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Diffuse grade II IDH−mutant gliomas are slow−growing brain tumors that progress into high−grade gliomas. They present intratumoral cell heterogeneity, and no reliable markers are available to distinguish the different cell subtypes. The molecular mechanisms underlying the formation of this cell diversity is also ill−defined. Here, we report that SOX9 and OLIG1 transcription factors, which specifically label astrocytes and oligodendrocytes in the normal brain, revealed the presence of two largely nonoverlapping tumoral populations in IDH1−mutant oligodendrogliomas and astrocytomas. Astrocyte−like SOX9+ cells additionally stained for APOE, CRYAB, ID4, KCNN3, while oligodendrocyte−like OLIG1+ cells stained for ASCL1, EGFR, IDH1, PDGFRA, PTPRZ1, SOX4, and SOX8. GPR17, an oligodendrocytic marker, was expressed by both cells. These two subpopulations appear to have distinct BMP, NOTCH1, and MAPK active pathways as stainings for BMP4, HEY1, HEY2, p−SMAD1/5 and p−ERK were higher in SOX9+ cells. We used primary cultures and a new cell line to explore the influence of NOTCH1 activation and BMP treatment on the IDH1−mutant glioma cell phenotype. This revealed that NOTCH1 globally reduced oligodendrocytic markers and IDH1 expression while upregulating APOE, CRYAB, HEY1/2, and an electrophysiologically−active Ca2+−activated apamin−sensitive K+ channel (KCNN3/SK3). This was accompanied by a reduction in proliferation. Similar effects of NOTCH1 activation were observed in nontumoral human oligodendrocytic cells, which additionally induced strong SOX9 expression. BMP treatment reduced OLIG1/2 expression and strongly upregulated CRYAB and NOGGIN, a negative regulator of BMP. The presence of astrocyte−like SOX9+ and oligodendrocyte−like OLIG1+ cells in grade II IDH1−mutant gliomas raises new questions about their role in the pathology.

Background Breast cancer is amongst the 10 first causes of death in women worldwide. Around 20% of patients are misdiagnosed leading to early metastasis, resistance to treatment and relapse. Many clinical and gene expression profiles have been successfully used to classify breast tumours into 5 major types with different prognosis and sensitivity to specific treatments. Unfortunately, these profiles have failed to subclassify breast tumours into more subtypes to improve diagnostics and survival rate. Alternative splicing is emerging as a new source of highly specific biomarkers to classify tumours in different grades. Taking advantage of extensive public transcriptomics datasets in breast cancer cell lines (CCLE) and breast cancer tumours (TCGA), we have addressed the capacity of alternative splice variants to subclassify highly aggressive breast cancers. Results Transcriptomics analysis of alternative splicing events between luminal, basal A and basal B breast cancer cell lines identified a unique splicing signature for a subtype of tumours, the basal B, whose classification is not in use in the clinic yet. Basal B cell lines, in contrast with luminal and basal A, are highly metastatic and express epithelial-to-mesenchymal (EMT) markers, which are hallmarks of cell invasion and resistance to drugs. By developing a semi-supervised machine learning approach, we transferred the molecular knowledge gained from these cell lines into patients to subclassify basal-like triple negative tumours into basal A- and basal B-like categories. Changes in splicing of 25 alternative exons, intimately related to EMT and cell invasion such as ENAH, CD44 and CTNND1, were sufficient to identify the basal-like patients with the worst prognosis. Moreover, patients expressing this basal B-specific splicing signature also expressed newly identified biomarkers of metastasis-initiating cells, like CD36, supporting a more invasive phenotype for this basal B-like breast cancer subtype. Conclusions Using a novel machine learning approach, we have identified an EMT-related splicing signature capable of subclassifying the most aggressive type of breast cancer, which are basal-like triple negative tumours. This proof-of-concept demonstrates that the biological knowledge acquired from cell lines can be transferred to patients data for further clinical investigation. More studies, particularly in 3D culture and organoids, will increase the accuracy of this transfer of knowledge, which will open new perspectives into the development of novel therapeutic strategies and the further identification of specific biomarkers for drug resistance and cancer relapse.

