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Biofluid biomarkers of age-related macular degeneration (AMD) are still lacking, and their identification is challenging. Metabolomics is well-suited to address this need, and urine is a valuable accessible biofluid. This study aimed to characterize the urinary metabolomic signatures of patients with different stages of AMD and a control group (>50 years). It was a prospective, cross-sectional study, where subjects from two cohorts were included: 305 from Coimbra, Portugal (AMD patients n = 252; controls n = 53) and 194 from Boston, United States (AMD patients n = 147; controls n = 47). For all participants, we obtained color fundus photographs (for AMD staging) and fasting urine samples, which were analyzed using (1)H nuclear magnetic resonance (NMR) spectroscopy. Our results revealed that in both cohorts, urinary metabolomic profiles differed mostly between controls and late AMD patients, but important differences were also found between controls and subjects with early AMD. Analysis of the metabolites responsible for these separations revealed that, even though distinct features were observed for each cohort, AMD was in general associated with depletion of excreted citrate and selected amino acids at some stage of the disease, suggesting enhanced energy requirements. In conclusion, NMR metabolomics enabled the identification of urinary signals of AMD and its severity stages, which might represent potential metabolomic biomarkers of the disease.

The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes. Findings in rodent models are consequently often difficult to extrapolate to human clinical trials. To overcome this ‘translational gap’, we and other groups are developing porcine disease models. Pigs share many anatomical and physiological traits with humans and thus hold great promise as translational animal models. Importantly, the toolbox for genetic engineering of pigs is rapidly expanding. Human disease mechanisms and targets can therefore be reproduced in pigs on a molecular level, resulting in precise and predictive porcine (PPP) models. In this short review, we summarize our work on the development of genetically (pre)diabetic pig models and how they have been used to study disease mechanisms and test therapeutic strategies. This includes the generation of reporter pigs for studying beta-cell maturation and physiology. Furthermore, genetically engineered pigs are promising donors of pancreatic islets for xenotransplantation. In summary, genetically tailored pig models have become an important link in the chain of translational diabetes and metabolic research.

Alongside the obesity epidemic, the prevalence of maternal diabetes is rising worldwide, and adverse effects on fetal development and metabolic disturbances in the offspring's later life have been described. To clarify whether metabolic programming effects are due to mild maternal hyperglycemia without confounding obesity, we investigated wild-type offspring of INSC93S transgenic pigs, which are a novel genetically modified large-animal model expressing mutant insulin (INS) C93S in pancreatic β-cells. This mutation results in impaired glucose tolerance, mild fasting hyperglycemia and insulin resistance during late pregnancy. Compared with offspring from wild-type sows, piglets from hyperglycemic mothers showed impaired glucose tolerance and insulin resistance (homeostatic model assessment of insulin resistance: +3-fold in males; +4.4-fold in females) prior to colostrum uptake. Targeted metabolomics in the fasting and insulin-stimulated state revealed distinct alterations in the plasma metabolic profile of piglets from hyperglycemic mothers. They showed increased levels of acylcarnitines, gluconeogenic precursors such as alanine, phospholipids (in particular lyso-phosphatidylcholines) and α-aminoadipic acid, a potential biomarker for type 2 diabetes. These observations indicate that mild gestational hyperglycemia can cause impaired glucose tolerance, insulin resistance and associated metabolic alterations in neonatal offspring of a large-animal model born at a developmental maturation status comparable to human babies.

Background MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01. Methods MYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively. Results MYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed. Conclusions An alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells. Electronic supplementary material The online version of this article (10.1186/s12885-018-4129-8) contains supplementary material, which is available to authorized users.

Olmesartan-associated enteropathy is a rare cause of severe enteropathy that should be considered in the differential diagnosis of patients with unexplained chronic diarrhoea. It may be difficult to recognise because of its clinical and histologic similarities to other clinical entities. The authors present the case of a 72-year-old woman with a 6-month clinical history of non-bloody diarrhoea and weight loss. Discontinuation of olmesartan resulted in clinical and histologic recovery, and therefore, physicians need to be aware of olmesartan-associated enteropathy in order to avoid unnecessary testing. Although rare, it is considered an emerging and underdiagnosed enteropathy. LEARNING POINTS Olmesartan-associated enteropathy is characterised by chronic diarrhoea (often severe) and weight loss that is unresponsive to a gluten-free diet. When a patient presents with unexplained chronic diarrhoea, a detailed medication review is needed. If duodenal biopsies reveal villous atrophy and coeliac disease is excluded, drug-induced enteropathy is likely. Clinical response and histologic improvement are expected after olmesartan is withdrawn.

