prodrugs. The toxicity was two-fold higher when expected if the cytotoxic effect of each prodrug was purely additive. Moreover, combined enzyme/prodrug treatment significantly increased the radiosensitivity of tumor cells . This fin vitro , ratio of target/therapeutic cells was 1:2 [Recently, additive cytotoxic effect of human amniotic fluid-derived stem cells (hAFSC) transfected with bacterial CD::HSVtk fusion gene on MDA-MB-231 cells was described . They ob was 1:2 . We appl was 1:2 . Moreove was 1:2 .in vitro studies partially depends on the setup of the experiment, duration of the treatment and prodrug concentration. When we used too high concentrations of prodrugs in combination, the synergic effect was suppressed (data not shown).In our experimental setup we confirmed observation of Boucher et al.  that seqThe sensitivity of tumor cells to the single treatment by CD(::UPRT)/5-FC or HSVtk/GCV probably also contributes to the extent of synergy. We performed double treatment on three tumor cell lines with different sensitivity to the particular approaches, and we observed different response.in vivo. We applied both types of therapeutic cells in two injections daily for two weeks. In order to minimize the stress of experimental animals both prodrugs were also administered at the same time. Maximal tolerable doses of prodrugs were administered intraperitoneally.Boucher et al.  supposedin vitro and in vivo. In the later study which focused on the safety of this approach, they demonstrated that therapeutic NSC expressing two prodrug-converting genes showed lower proliferation and viability in comparison to the CD-expressing NSC thus exerting increased safety of this approach. On the other hand, they admitted that therapeutic effect might be limited. As demonstrated in our previous study [Two recent papers ,11 descrus study , engineein vitro. Systemic combined treatment confirmed the efficacy of this approach by inhibition of proliferation of systemically administered MDA-MB-231/EGFP cells in mouse lungs.Although the extent of the synergy of combined GDEPT mediated by MSC depends on the sensitivity of tumor cells to the particular approach and on the experimental setup, cooperation of CD::UPRT-MSC and HSVtk-MSC exerted synergic cytotoxic effect on human breast adenocarcinoma cells MDA-MB-231/EGFP


By studying hearts obtained from global BK-null mice (BK-KO) in an ex vivo Langendorff perfusion setup we and others previously found evidence for mitochondrial BK channels (mitoBKs) in cardiomyocytes as infarct-limiting factors ,2. It isUsing the Cre/loxP recombination system we generated animals with a CM-restricted deletion of BK channels (CMBK-KO)  and of nBaseline infarct size of CMsGC-KOs did not differ from that of CMsGC-CTRs, however, 30 min of ischemia followed by 120 min of reperfusion provoked significantly more cardiac damage in global BK-KO and CMBK-KO mice than in age- and litter-matched BK-WTs and CMBK-CTRs, respectively.The BK blocker paxilline (applied 5 min after the onset of ischemia) did not affect the response to I/R of CMBK-KO hearts, whereas in hearts from CMBK-CTR mice we observed an increase in the cardiac damage. With the BK opener NS11021 (5 min before reperfusion) we observed a more drastic decrease in the infarct size in hearts from CMBK-CTR than in CMBK-KO mice.As expected, short repetitive episodes of ischemia applied directly after infarction (iPost) significantly reduced the myocardial damage in all WT/CTR hearts, whereas protection afforded by iPost was less pronounced in the absence of mitoBK channels and completely abolished in hearts lacking CM NO-sGC.Interestingly, cardioprotection elicited either by an intra-atrial injection of sildenafil (5 min before reperfusion) or by an i.p. injection of tadalafil (60 min before the ischemic insult) also seems to require mitoBK and NO-GC in the CM.In summary, the presented findings demonstrate that the lack of CM BK channels renders the heart more susceptible to I/R injury. Cardioprotection elicited by the BK opener NS11021 suggests that BK channels may be promising drug targets that interfere with the causes and/or consequences of myocardial ischemia. Interestingly, both CM NO-GC and BK are important to allow the protective signaling events triggered either by short, repetitive episodes of ischemia or by agents targeting PDE5. Further studies are needed to elucidate whether cardioprotection via NO-GC and BK are linked by a common cGMP pathway in the CM itself.Sandra Frankenreiter, Angelina Kniess, Peter Ruth, Andreas Friebe and Robert Lukowski are members of the DFG Research Unit 2060 “cGMP signalling in cell growth and survival”.


Stress research has progressively become more integrative in nature, seeking to unfold crucial relations between the different phenotypic levels of stress manifestations. This study sought to unravel stress-induced variations in expression of human microRNAs sampled in peripheral blood mononuclear cells and further assess their relationship with neuronal and psychological indices.We obtained blood samples from 49 healthy male participants before and three hours after performing a social stress task, while undergoing functional magnetic resonance imaging (fMRI). A seed-based functional connectivity (FC) analysis was conducted for the ventro-medial prefrontal cortex (vmPFC), a key area of stress regulation. Out of hundreds of microRNAs, a specific increase was identified in microRNA-29c (miR-29c) expression, corresponding with both the experience of sustained stress via self-reports, and alterations in vmPFC functional connectivity. Explicitly, miR-29c expression levels corresponded with both increased connectivity of the vmPFC with the anterior insula (aIns), and decreased connectivity of the vmPFC with the left dorso-lateral prefrontal cortex (dlPFC). Our findings further revealed that miR-29c mediates an indirect path linking enhanced vmPFC-aIns connectivity during stress with subsequent experiences of sustained stress. The correlative patterns of miR-29c expression and vmPFC FC, along with the mediating effects on subjective stress sustainment and the presumed localization of miR-29c in astrocytes, together point to an intriguing assumption; miR-29c may serve as a biomarker in the blood for stress-induced functional neural alterations reflecting regulatory processes. Such a multi-level model may hold the key for future personalized intervention in stress psychopathology. When confronted with stress a cascade of psychophysiological responses are set in motion, while adaptive coping and post-stress recovery largely depends on optimal neuronal functioning, requiring precise and rapid gene expression changes. This may be achieved through post-transcriptional regulation via microRNAs (miRNAs) , with eaSeveral studies reported miRNA expression changes in the brain upon exposure to stress –7, also Brain-imaging studies implicate the medial prefrontal cortex as a core area for processing of emotional events , 21. ItsIn our study, we sampled PBMCs before and 3-hours after social-stress induction in the fMRI-scanner. Neural indications were obtained from a seed-based functional-connectivity (FC) analysis of the vmPFC. Behavioral indications were based on repeated subjective stress ratings. We hypothesized that variations in stress-responsive miRNAs, presenting variability in expression among participants, would correspond with processes of affect regulation manifested in the subjective reports and attributed to the vmPFC connections. Gaining such understanding could help reveal indicants of individual susceptibility to stress; providing a foundation for understanding the link between cellular responses and neural circuit functioning underlying neuropsychiatric symptoms.The study was approved by the Tel Aviv Sourasky Medical Center (TASMC) ethical committee. 61 healthy male IDF soldiers (age 19–22) from the same unit, amidst the same military course, and before operational deployment participated in the study. All participants had normal or corrected-to-normal vision and provided written informed consent, approved by the ethical committee and conformed to the Code of Ethics of the World Medical Association (Helsinki Declaration). Molecular data was acquired from 49 participants, for which sufficient RNA samples were obtained. 4 participants were excluded from the fMRI data analysis due to signal artifacts . Participants had no reported history of psychiatric or neurological disorders, no current use of psychoactive drugs, no family history of major psychiatric disorders, and no previous exposure to abuse during childhood and/or potentially traumatic events before entering the study.The protocol consisted of 4 phases: acclimation (15-min), 1st MRI scanning session (65-min), intermission (90-min) and a 2nd MRI session (60-min). During acclimation, participants were given a 15-min resting period, were introduced to the experimental procedure and their first blood sample for miRNA profiling was drawn. The second blood sample was obtained at the end of the experiment. MRI testing, in the 1st scanning session, included two 'rest' scans (5 min each) interspersed by a control task and an arithmetic social stress task. During 'rest' conditions participants were instructed to stare at a fixation point. The 2nd MRI session included a 'rest' scan and memory task.Stress was induced using a serial subtraction arithmetic task –35. PartThe psychological effect was evaluated through repeated self-reports of stress ; physiological effects were evaluated via continuous heart-rate measure and salivary cortisol (marked as S). Subjective stress and cortisol were sampled at 4 time points: after the first 'rest' scan [R1/S1], after the control task [R2/S2], after the stress task [R3/S3] and 20-min following a second 'rest' scan and an anatomical scan [R4/S4] .To evaluate traces of stress observed in the self-reports, we calculated changes in the ratings 20-min after stress induction ended (R4), as compared to the ratings obtained immediately after the stress task (R3). We speculated that a decrease in the reported post-stress experience indicated more effective emotion regulation processing. Thus, participants were divided into two groups—Salivary cortisol levels were assayed using Coat-A-Count radioimmunoassay , inter- and intra-assay coefficient of variation (CV) 14.4%, 8.9%, respectively. Inter-assay % CVs of less than 15 and intra-assay % CVs of less than 10 are considered to indicate assays with good and reliable performance.Electrocardiography (ECG) was recorded continuously via a BrainAmp ExG MRI-compatible system . The sampling rate was 5000 Hz. Bipolar Ag/AgCl electrodes were attached to the sides of the chest. Preprocessing and RR-interval analysis included gradient artifacts removal using FASTR algorithm , implemeBlood was drawn twice in EDTA-tubes: upon arrival at the MRI facility, following acclimation, and at the end of the second scanning session, approximately 3-hours after the stress task. The interval was based on the notion that gene expression takes a few hours to complete, along with previous evidence regarding rapid expression changes in PBMCs in response to real-life events , 17, 18.stress-induced change were selected according to two parameters: all