IDH1-mutated gliomas are slow growing brain tumours, which progress into high-grade gliomas. They present intra-tumoural cell heterogeneity, but no good markers are available to distinguish the different cell subtypes. The molecular mechanisms underlying the formation of this cell diversity is also ill defined. Here we report that the SOX9 and OLIG1 transcription factors, which specifically label astrocytes and oligodendrocytes in the normal brain, reveal the presence of two largely non-overlapping tumoural populations in IDH1-mutated oligodendrogliomas and astrocytomas. Astro-like SOX9+ cells additionally stain for APOE, CRYAB, ID4, KCNN3, while oligo-like OLIG1+ cells stain for ASCL1, EGFR, IDH1, PDGFRA, PTPRZ1, SOX4, and SOX8. GPR17, an oligodendrocytic marker, was expressed by both cells. These two sub-populations appear to have distinct BMP, NOTCH1, and MAPK active pathways as stainings for BMP4, HEY1, HEY2, p-SMAD1/5 and p-ERK were higher in SOX9+ cells. We used primary cultures and a new cell line to explore the influence of NOTCH1 activation and BMP treatment on low-grade glioma cell phenotype. This revealed that NOTCH1 globally reduced oligodendrocytic markers and IDH1 expression while upregulating APOE, CRYAB, HEY1/2 and an electrophysiologically Ca2+-activated apamin-sensitive K+ channel (KCNN3/SK3). This was accompanied by reduction in proliferation. Similar effects of NOTCH1 activation were observed in non-tumoural human oligodendrocytic cells, which additionally induced strong SOX9 expression. BMP treatment reduced OLIG1/2 expression and strongly upregulated CRYAB and NOGGIN, a negative regulator of BMP. The presence of astro-like SOX9+ and oligo-like OLIG1+ cells in diffuse low-grade gliomas raise new questions about their role in the pathology.

Myelopoiesis involves differentiation of hematopoietic stem cells to cellular populations that are restricted in their self-renewal capacity, beginning with the common myeloid progenitor (CMP) and leading to mature cells including monocytes and granulocytes. This complex process is regulated by various extracellular and intracellular signals including microRNAs (miRNAs). We characterised the miRNA profile of human CD34+CD38+ myeloid progenitor cells, and mature monocytes and granulocytes isolated from cord blood using TaqMan Low Density Arrays. We identified 19 miRNAs that increased in both cell types relative to the CMP and 27 that decreased. miR-125b and miR-10a were decreased by 10-fold and 100-fold respectively in the mature cells. Using in vitro granulopoietic differentiation of human CD34+ cells we show that decreases in both miR-125b and miR-10a correlate with a loss of CD34 expression and gain of CD11b and CD15 expression. Candidate target mRNAs were identified by co-incident predictions between the miRanda algorithm and genes with increased expression during differentiation. Using luciferase assays we confirmed MCL1 and FUT4 as targets of miR-125b and the transcription factor KLF4 as a target of miR-10a. Together, our data identify miRNAs with differential expression during myeloid development and reveal some relevant miRNA-target pairs that may contribute to physiological differentiation.

International audience; Multiple myeloma (MM) is still an incurable hematological malignancy. Despite recent progress due to new anti-myeloma agents, the pathology is characterized by a high frequency of de novo or acquired resistance. Delineating the mechanisms of MM resistance is essential for therapeutic advances. We previously showed that long-term genotoxic stress induces the establishment of a senescence-associated secretory phenotype, a pro-inflammatory response that favors the emergence of cells with cancer stem-like properties. Here, we studied the short-term response of MM cells following treatment with various DNA damaging agents such as the energetic C-ion irradiation. MM cells are highly resistant to all treatments and do not enter apoptosis after they arrest cycling at the G2 phase. Although the DNA damage response pathway was activated, DNA breaks remained chronically in damaged MM cells. We found, using a transcriptomic approach that RAD50, a major DNA repair gene was downregulated early after genotoxic stress. In two gerosuppression situations: induction of hypoxia and inhibition of the mammalian target of rapamycin (mTOR) pathway, we observed, after the treatment with a DNA damaging agent, a normalization of RAD50 expression concomitant with the absence of cell cycle arrest. We propose that combining inhibitors of mTOR with genotoxic agents could avoid MM cells to senesce and secrete pro-inflammatory factors responsible for cancer stem-like cell emergence and, in turn, relapse of MM patients.