Ewing's sarcoma family of tumours comprises a group of very aggressive diseases that are potentially curable with multimodality treatment. Despite the undoubted success of current treatment, approximately 30% of patients will relapse and ultimately die of disease. The insulin-like growth factor 1 receptor (IGF-1R) has been implicated in the genesis, growth, proliferation, and the development of metastatic disease in Ewing's sarcoma. In addition, IGF1-R has been validated, bothin vitroandin vivo, as a potential therapeutic target in Ewing's sarcoma. Phase I studies of IGF-1R monoclonal antibodies reported several radiological and clinical responses in Ewing's sarcoma patients, and initial reports of several Phase II studies suggest that about a fourth of the patients would benefit from IGF-1R monoclonal antibodies as single therapy, with approximately 10% of patients achieving objective responses. Furthermore, these therapies are well tolerated, and thus far severe toxicity has been rare. Other studies assessing IGF-1R monoclonal antibodies in combination with traditional cytotoxics or other targeted therapies are expected. Despite, the initial promising results, not all patients benefit from IGF-1R inhibition, and consequently, there is an urgent need for the identification of predictive markers of response.

To the Editor: We read with great interest the article written by Raspollini et al. ([1][1]). The authors studied a series including 32 cases of uterine leiomyosarcomas, which showed a 50% rate of positive immunostaining for KIT. Based on these results,the authors suggested the possibility of

Metabolic biomarkers of pre- and postdiagnosis gestational diabetes mellitus (GDM) were sought, using nuclear magnetic resonance (NMR) metabolomics of maternal plasma and corresponding lipid extracts. Metabolite differences between controls and disease were identified through multivariate analysis of variable selected H-1 NMR spectra. For postdiagnosis GDM, partial least squares regression identified metabolites with higher dependence on normal gestational age evolution. Variable selection of NMR spectra produced good classification Models for both pre- and postdiagnostic GDM. Prediagnosis GDM was accompanied by cholesterol increase and minor increases in lipoproteins (plasma), fatty acids, and triglycerides. (extracts). Small metabolite changes comprised variations in glucose (up regulated), amino acids, betaine, urea, creatine, and metabolites related to gut microflora. Most changes were enhanced upon GDM diagnosis, in addition to newly observed changes in low-M-w compounds. GDM prediction seems possible exploiting multivariate profile changes rather than a,set of univariate changes. Postdiagnosis GDM is successfully classified using a 26-resonance plasma biomarker. Plasma and extracts display comparable classification performance, the former enabling direct and more rapid analysis. Results and putative biochemical hypotheses require further,confirmation in larger cohorts of distinct ethnicities.

Purpose To differentiate the plasma metabolomic profile of patients with age related macular degeneration (AMD) from that of controls, by Nuclear Magnetic Resonance (NMR) spectroscopy. Methods Two cohorts (total of 396 subjects) representative of central Portugal and Boston, USA phenotypes were studied. For each cohort, subjects were grouped according to AMD stage (early, intermediate and late). Multivariate analysis of plasma NMR spectra was performed, followed by signal integration and univariate analysis. Results Small changes were detected in the levels of some amino acids, organic acids, dimethyl sulfone and specific lipid moieties, thus providing some biochemical information on the disease. The possible confounding effects of gender, smoking history and age were assessed in each cohort and found to be minimal when compared to that of the disease. A similar observation was noted in relation to age-related comorbidities. Furthermore, partially distinct putative AMD metabolite fingerprints were noted for the two cohorts studied, reflecting the importance of nutritional and other lifestyle habits in determining AMD metabolic response and potential biomarker fingerprints. Notably, some of the metabolite changes detected were noted as potentially differentiating controls from patients diagnosed with early AMD. Conclusion For the first time, this study showed metabolite changes in the plasma of patients with AMD as compared to controls, using NMR. Geographical origins were seen to affect AMD patients´ metabolic profile and some metabolites were found to be valuable in potentially differentiating controls from early stage AMD patients. Metabolomics has the potential of identifying biomarkers for AMD, and further work in this area is warranted.