Cts were required to be between 15 and 35, and a fold-change variance > 2 was needed between individuals. The analysis revealed 6 miRNAs that presented stress-induced change: hsa-let-7b, miR-342-3p, hsa-miR-20b, hsa-miR-345, hsa-miR-30c, hsa-miR-29c. Out of these, we further examined hsa-miR-29c, based on the previously found expression in the human prefrontal cortex and its suggested involvement in CNS disorders  contrast at p<0.05, small volume corrected cluster utilizing an anatomical mask of the mPFC that included both sides of the medial frontal, orbital and rectal gyri and the anterior cingulate cortex, defined from the WFU Pickatlas toolbox for SPM . Next, FMediation was tested in a standard three variables path model, using SPSS INDIRECT macro script , in whicblack line and circle, see also .The division into stress d-change . An ANOVOur further investigation in mice subjected to a social defeat stress revealed a 1.8 fold increase in expression of miR-29 family in the PFC of stressed mice compared to controls see .A positive correlation .Our results indicate that increased miR-29c expression is linked with both enhanced vmPFC-aIns FC during stress  and subjOur study indicates the feasibility of detecting specific miRNA changes in PBMCs shortly following stress induction in healthy humans, further assessing their relationship with neuronal and psychological indices. Together our findings point to a potential neuro-molecular marker for the subjective experience of stress.stress-induced changes varying among participants, we focused on miR-29c, previously shown to be expressed in the human prefrontal cortex and involved in psychopathologies such as schizophrenia and bipolar disorder [Of the 6 miRNAs that presented disorder , and bradisorder , Huntingdisorder  and Parkdisorder  diseasesdisorder , and as disorder . We presdisorder , 49, seeWe further present the novel relationship between the epigenetic level of response and the subjective experience of stress; enhanced miR-29c expression corresponded with sustained stress feelings . The finThe importance of microRNA families lay in their common sequence or structure, which promotes similar functionality . MeasuriFollowing the finding of correlation between the miR29c and the enduring of perceived stress we speculated that stress sustainment may have resulted from a failure in spontaneous regulation and thereby the molecular-behavioral association was driven by changes in relevant neural circuits. Indeed, we found that fold changes in miR-29c were greater on the one hand with increased vmPFC-aIns connectivity and on the other with decreased vmPFC-dlPFC connectivity during stress induction. This opposite pattern demonstrates a relationship between neural and epigenetic changes in humans which might correspond to stress regulation mechanism . The aInThe additional finding of decreased vmPFC-dlPFC FC related to increased miR-29c expression may reflect the neuro-epigenetic regulation of both the sustained emotional response and the parallel disturbance of cognitive processing. A similar reduction in vmPFC-dlPFC FC was previously shown in the context of enhanced cognitive load; argued to be related to increased need to allocate attentional/cognitive processes, possibly to eliminate potential emotional processing interference . Of partOur results provide a neuromolecular account for inter-individual differences in post-stress recovery. It is yet unclear how miR-29c mediates this brain-behavior relationship, though one possible mechanism is through the miR-29 family's abundant expression in astorcytes . AstrocyWith regard to the temporal effect of miR-29c, we assume that stress-induced expression changes may occur minutes after induction, thus impacting one’s subjective experience. Previous evidence indeed points to rapid turnover as a common property of neuronal miRNAs . SecondlOur combined results point to miR-29c as a mediator of psychological stress, prompted by enhancement of vmPFC-aIns FC, simultaneously linked to decreased vmPFC-dlPFC FC and the assumed diminishment of evaluative-cognitive capacity needed to cease the emotional response. These findings, along with the presumed localization of miR-29c in astrocytes, lead to an intriguing notion: miR-29c may serve as a biomarker in the blood for plastic stress-induced alterations in functionally-distinct neural circuits, reflecting affective-cognitive regulation processes. Identifying components of epigenetic control related to regulatory brain function may provide the foundation for a new era in stress research and the future development of individually tailored early treatments for anxiety-related pathologies.S1 Fig(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Salmonella, which have been widely used for tumor therapy. Salmonella have mainly been applied as gene-delivery vectors, antitumor immune activators and tumor cell death inducers. This study will not only evaluate the therapeutic efficacy of Salmonella for the treatment of tumor but will also elucidate the mechanisms underlying the antitumor activities mediated by Salmonella, which involve host immune responses and cellular molecular responses.Lack of specificity of the therapeutic agent is a primary limitation in the treatment of a tumor. The use of preferentially replicating bacteria as therapeutic agents is an innovative approach to tumor treatment. This is based on the observation that certain obligate or facultative anaerobic bacteria are capable of multiplying selectively in tumors and inhibiting their growth. Bacteria have been employed as antitumor agents that are capable of preferentially amplifying within tumors and inhibiting their growth. Moreover, bacteria-derived factors have an immune-stimulation effect. Therefore, bacteria are able to transfer therapeutic genes into the tumor cells using their infective ability. Herein, we introduce the application of bacteria for tumor therapy and focus on  Salmonella are gram-negative, facultative anaerobes that are a common cause of intestinal infections. Due to substantial immunostimulation produced by Salmonella lipopolysaccharide (LPS) and other components, systemic infection with Salmonella induces proinflammatory cytokine expression and immune cell infiltration in the host [Salmonella is preferentially accumulated within tumors for at least one month, forming tumor-to-normal-tissue ratios exceeding 1000–10,000 to 1 [Salmonella can grow under both aerobic and anaerobic conditions, so they are able to colonize small metastatic tumors as well as larger tumors [Salmonella hinder tumor growth in a broad range of human and mouse tumors [Salmonella are good candidates for therapeutic antitumor agents, and genetically engineered Salmonella have been developed to express exogenous genes, aiming to enhance their antitumor effects [Recently, general tumor treatments including surgery, radiation therapy, and chemotherapy have shown some limitations. Surgery and radiation therapy are limited to localized tumors , and chethe host . Systemi000 to 1 . Further000 to 1 ,5. Salmor tumors ,7,8. Atte tumors ,10. Tumo effects ,5,11.Salmonella is composed of an immediate response mediated by the innate arm of the immune system followed by antigen-specific adaptive immunity [Salmonella-induced tumor inhibition occurs with alterations in myeloid cells consistent with maturation to macrophage effectors and a reduction in their suppressive capacity. The antitumor effectiveness of Salmonella relies on the induction of the innate immune response through the toll-like receptor-myeloid differentiation primary response gene (TLR-MYD88) signaling pathway [Salmonella induce cytokine production and antitumor activities via TLR4 signaling, which may help clarify the molecular mechanism of Salmonella-induced host antitumor responses [Salmonella treatment in vivo. IFN-dependent chemokines induced by Salmonella, such as monokine induced by IFN-γ (MIC) and IFN-inducible protein-10 (IP-10), are expected to recruit activated effector cells within the tumor. Taken together, we and other groups show that Salmonella significantly upregulates IFN-γ [The immune response against immunity . Togetheimmunity . Salmone pathway . This isesponses . The inces IFN-γ ,16,17 anes IFN-γ .Salmonella-induced immune responses, especially T cell activation by Salmonella, may include both anti-Salmonella-specific and tumor antigen-specific [et al. demonstrated that bacterial components, such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and flagellin, induced the expression of connexin 43 (Cx43) in tumor cells. Salmonella can cause melanoma cells to form gap junctions (Cx43) with adjunct immune dendritic cells. Consequently, the dendritic cells use peptides transferred from the tumor cells to stimulate T cells to recognize and kill the tumor cells at the primary site and prevent metastasis formation [Salmonella act both locally, by recruiting T cells that inhibit tumor growth, and systemically, where Salmonella promote development of the immune response via the cross-presentation of tumor antigen [Salmonella replication and lysis of tumor cells may induce cell-mediated immune responses to tumor cells by increased infiltration of CD8+ T cells in Salmonella-treated tumors [Salmonella-expressing cytokines, exhibiting an ability to modulate host immunity and inhibit tumor growth [et al., reported on tumor-specific antigen delivery into tumors using Salmonella to increase antigen levels and generate a proinflammatory tumor microenvironment [Salmonella is a powerful therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8+ T cells. We used wild-type, CD4+ T cell-deficient and CD8+ T cell-deficient mice to study the role of T cells in the antitumor immune responses induced by Salmonella. When systemically administered into mice bearing tumors, Salmonella significantly inhibited tumor growth by 50%. In contrast, in CD4+ T cell-deficient mice, there was only a 34%–42% inhibition of tumor growth [Salmonella-specific T cells that are induced during the infection are recruited in the flamed tumor site where they kill infected tumor cells [Salmonella actively infects tumor cells in the presence of an expanded pool of anti-Salmonella-specific immune responses. During the clearances of Salmonella-killed tumor cells, tumor debris could be generated in large quantities, be taken up by antigen-presenting cells including macrophage and dendritic cell, and presented to naïve T cells for stimulation of tumor-specific T cells. These results suggest that tumor-targeted therapy using Salmonella, which exerts tumoricidal effects and stimulates T cell activities, represents a potential strategy for the treatment of tumors  in humans with melanoma was reported. No antitumor responses were observed and VNP20009 was detected in tumor sites in only 12.5% of patients [Salmonella within the solid tumor. Salmonella have great potential as immune stimulants. However, following Salmonella administration, strong host immunity may develop. In addition, antibodies directed against Salmonella are also synthesized. The anti-Salmonella immune responses that exist in the host influence the tumor-targeting potential of Salmonella after systemic administration [Salmonella within the tumor contributes to the inhibition