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC‐3 prostate cancer cell lines, we showed that chemical or shRNA‐mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2‐mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC‐3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down‐regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2‐mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

International audience; Reduced expression of DICER, a key enzyme in the miRNA pathway, is frequently associated with aggressive, invasive disease, and poor survival in various malignancies. Regulation of DICER expression is, however, poorly understood. Here, we show that NF90/NF110 facilitates DICER expression by controlling the processing of a miRNA, miR-3173, which is embedded in DICER pre-mRNA. As miR-3173 in turn targets NF90, a feedback amplification loop controlling DICER expression is established. In a nude mouse model, NF90 overexpression reduced proliferation of ovarian cancer cells and significantly reduced tumor size and metastasis, whereas overexpression of miR-3173 dramatically increased metastasis in an NF90- and DICER-dependent manner. Clinically, low NF90 expression and high miR-3173-3p expression were found to be independent prognostic markers of poor survival in a cohort of ovarian carcinoma patients. These findings suggest that, by facilitating DICER expression, NF90 can act as a suppressor of ovarian carcinoma.

International audience; Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 degrees C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 degrees C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

The regulation of function of endothelial cell–cell junctions is fundamental in sustaining vascular integrity. The polycistronic microRNA (miR) complexes containing miR-23a-27a-24-2, and 23b-27b-24-1 are predicted to target the majority of major endothelial junctional proteins. We focus on miR-23a and miR-23b, and investigate the functional effects of these miRs on junctions. While miR-23a and 23b only differ by 1 nucleotide (g19) outside the seed region and thus are predicted to have the same targets, they function differently with miR-23a inhibiting permeability and miR-23b inhibiting angiogenesis. Both miRs target the junctional attractive molecule (tight junction protein 2) ZO-2 and the repulsive molecule junctional adhesion molecule C (JAM-C), although the inhibition of JAM-C by miR-23a is more profound than by miR-23b. The difference in potency is attributable to differences at g19 since a mutation of the t17, the g19 binding site of miR-23b in the 3′UTR of JAM-C restores identity. We also show that the pattern of expression of miR-23a and miR-23b and their targets are different. Thus, the paralogues miR-23a and miR-23b can have profoundly different effects on endothelial cell function due at least partially to selective effects on target proteins and differences in expression patterns of the miRs. This work exposes a hitherto unappreciated complexity in therapeutically targeting miRs.

International audience; Angiogenesis, the process of forming new blood vessels, is crucial in the physiological response to ischemia, though it can be detrimental as part of inflammation and tumorigenesis. We have previously shown that high-density lipoproteins (HDL) modulate angiogenesis in a context-specific manner via distinct classical signalling pathways, enhancing hypoxia-induced angiogenesis while suppressing inflammatory-driven angiogenesis. Whether additional novel targets exist to account for these effects are unknown. A microarray approach identified two novel genes, cyclic-adenosine-monophosphate-response-element-binding protein 3 regulatory factor (CREBRF) and tripartite motif-containing protein 2 (TRIM2) that were upregulated by reconstituted HDL (rHDL). We measured CREBRF and TRIM2 expression in human coronary artery endothelial cells following incubation with rHDL and exposure to either hypoxia or an inflammatory stimulus. We found that CREBRF and TRIM2 mRNA were significantly upregulated by rHDL, particularly in response to its phospholipid component 1-palmitoyl-2-linoleoyl-phosphatidylcholine, however, protein expression was not significantly altered. Knockdown of TRIM2 impaired endothelial cell tubulogenesis in vitro in both hypoxia and inflammation, implying a necessary role in angiogenesis. Furthermore, TRIM2 knockdown attenuated rHDL-induced tubule formation in hypoxia, suggesting that it is important in mediating the pro-angiogenic action of rHDL. Our study has implications for understanding the regulation of angiogenesis in both of these pathophysiological contexts by HDL.