Myelodysplastic syndromes (MDS) are a very heterogeneous group of conditions affecting the hematopoietic system. They are characterised by inefficient maturation of haematopoietic precursors with increased risk of evolution to acute myeloid leukaemia (AML) (Figure ​(Figure1)1) (1). A new study from Flandrin-Gresta et al (2) shows the importance of the molecular chaperone heat shock protein 90 (HSP90) and its ‘client’ proteins in the molecular evolution of MDS and indicates the therapeutic potential of HSP90 inhibitors in preventing this. There are exciting broader implications for use of HSP90 inhibitors earlier in the natural history of clinical malignancies. Figure 1 Potential for HSP90 inhibition in MDS and AML Standard of care for patients with MDS typically includes supportive therapy (including transfusions of the deficient cells), epigenetic drugs and/or cytotoxic chemotherapy depending on disease subtype, genetic abnormalities, International Prognostic Scoring System stage and performance status (1). Stem cell transplantation from the bone marrow of suitable donors may be a viable alternative. Treatment options were increased recently with approval of drugs such as lenalidomide, and there are other new agents and drug combinations currently undergoing clinical evaluation (1). Nevertheless, novel agents targeted to the molecular pathogenesis of MDS are urgently needed. Many molecular aberrations occur in MDS, ranging from chromosomal abnormalities, amplifications, deletions and mutations to epigenetic changes. Genes involved cover a variety of biological processes supporting malignancy and include TET2, ASXL1, RUNX1, IDH1, IDH2, EZH2, and those encoding various tyrosine kinases, as well as NRAS, KRAS, CBL and TP53 (3). The importance of HSP90 in cancer is well established, in particular its role in supporting the active conformation of many oncogenic client proteins (4). Pharmacologic inhibition of HSP90 causes degradation of these client proteins by the ubiquitin-proteasome pathway, profoundly inhibiting many signal transduction pathways and inducing cell cycle arrest and apoptosis (5). The therapeutically attractive ability of HSP90 inhibitors to deplete simultaneously many cancer-causing clients, reducing the molecular avenues via which drug resistance can develop, has led to these agents becoming of major pharmaceutical company interest, with ~20 inhibitors entering clinical trials (4). Phase I and II clinical studies have shown promising results for HSP90 inhibitors in malignancies addicted to particular HSP90 clients, especially HER2+ trastuzumab-refractory breast and EGFR-mutant and ALK-translocated non small cell lung cancer, as well as other opportunities in haematological cancers, including multiple myeloma (where HSP90 inhibition exploits the unfolded protein response) and leukaemias driven by HSP90 clients (eg FLT3 in AML and BCR-ABL in chronic myeloid leukaemia) (4). Previous studies in a large series of AML patients revealed an association between higher expression levels of HSP90 and poor prognosis together with enhanced activation of oncogenic signaling pathways involving PI3 kinase, AKT and ERK1/2 (6). Furthermore, HSP27, HSP70 and HSP90 are overexpressed in advanced MDS compared to early MDS and normal bone marrow, consistent with involvement of HSPs in the pathogenesis and evolution of this condition (7). In the present issue of Oncotarget, Flandrin-Gresta and colleagues now report the clinical and biological relevance of HSP90 and phosphorylated forms of its protein kinase clients AKT and FAK (focal adhesion kinase) in bone marrow mononuclear cells (dysplastic white cells) and CD34+ cells (myeloid stem cells) from a series of 177 patients with MDS, determined at diagnosis and in some cases after evolution to high grade MDS or to AML (2). First, they observe that HSP90, FAK, phospho-FAK and phospho-AKT are overexpressed in high risk MDS and are associated with shorter survival and increased risk of progression to AML. The expression levels of all of these proteins were significantly higher after transformation than at the time of diagnosis. Next, the authors show that the pharmacologic HSP90 inhibitor tanespimycin (17-AAG) reduces viability and markedly increases apoptosis in bone marrow mononuclear cells and CD34+ cells isolated from 39 patients. Furthermore, tanespimycin decreases expression of FAK, phospho-FAK and phospho-AKT. Somewhat surprisingly, HSP70 is not induced while HSP90 levels are decreased. Taken together, these results suggest a possible new clinical application for HSP90 inhibitors in MDS, either alone or combined with agents such as disease-relevant kinase inhibitors or epigenetic therapies (8). In addition, HSP90, phospho-FAK and phospho-AKT may be useful prognostic or predictive biomarkers in MDS. Further work is needed to understand the exact mechanisms by which HSP90 is involved in the pathogenesis of MDS and its progression to AML, including the contribution of chaperone support for FAK, AKT and quite possibly other HSP90 clients, including mutated FLT3. In addition, following termination of tanespimycin development, preclinical and clinical studies are now needed to assess the impact of improved, next-generation HSP90 inhibitors (4) in MDS. Of particular biological as well as therapeutic interest is that the use of HSP90 inhibitors to prevent the molecular evolution of MDS to AML would be the first example of their application to prevent progression from pre-malignant to fully malignant disease. Such a clinical use, potentially applicable to other cancers, would be consistent with the hypothesis that HSP90 is essential to support initial oncogenic transformation as well as subsequent malignant progression, for example by preventing destruction of metastable oncogenic driver client proteins. This would encourage treatment with HSP90 inhibitors earlier in the natural history of other human malignancies, with potential for even greater clinical impact.