of the Salmonella-mediated antitumor response. These results may explain the limited accumulation of Salmonella within tumor sites and the non-significant antitumor response after systemic administration in clinical trials [B cells play an important role in the antitumor activity mediated by y organs . A majory organs . The natpatients . This clstration . The losl trials .Salmonella have been reported to possess antitumor activities. The mechanisms underlying their direct antitumor activities are largely unknown. Previous studies demonstrated that the induction of tumor apoptosis was correlated with Salmonella accumulation in the tumor sites [Salmonella in the tumor induced apoptosis by multiple mechanisms, including competition for nutrients and stimulation of immune response [Salmonella-induced cell death exhibit heterogeneous morphological features [Salmonella-induced killing of cells [Salmonella may induce cell death via apoptosis and autophagic pathways by using an autophagy inhibitor (3-methyladenine) and an apoptosis inhibitor (Z-VAD-FMK). Autophagy is involved in the cell-defense elimination of bacteria [Salmonella-treated tumor cells [Salmonella can induce autophagic activities in addition to caspase-dependent cell death in tumor cells  signaling pathways [Salmonella presumably enhance the response to chemotherapy agents by increasing the passage of these drugs between neighboring tumor cells. The combination of Salmonella with 5-fluorouracil (5-FU) improved the antitumor efficacy compared with 5-FU monotherapy in pancreatic stem-like cells [Salmonella [Salmonella and the pleiotropic activities of chemotherapeutic agents, Salmonella in combination with chemotherapy appears to hold promise for the treatment of solid tumors.Avoiding side effects has emerged as a key issue in the development of safe ibiotics . Recentllmonella . These rg tumors . Our resing mice . Recentlpathways . Salmoneke cells . ChemothSalmonella immune responses that exist in the host influence the tumor-targeting potential of Salmonella after systemic administration. The loss of accumulation of Salmonella within the tumor contributes to the inhibition of the Salmonella-mediated antitumor response. The bacterial cell wall is negatively charged due to the presence of either teichoic acid in gram-positive bacteria or LPS in gram-negative bacteria. Positively charged polyelectrolytes can bond to negatively charged Salmonella surfaces to form thin films that circumvent preexisting immunity against Salmonella [Salmonella in polymer reduces the antigenicity of Salmonella following in vivo delivery. The polymer-modified Salmonella bacteria not only resist the binding of neutralizing antibodies in peripheral blood but also replicate in tumor sites, which is believed to stimulate non-specific antitumor immunity. In other words, polymer shields Salmonella from neutralizing antibodies. Polymer-modified Salmonella also display lower toxicity and improved efficacy and safety. Mice treated with Salmonella had a 9% lower average body weight compared with mice treated with PBS. In contrast, the body weights of mice treated with polymer-modified Salmonella were not significantly decreased. After Salmonella and polymer-modified Salmonella treatment, the levels of inflammatory cytokines including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured in the sera. The induction of inflammation cytokines  in mice treated with Salmonella was increased by 1.5–4.5-fold compared with induction by polymer-modified Salmonella treatment [Salmonella, perhaps allowing other chemotherapeutic drugs to bind to tumor-targeting Salmonella. Recently, the masking of Salmonella with a polymer and DNA resulted in the tumor-targeting gene transfer of Salmonella following in vivo delivery [Salmonella carrying the therapeutic gene, which exerts antitumor activities, represents a promising strategy for the treatment of tumors.The anti-lmonella . The encreatment . Polymerdelivery . These rSalmonella for tumor treatment. However, we believe that no powerful therapeutic agents are available to eradicate various types of tumor. Salmonella is a useful antitumor agent due to its tumor-targeting potential, antitumor capability, and ability to deliver therapeutic genes. Thus, Salmonella-mediated tumor therapy may have an important role in tumor treatment. These results will contribute to new research that gives thought to the complex interactions between bacteria and host immunity in order to maximize the chances of therapeutic success.In this study, we have described the utility of


MYCN is a transcription factor that plays key roles in both normal development and cancer. In neuroblastoma, MYCN acts as a major oncogenic driver through pleiotropic effects regulated by multiple protein encoding genes as well as microRNAs (miRNAs). MYCN activity is tightly controlled at the level of transcription and protein stability through various mechanisms. Like most genes, MYCN is further controlled by miRNAs, but the full complement of all miRNAs implicated in this process has not been determined through an unbiased approach. To elucidate the role of miRNAs in regulation of MYCN, we thus explored the MYCN-miRNA interactome to establish miRNAs controlling MYCN expression levels. We combined results from an unbiased and genome-wide high-throughput miRNA target reporter screen with miRNA and mRNA expression data from patients and a murine neuroblastoma progression model. We identified 29 miRNAs targeting MYCN, of which 12 miRNAs are inversely correlated with MYCN expression or activity in neuroblastoma tumor tissue. The majority of MYCN-targeting miRNAs in neuroblastoma showed a decrease in expression during murine MYCN-driven neuroblastoma tumor development. Therefore, we provide evidence that MYCN-targeting miRNAs are preferentially downregulated in MYCN-driven neuroblastoma, suggesting that MYCN negatively controls the expression of these miRNAs, to safeguard its expression. MYCN acts as a major driver oncogene in neuroblastoma, a pediatric tumor of the sympathetic nervous system. Genomic amplification of this gene occurs in about half of all high-stage neuroblastoma tumors and has been associated with poor survival. As such, MYCN amplification has been used as one of the first biomarkers for therapeutic decision-making.MYC proto-oncogenes  are transcription factors that play key roles in both normal development and cancer. The MYC proteins act as direct amplifiers of transcriptionally active genes through sequence-specific binding to consensus E-box DNA binding sites -3. In adNumerous signal transduction pathways and regulatory mechanisms keep the expression of MYC family members under tight control. This regulation occurs at multiple levels, including gene transcription, messenger RNA (mRNA) turnover, and protein activity and stability -3. miRNAin vitro [In previous studies, individual miRNAs targeting MYCN have been identified, using miRNA target reporter assays to confirm predicted miRNA target sites in the 3′ untranslated region (3′UTR) of MYCN . Eleven in vitro . AlthougHere, we performed a comprehensive, genome-wide exploration of the miRNA-MYCN interactome in neuroblastoma. We combined results from an unbiased and genome-wide high-throughput miRNA target reporter screen with miRNA and mRNA expression data from patients and identified 12 MYCN-targeting miRNAs in neuroblastoma tumor tissue. Subsequently, the dynamic regulation of MYCN-targeting miRNAs during neuroblastoma development was evaluated in a murine neuroblastoma progression model. We provide evidence that MYCN targeting miRNAs are preferentially downregulated in MYCN-driven neuroblastoma tumors, suggesting that MYCN negatively controls the expression of these miRNAs, and as such safeguards its own expression. Hence, our findings further clarify the role of miRNAs in the regulation of MYCN in neuroblastoma and describe a negative feedback loop from MYCN to its targeting miRNAs.Potential interactions of 470 miRNAs with the 3′UTR of MYCN were assayed in a high-throughput luciferase reporter screen. In brief, human embryonic kidney cells (HEK293T) were co-transfected with a reporter construct, containing the MYCN 3′UTR downstream of a luciferase reporter gene, and each of the individual miRNA mimics from a 470 miRNA mimic library. Based on the relative luciferase activities in two independent screens , an inteApplying this strategy, we identified 29 miRNAs with a high probability of targeting MYCN  . In brieIn summary, 12 of 29 MYCN-targeting miRNAs were inversely correlated to either MYCN expression or MYCN activity in MYCN non-amplified tumor samples, and therefore likely regulate MYCN in neuroblastoma Fig , Fig. 3.In a next step, we explored how expression levels of miRNAs that target MYCN are dynamically regulated during MYCN-driven tumor formation. To this end, we performed miRNA expression profiling of the TH-MYCN mouse model, a murine MYCN-driven neuroblastoma progression model . In brieThe majority of the MYCN-targeting miRNAs in neuroblastoma (10 out of 12) showed a significantly decreased expression during tumor development in TH-MYCN+/+ mice compared to normal development, while 2 of them showed no significantly different expression pattern Fig. , Fig. 4BMYCN;Dbh-iCre mouse model [MYCN;Dbh-iCre tumors compared to wild-type adrenals , three miR-29 family miRNAs  and miR-34a-5p.In a next step, we investigated the dynamic regulation of these miRNAs during the process of MYCN-driven tumor formation, using the extensively validated TH-MYCN mouse model. Notably, we observe that MYCN-targeting miRNAs are preferentially downregulated during MYCN-driven tumor development, suggesting that MYCN negatively regulates the miRNAs by which it is targeted, to safeguard its own expression. Growing evidence suggests that MYCN predominantly acts repressively on the overall miRNA composition ,21-24 anin vivo MYCN model systems showed that activation of MYCN is associated to increased expression of miR-19a-3p, miR-19b-3p and miR-494-3p. Indeed, miR-19a-3p and miR-19b-3p, members of the oncogenic miR-17-92 cluster, have been shown to be direct transcriptional targets of MYCN in neuroblastoma cells [ESR1 [in vitro SHEP-MYCN-ER model system, the upstream region of miR-34c-5p does not contain E-box sequences (data not shown), and miR-449a, residing in the second intron of CDC20B, was not identified by Shohet and colleagues [MYCN and miR-34c-5p expression in MYCN non-amplified versus amplified environment should not be surprising: activation of miR-34c-5p, either direct or indirect, could be one of the mechanisms through which normal physiological levels of MYCN affect apoptosis. In cases where MYCN exceeds its physiological levels and oncogene-induced apoptosis is triggered, a transforming cell will need to circumvent MYCN-mediated apoptosis, for instance via downregulation of miR-34c-5p, which seems to occur in TH-MYCN and LSL-MYCN;Dbh-iCre tumors.The integration of the screen results with miRNA expression data from MYCN non-amplified primary neuroblastoma tumors further identified 5 MYCN-targeting miRNAs with positive correlation to MYCN expression or activity. Subsequent analysis of these miRNAs in ma cells , and thels [ESR1 , a liganls [ESR1 , a markelleagues  in