MiR675-5p acts on HIF-1α to sustain hypoxic responses: A new therapeutic strategy for glioma

Hypoxia is a common feature in solid tumours. In glioma, it is considered the major driving force for tumour angiogenesis and correlates with enhanced resistance to conventional therapies, increased invasiveness and a poor prognosis for patients. Here we describe, for the first time, that miR675-5p, embedded in hypoxia-induced long non-coding RNA H19, plays a mandatory role in establishing a hypoxic response and in promoting hypoxia-mediated angiogenesis. We demonstrated, in vitro and in vivo, that miR675-5p over expression in normoxia is sufficient to induce a hypoxic moreover, miR675-5p depletion in low oxygen conditions, drastically abolishes hypoxic responses including angiogenesis. In addition, our data indicate an interaction of miR675-5p, HIF-1α mRNA and the RNA Binding Protein HuR in hypoxia-induced responses. We suggest the modulation of miR675-5p as a new therapeutic option to promote or abolish hypoxia induced angiogenesis

Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes

<p>Abstract</p> <p>Background</p> <p>Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed.</p> <p>Results</p> <p>We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach.</p> <p>Conclusion</p> <p>The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning.</p

The Dark That Matters: Long Non-coding RNAs as Master Regulators of Cellular Metabolism in Non-communicable Diseases

Non-coding RNAs are pivotal for many cellular functions, such as splicing, gene regulation, chromosome structure, and hormone-like activity. Here, we will report about the biology and the general molecular mechanisms associated with long non-coding RNAs (lncRNAs), a class of &gt;200 nucleotides-long ribonucleic acid sequences, and their role in chronic non-transmissible diseases. In particular, we will summarize knowledge about some of the best-characterized lncRNAs, such as H19 and MALAT1, and how they regulate carbohydrate and lipid metabolism as well as protein synthesis and degradation. Evidence is discussed about how lncRNAs expression might affect cellular and organismal metabolism and whether their modulation could provide ground for the development of innovative treatments

Correlation of Anti-Salmonella Antibodies Between Serum and Saliva Samples Collected From Finisher Pigs

Saliva samples obtained by using absorptive devices, can provide an alternative diagnostic matrix to serum for monitoring disease status in pigs. The aim of this study was to investigate the correlation of anti-Salmonella antibodies between serum and saliva samples collected from pigs. Twenty individual paired serum and saliva samples were collected from a single farm. Anti-Salmonella IgG was detected in individual serum samples using a commercial Salmonella ELISA kit, validated for sera. The same kit was used with a protocol modified by extending incubation time and increasing temperature to test individual saliva samples. Anti-Salmonella IgG antibodies in pig saliva were always detected at a lower level than in the matching serum samples. A correlation (rho = 0.66; p = 0.002) and a moderate agreement (K &gt; 0.62 p = 0.003) was found between individual Salmonella IgG in serum and saliva samples. Both correlation and the agreement levels are moderate. The size of this investigation was small, and further studies are necessary to further confirm these findings. The results of this work provide some evidence that saliva samples have the potential to be used for the diagnosis of Salmonella infection in pig farms

Hypoxia-Inducible Factor-1α Activity as a Switch for Glioblastoma Responsiveness to Temozolomide

RationaleThe activity of the transcription factor, hypoxia-inducible factor (HIF)-1α, is a common driver of a number of the pathways involved in the aggressiveness of glioblastomas (GBMs), and it has been suggested that the reduction in this activity observed, soon after the administration of temozolomide (TMZ), can be a biomarker of an early response in GBM models. As HIF-1α is a tightly regulated protein, studying the processes involved in its downregulation could shed new light on the mechanisms underlying GBM sensitivity or resistance to TMZ.MethodsThe effect of HIF-1α silencing on cell responsiveness to TMZ was assessed in four genetically different human GBM cell lines by evaluating cell viability and apoptosis-related gene balance. LAMP-2A silencing was used to evaluate the contribution of chaperone-mediated autophagy (CMA) to the modulation of HIF-1α activity in TMZ-sensitive and TMZ-resistant cells.ResultsThe results showed that HIF-1α but not HIF-2α activity is associated with GBM responsiveness to TMZ: its downregulation improves the response of TMZ-resistant cells, while blocking CMA-mediated HIF-1α degradation induces resistance to TMZ in TMZ-sensitive cells. These findings are in line with the modulation of crucial apoptosis-related genes.ConclusionOur results demonstrate the central role played by HIF-1α activity in determining the sensitivity or resistance of GBMs to TMZ, and we suggest that CMA is the cellular mechanism responsible for modulating this activity after TMZ treatment

A highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) strongly modulates cellular innate and adaptive immune subsets upon experimental infection

Highly pathogenic (HP) PRRSV isolates have been discovered within both PRRSV-1 and PRRSV-2 genotypes and investigated in recent years especially for their ability to cause extremely severe disease in conventional pig herds. The exacerbation of general and respiratory clinical signs has been attributed not only to an efficient replication (virulence) but also to the ability to dysregulate viral recognition and induce mechanisms of immune evasion or immune enhancement of humoral and cellular anti-viral responses differently from non-HP PRRSV isolates in terms of intensity and temporal onset. Thus, the understanding of the immunopathogenesis of HP PRRSV is a major concern for the study of virus biology and development of efficacious vaccines. The present study aims at addressing the modulation of relevant immune cell subsets by flow cytometry in the blood of 4- week-old pigs experimentally infected with the recently discovered PR40/2014 HP PRRSV-1.1 strain phenotypically characterized in Canelli et al. (2017) compared to pigs infected with a non-HP PRRSV isolate (PR11/ 2014) and uninfected controls. PR40 infected animals showed an early and marked reduction of pro-inflammatory CD172α+ CD14+CD16+ and CD14+CD163+ monocytes and TCRγδ+CD8α+/CD8α- lymphocytes when pigs were most infected, possibly due to a recruitment sustaining an acute inflammatory response in target tissues. The prolonged increased CD3+CD16+ NKT cell levels may sustain peripheral inflammation and/ or the anti-viral response. The late reduction (potential depletion) of γ/δ T lymphocytes and CD3+CD4+CD8α- naïve Th lymphocytes paralleled with the delayed increase of CD3+CD4+CD8α+ memory and CD3+CD4- CD8α/β+cytotoxic T lymphocytes. In addition, PR40 infection showed an early depletion of activated CD4+CD25+ T lymphocytes and Tregs together with an intense and lasting depletion of CD21+ B lymphocytes. Overall, these features demonstrate that the more severe clinical signs observed upon infection with the HP PR40 strain are sustained by remarkable changes in the peripheral blood distribution of immune cells and provide further insights into the immune regulation/immunopathogenesis induced by PRRSV-1 subtype 1 European isolates

Systematic Study for the Preparation of Au Based Catalyst for the Glucose Oxidation

Glucaric acid (GA) is one of the top 12 value added chemicals from biomass and this is due to the several applications that this molecule and its derivates can have in the industrial fields. For this reason, it is interesting to find a competitive way to produce it from lignocellulosic feedstock, more in details from glucose. Therefore, our research is focused on the synthesis of GA starting from Glucose in aqueous phase, using molecular oxygen as oxidant agent and gold nanoparticles supported materials as catalysts. All the tests have been carried out in a batch reactor and the catalysts have been prepared using the sol-immobilization method. The role of the stabilizer (polyvinyl alcohol-PVA) has been studied by varying systematically the amount of PVA in the colloidal synthesis and therefore how it could affect (i) the final morphology of the preformed supported metal nanoparticles and (ii) and the catalytic performance in terms of activity, yield and stability; high amount of PVA facilitates the formation of small nanoparticles (no PVA 7.8nm vs PVA2,4 2,61nm) but also block the active site of the catalyst, giving lower GA yield (22% vs 17%). For this reason, it has been evaluated two different method to partially remove the PVA, a washing step and several heat treatments, and seems that best results are obtained with the washing and the HT at 120℃ while at 200°C -250°C the average crystallite size increased giving a lower yield of G

Metformin and temozolomide, a synergic option to overcome resistance in glioblastoma multiforme models