[Background]: In KIT-expressing Ewing sarcoma cell lines, the addition of doxorubicin to imatinib increases apoptosis, compared with imatinib or doxorubicin alone. On the basis of these in vitro data, the authors conducted a phase 1-2 trial of doxorubicin with imatinib in patients with gastrointestinal sarcoma tumors refractory to high-dose imatinib therapy. [Methods]: Patients with metastatic gastrointestinal sarcoma tumor resistant to imatinib at 400 mg by mouth (p.o.) twice a day were eligible for this multicenter study, and received imatinib (400 mg p.o. every day [q.d.]) concomitantly with doxorubicin 15-20 mg/m2/weekly for 4 cycles (monthly cycles), followed by imatinib (400 mg p.o. q.d.) maintenance in nonprogressive patients. Spiral computed tomography and positron emission tomography with F18-fluorodeoxyglucose were done basally and after 2 months of therapy to evaluate response. An in vitro study assessed the effect of combining imatinib and doxorubicin. [Results]: Twenty-six patients with progressive gastrointestinal sarcoma tumor were entered in the study. Treatment was well tolerated. Three (14%) of 22 evaluable patients had partial responses per Response Evaluation Criteria in Solid Tumors, and 8 (36%) had clinical benefit (partial response or stable disease for ≥6 months). Median progression-free survival (PFS) was 100 days (95% confidence interval [CI], 62-138), and median survival was 390 days (95% CI, 264-516). Interestingly, PFS was 211 days (95% CI, 52-370) in patients with wild type (WT) KIT and 82 days (95% CI, 53-111) in non-WT patients (10 mutant, 6 not assessed). A synergistic effect on cell line proliferation and apoptosis was found with imatinib and doxorubicin combination. [Conclusions]: Low-dose chemobiotherapy combination showed promising activity in heavily pretreated gastrointestinal sarcoma tumor patients, especially in those with WT-KIT genotype. © 2010 American Cancer Society., This work was also supported by the Acción Transversal del Cáncer project, through an agreement between Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation, and the Centro de Investigación del Cáncer-IBMCC (USAL-CSIC).

Bone and soft tissue sarcomas are an infrequent and heterogeneous group of mesenchymal tumors, including more than a hundred different entities attending to histological patterns. Sarcomas are quite resistant to conventional chemotherapy (anthracycline and ifosfamide) with the exception of some subtypes, such as Ewing's sarcoma (ES). New drugs with proved efficacy against sarcomas include taxanes, gemcitabine, and ET-743. Preclinical studies have also identified key molecular events leading to the progression and development of sarcomas which are good candidates to targeted therapy. Inhibitors of the tyrosine kinase receptors, such as IGF-1R, c-kit, PDGFR, VEGFR, or the mTOR signaling pathway, proteasome, angiogenesis, and stress response proteins are under clinical evaluation against sarcomas. ES, a tumor characterized by chromosomal translocations that originate gene fusions (EWS-FLI1, EWS-ERG), is an example of a good chemotherapy responder tumor whose survival rate shows a plateau in recent years. Preclinical studies have identified that new targets such as HSP90 are of relevance to ES. On the other hand, recent studies showed the role of cancer stem cells (CSCs) in sarcomas and the relevance of the identification of reliable molecular markers and possible therapeutic targets. New therapeutic approaches could be directed against CSCs. This review describes more recent targeted therapy in sarcomas, with special emphasis on ES and the role of CSCs. We also emphasize the role of high throughput proteomic techniques in identifying new therapeutic targets. © 2008 Elsevier Inc. All rights reserved.