theirOf further notice, 17 out of 29 identified MYCN-targeting miRNAs were considered not to be relevant for MYCN regulation in the context of neuroblastoma, due to lack of inverse correlation to MYCN expression or activity, or due to unavailable expression data. Given the tissue specific nature of miRNA function, it is not excluded that these miRNAs regulate MYCN in an other cellular context, such as that of normal brain development  and a nuMYCN expression levels or activity in MYCN non-amplified neuroblastoma  cloned downstream of the Firefly luciferase gene  and 20 ng of pRL-TK control vector containing the Renilla luciferase gene (Promega) together with a library of 470 miRNA mimics (2.5 pmol) . Lipid based transfections were performed using 0.4 μl Dharmafect Duo reagent (Dharmacon). Forty-eight hours post-transfection, luciferase reporter gene activities were assayed using the Dual-Luciferase Reporter assay system (Promega) according to the manufacturer's protocol with minor changes (LARII and Stop & Glo buffer volumes were reduced to 50 μl). Firefly reporter gene activities were normalized to Renilla values and then log-transformed. Subsequently, robust z-scores were calculated and median centered to the distribution of robust z-scores from 36 analogous screens (of 17 selected cancer and disease associated genes) on a per miRNA basis in order to remove potential systematic bias. The resulting interaction scores are thus more negative for miRNAs that interact with the 3′UTR. In order to determine the interaction score cutoff that separates interactions from non-interactions, the scores for a set of validated miRNA interactions, probed in the MYCN screen or any of the analogous screens, were used together with the scores for a set of negative control interactions from an empty-3′UTR vector miRNA library screen to perform ROC-curve analysis and determine the point of highest accuracy . MYCN 3′UTR-miRNA library screen results were replicated in two independent experiments. For a more detailed description of the 3′UTR-miRNA library screen setup and data-analysis we refer to Van Peer et al. (in preparation).Potential miRNA target sites in the MYCN 3′UTR were identified as reported previously . In addihttp://www.ncbi.nlm.nih.gov/geo; Accession Number: GSE32664). Correlation of miRNA expression levels and MYCN mRNA levels was evaluated with Spearman's rank correlation coefficient. In order to find miRNAs that show negative correlation to MYCN activity and hence MYCN protein levels, mRNAs were ranked according to decreasing Spearman's rank correlation coefficient for each miRNA. Ranked gene lists were used for Gene Set Enrichment Analysis (GSEA) [MYC or MYCN in their name, the six gene sets that contain MYC(N) upregulated genes were used for this analysis: COLLER MYC TARGETS UP [160 MYCN non-amplified primary tumor samples of neuroblastoma patients were collected prior to therapy at the Ghent University Hospital , the University Children's Hospital Essen , the Hospital Clínico Universitario , the Academic Medical Center  and the National Children's Research Centre . Informed consent was obtained from the patients' relatives. mRNA data from 75 primary neuroblastoma tumors is available at the Gene Expression Omnibus (s (GSEA)  with selRGETS UP , DANG MYRGETS UP , DANG RERGETS UP , KIM MYCRGETS UP , LEE LIVRGETS UP , SCHUHMARGETS UP . Gene seIn this study we used data obtained with three different analytical miRNA platforms. Platform designs are based on different releases of the miRBase database for miRNA annotation, resulting in non-overlapping annotation of data sets. To allow for correct integration of miRNA data, we used miRBase Tracker, an in-house developed web tool for miRNA reannotation  and applThe MYCN-inducible SHEP-MYCN-ER cell model  was kindhttp://www.biogazelle.com/qbaseplus) [RT-qPCR reactions were performed and reported according to the MIQE guidelines . For quaaseplus) .q values lower than 32 were retained and normalized using global mean normalization, as previously described [TH-MYCN+/+ mice  were sacescribed . FurtherMYCN;Dbh-iCre mouse model is described elsewhere [The LSL-lsewhere  along wiAll statistical analyses were performed using R Bioconductor software (version 3.0.2). If not further specified in the results section, statistical significance was defined as p-value < 0.05 for all statistical tests.http://www.ncbi.nlm.nih.gov/geo) under accession number GSE32664. miRNA expression data from the TH-MYCN neuroblastoma progression model are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2618. Furthermore, the processed data can be visualized via the R2 microarray analysis and visualization platform (http://r2.amc.nl) under experiment “Exp Nb Hyperplasia TH-MYCN - Ghent - 24 - custom - mirbase19mm2”.mRNA data from 75 primary neuroblastoma tumors are available at the Gene Expression Omnibus (


Robust evidence indicates that these metagenes are: 1) positively correlated with distant metastasis-free survival (DMFS) of patients, 2) comprised of genes that regulate immune cell-specific biology, and 3) reflective of the relative abundance of discernible populations of tumor infiltrating leukocytes. In recent work we have leveraged the statistical associations between the immune metagenes and the DMFS of breast cancer patients to explore the underlying phenotypes that differ in their ability to potentiate long-term, immune-mediated tumor rejection. Using a tumor classification model that combines the prognostic attributes of three distinct immune metagenes, termed the B/P, T/NK and M/D metagenes, we have identified molecular subtypes of breast cancer that either permit or prohibit prognostication by the immune metagenes. On this basis, we have delineated the phenotypic attributes of breast cancer that distinguish two novel immunogenic tumor subtypes, which we have defined as: immune benefit-enabled (IBE) and immune benefit-disabled (IBD). Phenotypically, IBE tumors comprise of Basal-like tumors and highly-proliferative HER2-Enriched and Luminal-B subtypes, while IBD tumors comprise of Claudin-Low, Luminal-A, and low-proliferative HER2-Enriched and Luminal-B tumors. Prognostically, IBE tumors (n = 666) can be stratified by the immune metagene model into prognostic subgroups with high statistical significance , while IBD tumors cannot  consistent with the capacity for an innate anti-tumor immunity against IBE tumors, but not IBD tumors, that guards against distant metastasis. Furthermore, these observations were independent of adjuvant treatment, and may owe to differential activation of immunomodulatory pathways. Network analysis revealed that IBE/IBD differentially-expressed genes (q<0.01) underlie highly-significant pathway activation scores for TGF-beta signaling in IBD (p < 0.0001), and Interferon-gamma signaling in IBE (p < 0.0001). Furthermore, 15 of 19 genes comprising the previously described Immunologic Constant of Rejection (Marincola and colleagues) were significantly overexpressed in IBE tumors . Thus, we conclude that breast tumors can be dichotomized into two subtypes fundamentally distinct with respect to their potential for metastasis-protective immune responsiveness. These findings indicate new contexts for studying anti-tumor immunity and oncogenic mechanisms of immunosuppression in breast cancer. Whether IBE and IBD subtypes represent clinically-relevant contexts for assessing patient prognosis or evaluating the efficacy of immunotherapeutic treatments warrants further investigation (See Figure The abundance and functional orientation of tumor-infiltrating effector cells has long been observed to predict for reduced incidence of clinical metastasis and cancer-specific death. Using bioinformatics to mine large breast tumor microarray datasets, we and others have identified prognostic immune gene signatures, or


Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional  The latter can be further sub-divided into the BH3-only  and the multi-BH domain members .2 Pro-survival BCL-2 family members protect cells from dying through binding and neutralizing the pro-apoptotic BCL-2 family members. Upon an apoptotic stimulus, the levels of certain BH3-only proteins increase as a result of transcriptional and/or post-transcriptional upregulation. These BH3-only proteins activate the multi-domain members BAX and BAK either directly, or indirectly through neutralizing the pro-survival BCL-2 family members.4 Activated BAX and BAK oligomerize and form pores into the outer mitochondrial membrane, leading to the release of apoptogenic factors, such as cytochrome c, provoking the activation of the so-called caspase cascade with subsequent demolition of the cell.3The mitochondrial  apoptotic pathway is regulated by members of the BCL-2 family.5 During B- and T-cell development, BIM activity is required to eliminate autoreactive lymphocytes.7 Constitutive loss of BIM  leads to the accumulation of lymphocytes that infiltrate non-hematopoietic organs, such as lungs, kidneys, liver and salivary glands, produce autoantibodies and on a mixed C57BL/6x129SV background this causes severe autoimmune disease resembling systemic lupus erythematosus.5 BIM is also an important factor in peripheral T-cell apoptosis during the shutdown of an immune response.9 BIM is a tumor suppressor in mantle cell lymphoma, where the gene is lost,10 as well as Burkitt's lymphoma and renal carcinoma in which the gene is silenced.12 Loss of BIM renders cells resistant to several pro-apoptotic stimuli, such as withdrawal of growth factors, treatment with calcium ionophores5 or ER stress.13BIM is a critical initiator of the mitochondrial apoptotic pathway, particularly in hematopoietic cells.Bim gene was constitutively inactivated in all cell types.5 The complete absence of a gene product during the entire life of an animal can lead to phenotypes that are not cell-autonomous and may also lead to compensatory events . Therefore techniques have been developed so that a gene of interest can be conditionally inactivated in a tissue-specific and/or temporally controllable manner using the Cre/Lox system.14 A further refinement of this technique consisted in engineering an inducible Cre recombinase by fusing it with a modified hormone-binding domain of the estrogen receptor (CreER).15 In the absence of 4-hydroxytamoxifen (4-OHT), the CreER protein is sequestered in an inactive state in the cytosol. Upon administration of 4-OHT, the ER domain of the CreER fusion protein changes conformation, prompting the translocation of the fusion protein into the nucleus where the recombinase can delete DNA sequences flanked by loxP sites.15This information about BIM's physiological function was derived from the study of a mouse strain in which the Bim allele allowing for temporally and spatially controllable deletion of this critical apoptosis initiator. In addition, we developed a novel strain, which expresses the CreER fusion protein under the control of the pan-hematopoietic Vav promoter.16 We show that the deletion of Bim in the adult mouse through activation of the CreER recombinase, using the Vav-CreER or the ubiquitously expressed Rosa-CreER transgene, caused hematopoietic abnormalities that were similar to those found in the constitutive Bim knockout animals. These novel