The relatively high levels of aneuploidy identified in IVF embryos recently have raised the opportunity to improve pregnancy rates and liveborn outcomes with IVF by identifying and transferring only one chromosomally balanced embryo. With the increased use of CGH, it has been observed that any of the chromosomes can be involved in aneuploidy and even de novo segmental aneuploidies may contribute to implantation failure and miscarriage in a typical IVF cycle. The previous limitations in FISH complexity had meant that the full benefits of aneuploidy screening were not initially realised. The impact of reliable whole chromosome amplification methods and the availability of quality controlled commercial microarrays has seen strong uptake of total chromosome screening by many clinics around the world with substantial changes in implantation rates for the selected embryos reported in most cases. In spite of these benefits, the cost of the aCGH process appears to have limited the availability for a significant number of patients. The price of arrays has reduced in the last few years, but this remains a substantial cost burden in many situations. An alternative approach that can still meet the criteria of total chromosome screening but is potentially more cost-effective could bring the benefits of total chromosome screening to an even wider patient group. Partial genome sequencing (PGS) or low-pass sequencing is one alternative that can be used to rapidly karyotype whole-genome-amplified samples from human blastocysts. We compared these with array-based comparative genomic hybridization (aCGH) karyotypes. Readily available open-source software was customised and then used to map the individual Next Generation Sequencing (NGS) reads to chromosomes providing considerable flexibility for resolving both total chromosomes as well as segmental variations. Qualitatively, there was complete diagnostic agreement between the two approaches. Quantitatively, the sequencing approach also offered simple implementation of objective measures of levels of mosaicism within the multi-cell trophectoderm biopsy piece aiding final decision-making regarding suitability for transfer of the nominated (single) embryo. Depending on individual laboratory capabilities, either technique can have shortened protocols that enable next day transfer of one euploid embryo. Standard protocols for aCGH or PGS can fit timing wise with cleavage stage biopsy. Embryos biopsied on day 5 of cycle will only be transferrable the next day, while slower developing embryos biopsied on day 6 may need to be frozen and transferred at a later time. While reports of day 3 blastomere biopsy show improved pregnancy rates after screening, the final outcomes do not appear to be as good as blastocyst stage biopsy, thus not realising the full potential of screening. Applied correctly, simplified, cost-effective routine aneuploidy analysis via aCGH or NGS promises high live birth rates per single transferred embryo.

Near-infrared Raman spectroscopy, an optical technique that is able to interrogate biological tissues, has been used to study bladder and prostate tissues, with the objective being to provide a first approximation of gross biochemical changes associated with the process of carcinogenesis. Prostate samples for this study were obtained by taking a chip at TURP, and bladder samples from a biopsy taken at TURBT and TURP, following ethical approval. Spectra were taken from purchased biochemical constituents and different pathologies within the bladder and the prostate. We were then able to determine the biochemical basis for these pathologies by utilising an ordinary least-squares fit. We have shown for the first time that we are able to utilise Raman spectroscopy in determining the biochemical basis for the different pathologies within the bladder and prostate gland. In this way we can achieve a better understanding of disease processes such as carcinogenesis. This could have major implications in the future of the diagnosis of disease within the bladder and the prostate gland.

Objective : To determine the technical feasibility of sural nerve grafting for restoration of cavernous nerve continuity after radical retropubic prostatectomy by the laparoscopic approach. Materials and methods : 15 potent men (mean age: 56±4 years) underwent laparoscopic radical prostatectomy with deliberate wide uni- or bilateral neurovascular bundle resection. After prostatectomy, but before the vesicourethral anastomosis, an autologous sural nerve graft was interposed between the divided end of the cavernous nerves using a laparoscopic approach. Results : All 15 procedures could be completed laparoscopically. Visual cooptation between the nerve graft and the cut ends of the neurovascular bundles was possible in all 15 cases. Surgical time for the entire procedure ranged between 210 to 275 min with 30 to 60 min of this time required for the nerve harvest and graft. Conclusion : This experience demonstrates that sural nerve graft replacement is technically feasible by the laparoscopic approach. The magnified optics, bloodless surgical field, and improved instrumentation create an optimal environment for sural nerve grafting to the cut ends of the neurovascular bundles. Quality of life survey data at 12 to 18 month follow-up will be needed to assess functional return.