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor survival. Cytoreduction in association with radiotherapy and temozolomide (TMZ) is the standard therapy, but response is heterogeneous and life expectancy is limited. The combined use of chemotherapeutic agents with drugs targeting cell metabolism is becoming an interesting therapeutic option for cancer treatment. Here, we found that metformin (MET) enhances TMZ effect on TMZ-sensitive cell line (U251) and overcomes TMZ-resistance in T98G GBM cell line. In particular, combined-treatment modulated apoptosis by increasing Bax/Bcl-2 ratio, and reduced Reactive Oxygen Species (ROS) production. We also observed that MET associated with TMZ was able to reduce the expression of glioma stem cells (GSC) marker CD90 particularly in T98G cells but not that of CD133. In vivo experiments showed that combined treatment with TMZ and MET significantly slowed down growth of TMZ-resistant tumors but did not affect overall survival of TMZ-sensitive tumor bearing mice. In conclusion, our results showed that metformin is able to enhance TMZ effect in TMZ-resistant cell line suggesting its potential use in TMZ refractory GBM patients. However, the lack of effect on a GBM malignancy marker like CD133 requires further evaluation since it might influence response duration

Baseline total metabolic tumor volume is prognostic for refractoriness to Iimunochemotherapy in DLBCL: results from GOYA

Introduction A good response to initial therapy is key to maximizing survival in patients with diffuse large B-cell lymphoma (DLBCL), but patients with chemorefractory disease and early progression have poor outcomes. Patients and Methods Data from the GOYA study in patients with DLBCL who received first-line rituximab or obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) were analyzed. Positron emission tomography/computed tomography (PET/CT)-derived characteristics associated with total metabolic tumor volume (TMTV) and clinical risk factors for primary chemorefractory disease and disease progression within 12 months (POD12) were explored. Results Of those patients fulfilling the criteria for analysis, 108/1126 (10%) were primary chemorefractory and 147/1106 (13%) had POD12. Primary chemorefractory and POD12 status were strongly associated with reduced overall survival. After multivariable analysis of clinical and imaging-based risk factors by backward elimination, only very high TMTV (quartile [Q] 1 vs. Q4 odds ratio [OR]: 0.45; P = .006) and serum albumin levels (low vs. normal OR of 1.86; P = .004) were associated with primary chemorefractoriness. After additionally accounting for BCL2/MYC translocation in a subset of patients, TMTV and BCL2/MYC double-hit status remained as significant predictors of primary chemorefractoriness (Q1 vs. Q4 OR: 0.32, P = .01 and double-hit vs. no-hit OR of 4.47, P = .02, respectively). Risk factors including very high TMTV, high sum of the product of the longest diameters (SPD), geographic region (Asia), short time since diagnosis, extranodal involvement and low serum albumin were retained for POD12. Conclusion PET-derived TMTV has prognostic value in identifying patients at risk of early treatment failure

Persistent immune stimulation exacerbates genetically driven myeloproliferative disorders via stromal remodeling

Systemic immune stimulation has been associated with increased risk of myeloid malignancies, but the pathogenic link is unknown. We demonstrate in animal models that experimental systemic immune activation alters the bone marrow stromal microenvironment, disarranging extracellular matrix (ECM) microarchitecture, with downregulation of secreted protein acidic and rich in cysteine (SPARC) and collagen-I and induction of complement activation. These changes were accompanied by a decrease in Treg frequency and by an increase in activated effector T cells. Under these conditions, hematopoietic precursors harboring nucleophosmin-1 (NPM1) mutation generated myeloid cells unfit for normal hematopoiesis but prone to immunogenic death, leading to neutrophil extracellular trap (NET) formation. NET fostered the progression of the indolent NPM1-driven myeloproliferation toward an exacerbated and proliferative dysplastic phenotype. Enrichment in NET structures was found in the bone marrow of patients with autoimmune disorders and in NPM1-mutated acute myelogenous leukemia (AML) patients. Genes involved in NET formation in the animal model were used to design a NET-related inflammatory gene signature for human myeloid malignancies. This signature identified two AML subsets with different genetic complexity and different enrichment in NPM1 mutation and predicted the response to immunomodulatory drugs. Our results indicate that stromal/ECM changes and priming of bone marrow NETosis by systemic inflammatory conditions can complement genetic and epigenetic events towards the development and progression of myeloid malignancy