strains  will allow further detailed genetic investigations of cell death and tumourigenesis.We developed a conditional 5 Mice constitutively deficient for BIM show many abnormalities, including increased white blood cell (WBC) counts, splenomegaly and defects in thymic T-cell selection.6 As these defects could potentially be affected by the absence of BIM (i) in non-hematopoietic cells or (ii) be dependent on absence of BIM during embryonic development, we decided to generate mice with a conditional Bim allele (Bimfl), to allow specific deletion of Bim in a time- and/or tissue-specific manner. Coding exons 2, 3 and 4 of Bim17 were flanked by loxP sites.18 As expected, BIM protein expression and hematopoietic cell composition, WBC counts, spleen weights and thymic cell subset distribution, were comparable between Bimfl/fl and wt mice   that deletion of the floxed Bim allele recapitulates the phenotype observed in the constitutive Bim knockout mice.The BH3-only protein BIM is the most critical initiator of apoptosis in hematopoietic cell development and homeostasis. wt mice . Crossin embryo)  and the  embryo)  and sple embryo) , as well embryo) . These rBim allele is the possibility to delete the gene in a temporally and cell type-controllable manner. In order to delete Bim at a predetermined time specifically in hematopoietic cells of adult mice, we generated a new transgenic mouse model, in which the tamoxifen-inducible CreERT2 recombinase20 is expressed under the control of the pan-hematopoietic Vav promoter (Vav-CreER).16 To induce Bim deletion, 12–20-week-old Bimfl/fl/Vav-CreER-tg mice were administered 4-OHT by oral gavage. Four weeks after the treatment, lymph nodes, spleens and thymi were collected from 4-OHT-treated Bimfl/fl, Vav-CreER, Bimfl/fl/Vav-CreER as well as Bim−/− mice, and BIM protein levels were measured by intracellular FACS analysis  alleles or CreER (Vav-CreER) transgene had no impact on BIM protein expression when CreERT2 was not activated. This indicated that both Bimfl alleles had been successfully recombined in Bimfl/fl/Vav-CreER hematopoietic cells of tamoxifen-treated mice, explaining why the BIM protein was no longer present in these cells. To further validate the efficiency of the Vav-CreER strain, we crossed this strain to mice harboring a floxed allele of Mcl-1 (Mcl1fl) and treated Mcl-1fl/+/Vav-CreER-tg animals with three doses of 4-OHT. As the deletion of the floxed Mcl-1 allele leads to the expression of the hCD4 reporter,22 we analyzed the blood of Mcl-1fl/+/Vav-CreER-tg two days post treatment by flow cytometry for hCD4 expression  of 4-OHT-treated Bimfl/fl/Rosa-CreER-tg mice. As expected, BIM protein was almost undetectable in these organs, whereas treatment of Rosa-CreER-tg mice with 4-OHT had no impact on BIM protein levels .These results demonstrate that recombination of the floxed 5 we analyzed the blood from Bimfl/fl/Vav-CreER-tg and Bimfl/fl/Rosa-CreER-tg mice 3–4 weeks after 4-OHT treatment  and single-positive  thymocytes and reduced proportions of double-positive (CD4+CD8+) thymocytes compared with control (wt) mice  appears to occur at similar rates in both strains.As loss of BIM in the entire mouse leads to increased WBC numbers,reatment . As antit levels . The larwt) mice . This in5 We therefore compared the response of thymocytes from Bim−/−, Bimfl/fl/Vav-CreER-tg, Bimfl/fl/Rosa-CreER-tg, Bimfl/fl, Vav-CreER and Rosa-CreER mice, which had been treated with 4-OHT four weeks prior to organ isolation, to diverse cytotoxic stimuli. Thymocytes of the different genotypes were cultured in medium  or treated with BIM-dependent (Ionomycin=Iono) and BIM-independent (phorbol ester=PMA) apoptotic stimuli (24), untreated (medium) and Ionomycin-treated thymocytes from 4-OHT-treated Bim−/−, Bimfl/fl/Vav-CreER-tg and Bimfl/fl/Rosa-CreER-tg mice all showed a marked survival advantage when compared with thymocytes from 4-OHT-treated Bimfl/fl, Rosa-CreER-tg and Vav-CreER-tg mice. This demonstrates that constitutive or induced deletion of Bim in thymocytes leads to a similar resistance to these apoptotic stimuli, and that the observed change in thymocyte distribution is a consequence of the loss of BIM and not due to other abnormalities caused by Cre-mediated recombination.BIM-deficient thymocytes are resistant to a variety of pro-apoptotic stimuli. stimuli . Althougfloxed Bim allele, which allows the deletion of this pro-apoptotic BH3-only protein in a cell type-restricted and temporally controllable manner. Using the well-established Rosa-CreER and newly developed Vav-CreER transgenic strains, deletion of Bim in the adult mice results in the same phenotype as that observed in the constitutive Bim knockout mice. It thus appears that the hematopoietic phenotype associated with the constitutive loss of BIM in all cell types reported previously5 is intrinsic to the hematopoietic system rather than a consequence of an unrecognized developmental defect due to the absence of BIM.We report here the development of a new Bim allele is functional and can be efficiently deleted by CreER recombinases. Importantly, these Bimfl/fl mice have recently been used in two other studies,25 in which Bim was specifically deleted in the regulatory T-cell lineage (Treg) only. This new mouse strain will thus be a valuable tool to further dissect the role of BIM in various cell types.Our studies clearly show that the floxed Vav-CreER transgenic strain showed the same deletion efficiency of the floxed Bim alleles 4 weeks after 4-OHT treatment as the very well-characterised and widely used Rosa-CreER strain.23 However, when we tested the loss of BIM protein 5 days after 4-OHT treatment, we observed residual BIM protein in the lymphoid cells from the Bimfl/fl/Vav-CreER mice, whereas it was almost completely absent in the cells from the Bimfl/fl/Rosa-CreER animals at this time point (data not shown). This indicates that deletion of Bimfl alleles occurs more rapidly and probably also more efficiently in the Bimfl/fl/Rosa-CreER strain than in the Bimfl/fl/Vav-CreER strain, possibly because of the lower expression of CreER in the Vav-CreER strain.Importantly, our new Rosa-CreER strain is accompanied by a higher toxicity observed in the animals upon treatment with 4-OHT. Accordingly, treatment of Rosa-CreER or Vav-CreER transgenic mice with 4-OHT for 5 consecutive days led to substantial destruction of lymphoid organs in the former, whereas these organs remained unaffected in the latter . This was never observed in Bimfl/fl/Vav-CreER-tg mice. Note that only mice with no evidence of Bimfl recombination prior to tamoxifen administration were used in this study. Pertinently, we observed no abnormalities in splenic weights and thymocyte sub-population distribution in Bimfl/fl/Vav-CreER-tg and Bimfl/fl/Rosa-CreER-tg animals prior to 4-OHT treatment (data not shown). We therefore conclude that the occasional leakiness of CreER recombinase activity in the Rosa-CreER-tg mice must be limited to embryonic development and that despite this issue , both this inducible CreER mouse strain and the Vav-CreER-tg strain are suitable tools for the inducible deletion of floxed genes.A significant leakage in CreER recombinase activity was observed in the Bimfl, Mcl-1fl and the RosaCreER mice, all on a C57BL/6 background, has been described previously.26Vav-CreER transgenic mice were generated by replacing the hCD4 sequence of the Vav hematopoietic vector27 with the CreERT220 sequence using Sfi1/Not1 restriction sites. The Vav-CreER construct was linearized with HindIII before pro-nuclear injection of the DNA into zygotes derived from C57BL/6 mice. Positive offspring were identified by PCR for the genomic integration of the Vav-CreER transgene.Experiments with mice were conducted according to the guidelines of The Walter and Eliza Hall Institute Animal Ethics Committee. The generation of the conditional 28To activate the latent CreER recombinase, mice were given 200 mg/kg tamoxifen  in peanut oil/10% ethanol each day for 5 days by oral gavage.2, 1 mM EGTA, 1% Triton X-100, 10% glycerol). Antibodies used include: rat anti-BIM , mouse anti-β-actin . Horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rat IgG antibodies  served as secondary reagents and the enhanced chemoluminescence  system was used for detection. WBC analysis was performed with an ADVIA blood analyzer .Cell extracts for western blot analysis were prepared in ONYX lysis buffer  from lymph nodes, spleen and thymus were isolated from mice of the indicated genotypes, fixed and permeabilized by suspension in 100 μl of BD Cytofix/Cytoperm  solution for 20 min on ice. Cells were then washed twice in Perm/Wash buffer (BD Biosciences) and stained with Alexa-647-conjugated BIM antibody . Analysis was performed in a FACSalibur (BD Biosciences).Cells . P-values <0.05 were considered to indicate a statistically significant difference. n indicates the number of mice analyzed for each genotype.Statistical comparisons were made using a two-tailed Student's


Recent studies in melanoma mouse models revealed that the anti-tumor activity of CTLA-4 blockade is mediated by FcgRIV-expressing macrophages in the tumor microenvironment (TME) via in-trans depletion of tumor-infiltrating regulatory T cells (Tregs). We speculated that a similar mechanism might operate in melanoma patients responding to Ipilimumab. To investigate this hypothesis, we interrogated peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding (R) and 14 non-responding (NR) to Ipilimumab. Our findings show, for the first time, that Ipilimumab leads to the depletion in vitro of Tregs via an antibody-dependent-cellular-cytotoxicity (ADCC) mechanism, selectively mediated by FcgRIIIA (CD16)-expressing, nonclassical monocytes (CD14+CD16++). In contrast, classical CD14++CD16- monocytes, lacking the FcgRIIIA expression, are unable to deplete Tregs in an ADCC assay. Interestingly, patients responding to Ipilimumab displayed significantly higher baseline peripheral frequencies of nonclassical monocytes than nonresponder patients. Evaluation of matched melanoma metastases from pre- and post-Ipilimumab time-points by IHC revealed that, in the TME, responders had the highest CD68+/CD163+ macrophage ratios at baseline, and showed decreased infiltration of Tregs after treatment. Notably, baseline Treg infiltration was comparable between the two groups. Our findings provide novel mechanistic insight into the clinical activity of Ipilimumab, highlighting the contribution of host-dependent factors into the final outcome of antibody-based immune-modulatory therapies and identify nonclassical monocytes as a potential biomarker of response.Enhancing immune responses with immune-modulatory monoclonal antibodies (mAbs) directed to inhibitory immune-receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as CTLA-4 or PD-1/PD-L1. Treatment with Ipilimumab (YervoyWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