Background MicroRNAs (miRNAs) are coordinators of cellular differentiation, including granulopoiesis. Although differential expression of many miRNAs is associated with the maturation of granulocytes, analysis of differentially expressed miRNAs and their cellular localization across all stages of granulopoiesis, starting from hemopoietic stems cells, is not well characterized. Methods We analyzed whole cell miRNA and mRNA expression during granulopoiesis using Taqman low-density and Affymetrix arrays respectively. We also performed nuclear and cytoplasmic fractionation followed by Taqman low-density array and/or quantitative PCR to identify nuclear-enriched miRNAs in hemopoietic stem/progenitor cells, promyelocytes, myelocytes, granulocytes and several hemopoietic cell lines. Anti-correlation between the expression of miRNA and target pairs was used to determine putative miRNA targets. Results Analyses of our array data revealed distinct clusters of differentially expressed miRNAs that are specific to promyelocytes and granulocytes. While the roles of many of these miRNAs in granulopoiesis are not currently known, anti-correlation of the expression of miRNA/mRNA target pairs identified a suite of novel target genes. Clusters of miRNAs (including members of the let-7 and miR-17-92 families) are downregulated in hemopoietic stem/progenitor cells, potentially allowing the expression of target genes known to facilitate stem cell proliferation and homeostasis. Additionally, four miRNAs (miR-709, miR-706, miR-690 and miR-467a*) were found to be enriched in the nucleus of myeloid cells and multiple hemopoietic cell lines compared to other miRNAs, which are predominantly cytoplasmic-enriched. Both miR-709 and miR-706 are nuclear-enriched throughout granulopoiesis and have putative binding sites of extensive complementarity downstream of pri-miRNAs. Nuclear enrichment of miR-467a* is specific to hemopoietic stem/progenitors and promyelocytes. These miRNAs are also nuclear-enriched in other hemopoietic cell lines, where nuclear sequestering may fine-tune the expression of cytoplasmic mRNA targets. Conclusions Overall, we have demonstrated differentially expressed miRNAs that have not previously been associated with hemopoietic differentiation and provided further evidence of regulated nuclear-enrichment of miRNAs. Further studies into miRNA function in granulocyte development may shed light on fundamental aspects of regulatory RNA biology and the role of nuclear miRNAs.

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Case Report A 41-year-old man with a 6-year history of hypertension under excellent control with three antihypertensive medications was involved in a high-speed motor vehicle rollover accident and was brought to the emergency department. A CT performed with chestabdomen-pelvis protocol and IV contrast enhancement revealed no abnormalities (Fig. 1A). The patient was admitted to the general surgical unit because of right hip and flank pain thought secondary to the muscle contusions confirmed at clinical examination. Two days later, the patient complained of severe sudden epigastric pain. ECG and abdominal radiographs revealed no abnormalities. CT was repeated in search of delayed injury (Figs. 1B and 1C). Conventional angiography and stenting were performed 5 days later (Fig. 1D). The abdominal symptoms resolved after stent placement. The diagnosis was posttraumatic celiac artery dissection.

Raman spectroscopy is an optical technique able to interrogate biological tissues, giving us an understanding of the changes in molecular structure that are associated with disease development. The Kerr-gated Raman spectroscopy technique uses a picosecond pulsed laser as well as fast temporal gating of collected Raman scattered light. Prostate samples for this study were obtained by taking a chip at the transurethral resection of the prostate (TURP), and bladder samples from a biopsy taken at transurethral resection of bladder tumor (TURBT) and TURP. Spectra obtained through the bladder and prostate gland tissue, at different time delays after the laser pulse, clearly show change in the spectra as depth profiling occurs, eventually showing signals from the uric acid cell and urea cell, respectively. We show for the first time, using this novel technique, that we are able to obtain spectra from different depths through both the prostate gland and the bladder. This has major implications in the future of Raman spectroscopy as a tool for diagnosis. With the help of Raman spectroscopy and Kerr gating, it may be possible to pick up the spectral differences from a small focus of adenocarcinoma of the prostate gland in an otherwise benign gland, and also stage the bladder cancers by assessing the base of the tumor post resection.

Introduction Heterogeneity within the CML patient population is evidenced by variable response dynamics and tolerability between patients treated with tyrosine kinase inhibitors (TKIs). Clinical risk scores have utility however do not inform upon the biology underpinning differential response. OCT1 activity has been established as a predictor of imatinib response but is not predictive for second generation TKIs nilotinib or dasatinib. In CML, microRNAs (miRNAs) have been implicated in BCR-ABL mediated signalling pathways and alter rapidly after TKI treatment. Indeed, we have previously established that miR-142-3p decreases within 14 days of TKI initiation and correlates with the SOKAL score. Method We performed miRNA profiling on 75 newly diagnosed, clinical trial CML patients (56% male, 44% female, median age 51 years) with greater than 24 months follow-up. RNA was extracted from peripheral blood mononuclear cells. The expression of 800 mature miRNA was analysed using the nCounter Human v2 miRNA Expression Assay (NanoString Technologies). In addition, IC50 values for imatinib and nilotinib were determined on all samples as previously described. Results The miRNA expression levels obtained on this platform showed a wide dynamic range and substantial interpatient variation. This included miRNA previously reported in CML in vitro and patient studies: interquartile ranges were 52414 to 121632 for miR-142-3p, 3120 to 15599 for miR-451a, 2658 to 12023 for miR-150, 828 to 1461 for miR-181a-5p. In addition, we were able to identify miRNAs that were associated with nilotinib IC50 and imatinib IC50. Discussion The current study highlights the variability evident at the miRNA level between newly diagnosed CML patients. It provides a rationale to further examine the role of miRNA in CML pathogenesis, TKI response and define patient heterogeneity from diagnosis. Disclosures: White: Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding. Hughes:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