TAM;Bcl-xLMycER model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of TAM;Bcl-xLMycER tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional  Progression of normal cells into malignancies in humans is dependent upon acquisition of a number of functional cancer “hallmarks” including uncontrolled proliferation, suppressed cell death, increased invasion, angiogenesis, and reprogramming of biosynthetic metabolism . HoweverAPCmin-driven model of colon cancer loss of CTS L results in increased tumor multiplicity [in vitro angiogenesis assays [The human cysteine cathepsins are family of 11 proteases, all of which share a conserved active site . Increasiplicity . For exas assays  yet genes assays . Similars assays  but not s assays . Nonethes assays ). It is myc gene, c-Myc, is a highly pleiotropic transcription factor that regulates expression of many, diverse genes [myc oncogene is implicated in driving the relentless growth of many human cancers  via itsview see , 24, angview see , 26 and view see . Each ofgression , a capabpoptosis . Here weCysteine cathepsin proteases, particularly CTS B and L, are frequently upregulated in various types of human cancers . Moreovein vivo is associated with an increase in cysteine cathepsin L, and to determine whether, and at what stage(s) of disease progression, such CTS L might be functionally important. Activation of 4-OHT-dependent c-MycERTAM in the beta-cell compartment of pancreatic islets of the MycERTAM;Bcl-xL model of pancreatic neuroendocrine tumors (PNET) results in synchronous onset and progression of beta-cell proliferation, culminating in highly angiogenic and invasive tumors [in vivo [Deregulated activity of transcriptional factor Myc is implicated in a large number of cancers and often associated with more aggressive tumors. We sought to establish whether Myc-induced tumorigenesis in pancreatic endocrine tumors e tumors , 28. Fol[in vivo .MycERTAM;Bcl-xL islets following 2, 8, 24 hours and 21 days of Myc activation [cathepsin L mRNA expression among the four cysteine cathepsin genes expressed in the MycERTAM;Bcl-xL islets (CACGTG) element at position -458 of the cathepsin L promoter. We reason that the transient induction of CTS B, C and S reflects the Myc-induced recruitment of infiltrating myeloid cells [[Analysis of a beta-cell Myc target gene database obtained from laser-captured L islets . Such red cells [, 32 and d cells [.CTS L mRNA expression in the early stages of Myc-induced tumorigenesis correlates with increased cysteine cathepsin L enzymatic activity, we used DCG-04, a chemical activity-based probe (ABP) specific to the cysteine cathepsins [in vitro and in vivo. Myc was activated in MycERTAM;Bcl-xL mice for 3 or 7 days, corresponding to the early stages of MycERTAM;Bcl-xL PNET tumor progression [Bcl-xL animals to seven-day treatment with TAM , which was administered intravenously into MycERTAM;Bcl-xL mice treatment 7 days. We detected an increase in total cystein cathepsin activity in the hyperplastic islets following Myc activation in vivo  was administered to sections —Myc-ON. or model . MoreoveMycERTAM;Bcl-xL models appear to have similar properties [MycERTAM;Bcl-xL and RT2 models [MycERTAM;Bcl-xL PNET cancer cells are driven by distinct oncogenes, the SV40 antigen and activated c-Myc, respectively.Progression of c-Myc-induced PNET tumorigenesis is accompanied by the recruitment of a variety of inflammatory cells to the neoplastic site, which serve to support tumor growth, angiogenesis and invasion , 32. The2 models . HoweverMycERTAM;Bcl-xL PNET progression, we bred the MycERTAM;Bcl-xL animals into the cathepsin L-deficient background. The resulting MycERTAM;Bcl-xL;Cathepsin L KO  animals were phenotypically similar to CTSL KO littermates [TAM activation. To assess the effect of CTS L-deficiency on the early stages of Myc-induced beta-cell tumourigenesis, Myc;Bcl-xL;CTSLKO and Myc;Bcl-xL animals were subjected to treatment with TAM for the duration of three days. This treatment induced proliferation in the beta-cell compartment of pancreatic islets accompanied by tumor angiogenesis [in vivo administration of a broad-spectrum inhibitor of cysteine cathepsin activity—JPM-OEt [RT2 model is the cell-cell adhesion glycoprotein E-cadherin [MycERTAM;Bcl-xL model, E-cadherin downregulation by Myc correlates with the abrupt loss of intercellular contacts within the islet, macroscopic islet expansion, and local invasion of the pancreas [Myc;Bcl-xL pancreatic islets revealed rapid and sustained loss of cell surface E-cadherin staining within the pancreatic islets following three days of c-Myc activation, independent of cysteine cathepsin activity  animals following three days of Myc-activation revealed that while the Bcl-xL-negative Myc-expressing islets rapidly involute following Myc-activation as described in [MycERTAM;Bcl-xL;CTSLKO islets . While cathepsin L-deficiency had no profound effect on pancreatic tissue pathology in the absence of MycERTAM activation  of tumors collected from the MycERTAM;Bcl-xL mice. We observed no dosage-specific effect of cathepsin L deficiency, as CTSL heterozygous animals developed tumors indistinguishable in their histopathological features from the lesions arising from the CTSL WT background. Intriguingly, we detected a profound deficit in Myc-induced beta-cell proliferation in CTSL-deficient animals after 2-weeks of MycERTAM activation (0.19% of proliferating tumor cells in the CTSLKO mice compared to 21.6% in the CTSL WT lesions)   that wasLKO mice .MycERTAM;Bcl-xL and MycERTAM;Bcl-xL;CTSL-deficient animals subjected to 14 days of Myc-activation in vivo revealed a significant increase in the punctate pattern of LC3 expression in the CTSLKO tumors  in the cathepsin L-deficient background was significantly reduced by ~90% when compared to control tumors with WT CTS L [MycERTAM;Bcl-xL model, deficiency in cathepsin L activity also had a profound effect on late stages of disease progression, manifested by ~97% of growth inhibition and partial collapse of the developed lesions. We conclude that cathepsin L is required for tumor maintenance and progression, rather than tumor initiation, during neuroendocrine tumorigenesis involving Myc-driven proliferation.The distinction between tumor initiating lesions and factors required for tumor maintenance is an important issue in tumor biology. In the constitutive model of PNET progression—WT CTS L . In the Glut-1, induced over-expression of metabolic enzymes, and the use of alternative sources of energy such as glutamine [in vitro and in vivo [Highly proliferative tumor cells require a constant supply of nutrients to support growth, usually achieved by increased uptake of glucose and a high glycolytic rate. C-Myc orchestrates tumor metabolic adaptation by several mechanisms, including upregulation of the glucose transporter lutamine , 46. In  in vivo , 48. On  in vivo , 50. Alt in vivo , 52. Thu in vivo  or knock in vivo  leads toin vivo. We show that deletion of cathepsin L in Myc-driven pancreatic neoplasias results in both a reduction in autolysosomal formation and an increase in tumor cell death. Though our current data does not formally demonstrate a causal relationship between cathepsin L deletion and alteration of autophagy in Myc-driven lesions, or that the increase in tumor cell death detected in late stages of tumor progression in the MycERTAM;Bcl-xL;CTSL-deficient neoplasias is a direct consequence of altered autophagic flux in vivo, it is reasonable to suggest that these events are intertwined [To our knowledge, the work presented herein is the first report demonstrating a link between the c-Myc oncogene and the lysosomal protease cathepsin L in tumor cells ertwined . The exaTAM and constitutive Bcl-xL in their pancreatic β cells pIns-MycERTAM;RIP7-Bcl-xL  mice have been previously described and characterized [TAM was activated in beta-cells in situ by daily i.p. injection of Tamoxifen (TAM) (1mg/mouse/day) dissolved in peanut oil (Sigma). TAM is metabolized in vivo to 4-OHT and has an equivalent effect to 4-OHT when administered to MycERTAM;Bcl-xL mice. The cathepsin L deficient animals were previously reported [In vivo labeling and detection of cathepsin activity with BODIPY 530/550-DCG-04 was performed as described [in vivo, the animals were subjected to 50mg/kg/day injection with the inhibitor JPM-OEt or control vehicle (30% DMSO/70% PBS) in conjunction with TAM injections for the duration of 5 days as previously described [2.This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco, protocol number AN076148. The transgenic mice expressing switchable MycERcterized . MycERTA2O2 in tap water for 5 minutes. Avidin/Biotin blocking and blocking of endogenous mouse immunoglobulins in the tissue were performed respectively by Vector Avidin/Biotin blocking kit (SP-2001) and Vector MOM basic kit (Cat. No. BMK-2202), both were used according to the manufacture’s protocols. The primary antibody used: anti-LC3 5F10 mAb . Sections were incubated with ABC reagent VACTASTAIN Elite ABC Standard Kit (PK-6100) and developed with diacylbutyrate (DAB). DAB-stained slides were counterstained lightly with hematoxylin.Briefly, paraffin-embedded sections (10 μm) were deparaffinized and rehydrated in a series of graded alcohols and blocked for endogenous peroxidase activity. Antigen retrieval was performed in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 3% HFor the OCT-embedded tissues, 10 μm sections were fixed in 1% paraformaldehyde. The primary antibodies used: anti-mouse VEGF A , anti-Meca-32 , anti-Ki67 , anti-murine CD45 , rabbit polyclonal anti-LAMP1 , rabbit polyclonal anti-active-caspase 3 , and anti-cystein cathepsins antibodies—Cathepsin B (AF965), Cathepsin S (AF1183), Cathepsin C (AF1034), and Cathepsin L (AF1515) . All were applied in blocking buffer  for 2–16 hours. Secondary antibodies were from Dako and Molecular Probes. The cells were counterstained with DNA-binding dye—DAPI .Fluorescent images were obtained using Axiovert 100 inverted microscope (Zeiss) or Leica DM5500 microscope with cameras DFC360X (fluorescent detection) and DFC 295 (colorimetric detection). Confocal images were obtained using a LSM 700 (Zeiss). Immunohistochemical analysis for each antigen was performed at least in duplicate. The quantitative analyses of immmunostained sections were performed automatically by Fuji NIH software, with algorithms provided by the Bioimaging and Optics platform at EPFL.Endothelial cell proliferation was quantified in tissue sections by counting randomized fields of endothelial cells (Meca32+) and calculating the percentage of Ki67+ cells. At least three animals were assayed of each genotype all analyses done in duplicate; ten randomized fields per analysis were considered. Statistical significance was assessed using the Student T-test.http://rsb.info.nih.gov/ij) as follows. Ten serial H&E-stained sections of the entire pancreata from the tumor-bearing animals were scanned with a Leica DM5500 at 20x magnification. The tumors were manually outlined with Freehand Selection tool. The software then calculates the outlined area. At least 17 tumors for each genotype were analyzed. Each collected tumor was treated as an independent event. Analysis of BODIFY fluorophore mean intensity was performed with the Fiji Software package. The tumors were manually outlined with Freehand Selection tool. The software then calculates the mean intensity of the selected area. At least eight tumors for each treatment were analyzed.Analysis of size was performed with the Fiji Software package . Protein concentration was established by the Bradford assay. The equal amount of protein lysates were labeled with biotinylated DCG-04 ABP [For escribed  were homG-04 ABP  equally G-04 ABP .These procedures were described in .http://rsb.info.nih.gov/ij) according to algoriphm defined by Bioimaging & Optics platform (PT-BIOP) at EPFL.Data were presented as the means ± standard error of the mean (SEM). The statistical analysis was calculated using GraphPad Prism5 software package. The quantification of tumor proliferation and apoptosis was performed by the Fiji Software package , which increases following administration of 4-OHT (Myc-ON) at the timepoints indicated. Two independent islet purifications are presented for MycERTAM;Bcl-xL animals treated for 7 days. The activity bands corresponding to cysteine cathepsins (CTS) L, B and C are indicated. Molecular weight marker is indicated on the side panel. Note the lack of cathepsin L and cathepsin B activation in samples collected from control Bcl-xL background animals.Cathepsin activity profiles for the pancreatic islets from (TIF)Click here for additional data file.S2 FigMycERTAM;Bcl-xL animals were either untreated or treated daily with TAM for 5 days in conjunction with control vehicle (DMSO) or the broad spectrum cathepsin inhibitor JPM-OEt (JMP). At the times indicated, animals were sacrificed and pancreata isolated, sectioned and stained with anti-VEGF-A antibody (red) and Meca-32 (green). Images collected from two independent animals are presented. The panels are representatives of at least three animals assayed at each data point, immunohistochemical analyses done in duplicate; seven randomized fields per analysis were considered. Scale bars represent 100 μm. (B) Immunohistochemical analysis of E-cadherin in the pancreata collected from the animals described above. The areas positive for E-cadherin expression in inhibitor-treated mice are indicated by arrows. I-islet area is outlined by dotted line. The panels are representatives of at least three animals assayed at each data point, immunohistochemical analyses done in duplicate; five randomized fields per analysis were considered. Scale bars represent 20 μm.(A) Inhibition of cathepsin activity does not interfere with Myc-induced redistribution of VEGF-A in islets. (TIF)Click here for additional data file.S3 FigMycERTAM;Bcl-xL and MycERTAM;Bcl-xL;CTSLKO animals subjected to 3-day-treatment with TAM. . Tissues collected from MycERTAM;CTSLWT islets were used as a negative control for the staining. Three animals were assayed of each genotype; seven randomized fields per analysis were considered. Scale bars represent 50 μm.Immunohistochemical analysis of Bcl-xL expression in pancreatic tissues collected from (TIF)Click here for additional data file.


N-acetyl galactosamine , a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt  gapmer ASOs, ∼60-fold enhancement in potency relative to the parent MOE (2′-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.Triantennary


Obesity increases the risk for nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. However, the underlying mechanisms involved in the disease process remain unclear. Recently, we have developed a transgenic obese mouse model (Mito-Ob) by prohibitin mediated mitochondrial remodeling in adipocytes. The Mito-Ob mice develop obesity in a sex-neutral manner, but obesity-associated adipose inflammation and metabolic dysregulation in a male sex-specific manner. Here we report that with aging, the male Mito-Ob mice spontaneously develop obesity-linked NASH and hepatocellular carcinoma (HCC). In contrast, the female Mito-Ob mice maintained normal glucose and insulin levels and did not develop NASH and HCC. The anti-inflammatory peptide ghrelin was significantly upregulated in the female mice and down regulated in the male mice compared with respective control mice. In addition, a reduction in the markers of mitochondrial content and function was found in the liver of male Mito-Ob mice with NASH/HCC development. We found that ERK1/2 signaling was significantly upregulated whereas STAT3 signaling was significantly down regulated in the tumors from Mito-Ob mice. These data provide a proof-of-concept that the metabolic and inflammatory status of the adipose tissue and their interplay at the systemic and hepatic level play a central role in the pathogenesis of obesity-linked NASH and HCC. No significant difference in PHB protein level was found in the liver from wild type and transgenic mice suggesting that PHB is not overexpressed in the liver of Mito-Ob mice  on their liver surfaces around 10–12 months of age, which progressively grew into multifocal larger tumors (6–8 mm) by 12–14 months of age , with a 1Glucose and lipid toxicity as well as chronic low-grade inflammation are known to cause mitochondrial dysregulationTaken together, these data suggest that the hepatic phenotype in male Mito-Ob mice mimics obesity-associated mitochondrial dysregulation in steatohepatitis and HCC in humansTo further explore the link between mitochondrial dysregulation and HCC development in male Mito-Ob mice, we measured oxidative DNA damage using the ROS antibody. Hepatic oxidative DNA damage was significantly increased in the liver of tumor bearing Mito-Ob mice compared with Mito-Ob mice without tumors and wild type mice . An incrIn this context, it is important to note that mitochondrial impairment and hepatic cell deaths are common features of NASH151The pathogenesis of NASH and HCC often associated with increased hepatic inflammation. Because male Mito-Ob mice showed reduced serum ghrelin level and increased hepatic inflammation, therefore, we determined the expression level of ghrelin in the liver from Mito-Ob mice by western immunoblotting. Similar to serum ghrelin levels, tumor bearing liver from male Mito-Ob mice showed significantly reduced ghrelin level in comparison with liver from Mito-Ob mice without tumor and wild type control mice . WhereasTo get insight into the cell signaling pathways involved in HCC development in Mito-Ob mice, we determined the activation level of PI3K/Akt, MAPK/ERK and STAT3 signaling pathways because they have been implicated in the pathogenesis of HCC126This study reports a role of obesity related hyperinsulinemia and chronic low-grade inflammation in the development of NASH and HCC, independent of diet and carcinogen respectively. This pathological progression is marked by adipose inflammation, an inverse alteration of serum insulin and ghrelin levels, an increase in hepatic lipid accumulation, macrophage and lymphocyte infiltration, and a reduction in hepatic mitochondrial content and function, with a parallel increase in hepatic DNA damage, cell death and compensatory proliferation . Of notehow and why do obesity-associated abnormalities lead to HCC development in male Mito-Ob mice? This may in part be due to a significant reduction in ghrelin levels in male Mito-Ob mice because emerging evidences suggest that ghrelin is an important anti-inflammatory peptide434445In this context, it is important to note that there are a number of obese rodent models available that display obesity-associated metabolic dysregulation and NAFLD. However, the majority of them do not develop obesity-linked HCC spontaneously like male Mito-Ob mice, except TSOD miceFurthermore, a parallel decrease in liver ghrelin level in Mito-Ob mice during disease progression as observed in this study may be due to a sex-dimorphic effect of PHB on ghrelin production from macrophages, as macrophages are known to produce ghrelin45Phb−/−) mice have been reported earlier, which also develop HCC by 35–46 weeks of agePhb−/− mice has been attributed to the tumor suppressor function of PHBPhb−/− mice may in part be due to compromised mitochondrial function. Nevertheless, because PHB has a role as transcriptional co-regulator as well as in membrane signaling50Our data suggest an important role of the oncogenic determinant p-ERK in combination with p-STAT3 down regulation and mitochondrial dysregulation in the development of HCC in male Mito-Ob mice. Whether these changes are related to each other or work independently in HCC development remains to be determined. Glucotoxicity, lipotoxicity, and chronic low-grade inflammation are known to cause mitochondrial dysregulation151617In summary, our data suggest an extensive interplay between the metabolic and inflammatory status at the adipose tissue and systemic levels, as well as at the hepatic tissue level in the progression of obesity-linked NAFLD to NASH and HCC . Thus, tMonoclonal anti-PHB antibody was purchased from Cell Signaling Technology . Anti-ERK pan, anti-phosphoERK (pERK), anti-Akt pan, anti-phopsho-Akt (pAkt) antibodies were purchased from Cell Signaling Technology . Polyclonal anti-ghrelin, anti-mitochondrial transcription factor A (mtTAF), anti-PPARγ-coactivator-1α (PGC-1α) and anti-nuclear respiratory factor-2 (Nrf-2) antibodies were obtained from Abcam Inc. . Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology  and Cell Signaling Technology . All other reagents were purchased from Sigma-Aldrich  unless stated otherwise.All experiments involving animals were performed as per the study protocols (#12-007/1/2/3) approved by the Animal Care and Use Committee at the University of Manitoba and following the guidelines of the Canadian Council of Animal Care (CCAC). The generation and initial characterization of Mito-Ob mice has been reported in our previous study35Visceral adipose tissue and the liver from 9–12 month old Mito-Ob and their wild type littermates (as applicable) were fixed in buffered formaldehyde  and subsequently dehydrated, embedded in paraffin, and 5 μm sections were stained with hematoxylin-eosin or a protein specific antibody for immunohistochemistry35Liver expression level of mitochondrial proteins including PHB and cell signaling proteins were determined by Western immunoblotting. In brief, the total liver tissue lysates from Mito-Ob and wild-type mice containing equal amount of proteins (~20 μg/lane) were separated by SDS-PAGE and subsequently analyzed by Western immunoblotting using a protein-specific primary antibody and a HRP-conjugated respective secondary antibody as described before35Adipokines and hormones in the mouse serum were measured using mouse Bio-Plex ProTM Assays Diabetes panel and Bio-Plex 200TM multiplex suspension array systems  as per the manufacturer’s protocols35Apoptotic cell death using TUNEL assay  and cell proliferation using anti-Ki67 antibody  were measured following the manufacturer’s protocol described earlier52mtDNA copy number in adipose tissue was determined by real-time PCRAll statistical analyses were performed using GraphPad Prism 6. Experimental results are shown as mean ± SEM. The two-tailed Student’s unpaired t-test was performed to compare sex-matched Mito-Ob and wild-type littermates. One-way ANOVA with Dunnett’s t-test was performed when comparisons were made between Mito-Ob mice with and without tumors with a control group. Two-way ANOVA was performed with sex and genotype as factors. P-values < 0.05 were considered significant.How to cite this article: Ande, S. R. et al. Prohibitin-induced, obesity-associated insulin resistance and accompanying low-grade inflammation causes NASH and HCC. Sci. Rep.6, 23608; doi: 10.1038/srep23608 (2016).