MicroRNAs (miRNAs) regulate post-transcriptional gene expression during development and disease. We have determined the miRNA expression levels of early- and end-stage hypertrophic cardiomyopathy (HCM) in a severe, transgenic mouse model of the disease. Five miRNAs were differentially expressed at an early stage of HCM development. Time-course analysis revealed that decreased expression of miR-1 and miR-133a commences at a pre-disease stage, and precedes upregulation of target genes causal of cardiac hypertrophy and extracellular matrix remodelling, suggesting a role for miR-1 and miR-133a in early disease development. At end-stage HCM, 16 miRNA are dysregulated to form an expression profile resembling that of other forms of cardiac hypertrophy, suggesting common responses. Analysis of the mRNA transcriptome revealed that miRNAs potentially target 15.7% upregulated and 4.8% downregulated mRNAs at end-stage HCM, and regulate mRNAs associated with cardiac hypertrophy and electrophysiology, calcium signalling, fibrosis, and the TGF-β signalling pathway. Collectively, these results define the miRNA expression signatures during development and progression of severe HCM and highlight critical miRNA regulated gene networks that are involved in disease pathogenesis.

1. The continental philosophers still continue to adopt the electric theory of galvanism proposed by Volta, whilst those in Britain as uniformly follow some modification of the chemical theory proposed by Dr. Wollaston. From this diversity of opinion we may safely conclude, that the experimental proofs for the truth of either theory are not sufficiently powerful, to command the assent of all capable of appreciating the weight of such evidence. I have therefore ventured to lay before the Society the following experiments and observations; as they appear to me to establish the truth of some modification of the chemical theory, and to demonstrate the fallacy of the principles on which the electric theory rests. 2. The fundamental principle assumed by Volta, and supported by his followers, is, that if dissimilar metals be brought into contact they are instantly thrown into opposite electric states. This he conceives to be a new law of nature, and claims to himself the honour of the discovery. He conceives that its truth is proved by the following experiment.

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

After observing that the theory of galvanism originally proposed by Volta, and generally termed the Electric theory, is still the uni­versally received doctrine among continental philosophers, the author adduces several experiments proving the fallacy of the principles on which that theory is founded. He points out the inconclusiveness of the reasoning by which it has been inferred that dissimilar metals, by being simply placed in contact with one another, are instantly thrown into opposite electric states; for in all the experiments which have been made with a view of establishing this fundamental prin­ciple of the electric theory, the metals have been exposed to the oxi­dizing action of the air, which is a constant source of electric disturb­ance, and the operation of which has been strangely overlooked. The author found, by forming galvanic circles with two different metals and an interposed acid, that when he used different kinds of acid, or varied the degree of their dilution, the electro-magnetic effects, as measured by a delicate galvanometer, bear no sort of relation to the conducting power of the fluid, as is assumed in the Voltaic hypothesis. He deduces the same conclusion from experiments made with an ap­paratus by which the fluid is confined in a rectangular box, divided by a membranous diaphragm into two compartments, so as to allow of the addition of an acid to the fluid contained in one of the com­partments, and thereby limiting its action to one of the metallic surfaces. By means of another contrivance, the author ascertained that of two different metals, the one which, when acted upon by an acid, combines with the greatest quantity of oxygen, as measured by the volume of hydrogen disengaged, is always positive with respect to the other metal. Even two pieces of the same metal, differing in hardness, will be acted upon by the same acid in different degrees, and may thus be brought into different states of electricity. In ge­neral it is the harder of the two pieces of metal which becomes po­sitive ; but with steel the reverse obtains. It would appear, how­ever, that with the same pairs of metallic discs, the direction of the electric current is determined by the nature of the acid employed: thus nitrous acid, acting upon zinc, copper, or iron, gives rise to a current in a direction opposite to the current which is produced by the sulphuric, nitric, or muriatic acids. Variations in the tempera­ture of the metals will also occasion diversities in the results, not hitherto satisfactorily explained on any theory. From one experi­ment the author is led to infer that an acid is capable of combining with a pure metal, without the latter being previously reduced to the state of an oxide.