The guided growth, visualized in real-time with a simpleoptical microscope, undergoes through a metastable solvatomorph formation in polaraprotic solvents. The presently discovered crystallization leads to the fabricationof mm2-sized surfaces composed of perovskite nanowires havingcontrolled sizes, cross-sectional shapes, aspect ratios and orientation which havenot been achieved thus far by other deposition methods. The automation of thisgeneral strategy paves the way towards fabrication of wafer-scale perovskitenanowire thin films well-suited for various optoelectronic devices, e.g. solarcells, lasers, light-emitting diodes and photodetectors.Spatial positioning of nanocrystal building blocks on a solid surface is aprerequisite for assembling individual nanoparticles into functional devices. Here,we report on the graphoepitaxial liquid-solid growth of nanowires of thephotovoltaic compound CH Therefore, the forecasted low fabrication costis one of the main arguments of researchers and investors for considering organometallichalide perovskites as a promising material for the development of next-generationphotovoltaics3 solvatomorphs proceeds exclusively in polar aprotic solvents suchas dimethylformamide (DMF), dimethylacetamide (DMAc) and dimethyl sulfoxide (DMSO), atnear room temperature. Moreover, for the first time, the kinetics of the nanowire growthwas visualized with a simple optical microscope in real-time. We envision thatstandardization of this general strategy will allow the reproducible fabrication ofwafer-scale perovskite nanowire thin films with highly controlled crystallite dimensionsand crystallinity. Their integration into various optoelectronic devices could help infurther boosting their performances.The liquid phase growth pattern is determined by the formation of nucleation centersrandomly distributed over the substrate, therefore there is little control on the areacoverage, pinholes, aspect ratio and orientation of the nanowires93 in polar aproticsolvents  results in the formation of perovskite nanowiresorganized in dandelion-like arrangement on a silicon surface. As can be seen from theoptical microscopy and scanning electron microscopy (SEM) images in We show that slip-coating of saturated solution of MAPbI3 has been reported to be unstable in the majority of ordinary solvents,including water, and to decompose at moderate temperatures(~400 K) in addition223 crystallites in a single step. Thenanowires were crystallized in the arrays of nanofabricated channels owing to the strongguiding effect of the nano-grooves28293 dissolvedin DMF onto the arrays of nanostructured trenches etched in a SiO2 substrate. Therefore, it can be regarded asa solvatomorph phase (i.e. metastable precursor phase) of the MAPbI3perovskite. Unlike MAPbI3 itself, this solvatomorph phase does not show thecharacteristic photo-luminescence under excitation with the green monochromaticincoherent light(λex = 546 nm) . It is important to realize, thatthese are metastable precursor compounds, hence the final MAPbI3 perovskitephase is formed by a subsequent solvent evaporation-induced recrystallization of thesolvatomorph phase, which is equivalent to the dehydration processes observed forinstance in many oxo-hydroxo compounds  formed by thepolar aprotic solvent. Nanoscale crystallite dimensions allow the solvent to escapewithout inducing significant shrinkage-induced cracks or damage in the crystal habit.Ultimately, the precursor phase transforms to the final, grey-silver MAPbI3phase and maintaining the elongated crystal shape  . Interesompounds . The anaompounds . We havearticles . The subarticles . This isarticles . Furtherth stops. The synal shape .The process yields nanowires with well-defined sizes, cross-sections and spatialdistributions. With this fairly simple technique, extremely long (up to few mm) andnarrow (down to 10 nm) V-shaped  and rectstate-of-the-art planar or mesoscopic configurationsis yet to be determined (2 or organic resin ofZEP520A (o degree angle) simultaneously  under pW light intensities. Here, the hybriddevices prepared by the graphoepitaxial liquid-solid growth of aligned, millimeter-longnanowires of MAPbI3 in open nanofluidic channels showed similar linearI-V characteristics, with characteristic response times of 2–5seconds  and thin (~few nm) nanowireswith a morphology defined by the shape of nanostructured open fluidic channels.Moreover, optimized spacing, shape and size in the periodic pattern may result inlarge-area, wafer-scale superstructures with advantageous optical properties forphotonic, optoelectronic, radio-frequency and non-linear optic applications. Ultimately,the graphoepitaxial nanowire growth enables well-engineered angular restriction designs,which can improve the photon reabsorption process and thus maximize the performance ofperovskite nanowire based optoelectronic deviceAs an example, we fabricated series of perovskite nanowire based photodetector devicesusing e-beam lithography . The lonseconds . The bes .


Vijay Kuchroo's lab), which expressed a truncated, phagocytosis deficient KIM-1 molecule, by deleting the mucin domain, (the domain where most mutations leading to human disease occur), while retaining most of the protein intact [Δmucin mice, decreased apoptotic cell clearance, increased pro-inflammatory cytokine secretion and increased immune cell infiltration culminated in more severe injury, when compared to wild-type mice, indicating intact KIM-1 plays a protective role in AKI [To address these questions, we characterized a mouse model KIM-1n intact . Utilizin intact . In addin intact . Indeed,e in AKI .in vitro by adding apoptotic cells to the apical surface of cultured PTCs and monitoring phagocytosis (Figure In addition to acting as non-professional phagocytes, PTCs constitutively express MHC II and can activate naïve T cells; thus, PTCs are also semi-professional antigen presenting cells . To detes Figure . ApoptotΔmucin PTCs [In vivo, kidneys from wild-type mice have greater numbers of Tregs and reduced numbers of CD4+ and CD8+ T cells, compared to KIM-1Δmucin mice [We next examined if phagocytosed material was presented to MHC I and II. KIM-1 expressing PTCs present antigens at a higher level than did KIM-1cin PTCs . Despitecin PTCs . Removalcin PTCs . In vivocin mice . Thus, mOur earlier work demonstrated that long-term expression of KIM-1, even at low levels, is maladaptive, inducing spontaneous and progressive interstitial inflammation, fibrosis and chronic kidney disease in the mouse . In modevs chronic injury. AKI is associated with an abundance of apoptotic cells and necrotic debris, while CKD is associated with a more mild but sustained injury, breakdown of the glomerular filtration barrier, and marked increases in modified serum proteins, lipids and other factors entering into the tubule lumen which can be taken up by KIM-1 expressing cells [What can explain the dichotomy between the protective role of KIM-1 in acute and maladaptive response in chronic kidney injuries? We believe the key to answering this question lies in the differences in the pathophysiology of acute ng cells . These lTaken together, these studies demonstrate that KIM-1 acts as a “double-edged sword” in the injured kidney. In acutely injured kidneys, KIM-1 facilitates repair by removal of luminal debris, leading to anti-inflammatory antigen presentation and down-regulation of the PTC immune response via NFκB suppression. By contast, in the chronically injured kidney, prolonged KIM-1 expression stimulates inflammation and fibrosis. Future studies are required to elucidate whether KIM-1′s phagocytic function to remove lumenal cellular debris or its ability to down-modulate inflammation is most important for its protective effect in AKI. The development of phospho-specific antibodies would enable the further exploration of divergent KIM-1 signaling in AKI and CKD, shedding light on potential mechanisms of KIM-1 action in the chronic setting. It is evident, however, that small molecules or biologics which can up or down-modulate KIM-1 activity should be explored as potential therapeutics in AKI and CKD.