A 4-gene prognostic index for enhancing acute myeloid leukaemia survival prediction.

Despite advancements in utilizing genetic markers to enhance acute myeloid leukaemia (AML) outcome prediction, significant disease heterogeneity persists, hindering clinical management. To refine survival predictions, we assessed the transcriptome of non-acute promyelocytic leukaemia chemotherapy-treated AML patients from five cohorts (n = 975). This led to the identification of a 4-gene prognostic index (4-PI) comprising CYP2E1, DHCR7, IL2RA and SQLE. The 4-PI effectively stratified patients into risk categories, with the high 4-PI group exhibiting TP53 mutations and cholesterol biosynthesis signatures. Single-cell RNA sequencing revealed enrichment for leukaemia stem cell signatures in high 4-PI cells. Validation across three cohorts (n = 671), including one with childhood AML, demonstrated the reproducibility and clinical utility of the 4-PI, even using cost-effective techniques like real-time quantitative polymerase chain reaction. Comparative analysis with 56 established prognostic indexes revealed the superior performance of the 4-PI, highlighting its potential to enhance AML risk stratification. Finally, the 4-PI demonstrated to be potential marker to reclassified patients from the intermediate ELN2017 category to the adverse category. In conclusion, the 4-PI emerges as a robust and straightforward prognostic tool to improve survival prediction in AML patients.

The Expression of NTAL and Its Protein Interactors Is Associated With Clinical Outcomes in Acute Myeloid Leukemia.

Non-T cell activation linker (NTAL) membrane protein depletion from lipid rafts by alkylphospholipids or downregulation by shRNA knockdown decreases cell viability through regulation of the Akt/PI3K pathway in mantle cell lymphoma and acute promyelocytic leukemia cells. Here, we confirmed that the knockdown of NTAL in acute myeloid leukemia (AML) cell lines was associated with decreased cell proliferation and survival. Similarly, a xenograft model using AML cells transduced with NTAL-shRNA and transplanted into immunodeficient mice led to a 1.8-fold decrease in tumor burden. Using immunoprecipitation, LC-MS/MS analysis, and label-free protein quantification, we identified interactors of NTAL in two AML cell lines. By evaluating the gene expression signatures of the NTAL protein interactors using the PREdiction of Clinical Outcomes from Genomic Profiles database, we found that 12 NTAL interactors could predict overall survival in AML, in at least two independent cohorts. In addition, patients with AML exhibiting a high expression of NTAL and its interactors were associated with a leukemic granulocyte-macrophage progenitor-like state. Taken together, our data provide evidence that NTAL and its protein interactors are relevant to AML cell proliferation and survival and represent potential therapeutic targets for granulocyte-macrophage progenitor-like leukemias.

Outcome misclassification: Impact, usual practice in pharmacoepidemiology database studies and an online aid to correct biased estimates of risk ratio or cumulative incidence.

PURPOSE: It is well documented that outcome misclassification can bias a point estimate. We aimed to understand current practice in addressing this bias in pharmacoepidemiology database studies and to develop an open source application (app) from existing methodology to demonstrate the impact and mechanism of this bias on results. METHODS: Studies of an exposure and a clinical outcome were selected from all Pharmacoepidemiology and Drug Safety publications during 2017 and any reference to outcome misclassification described. An app to correct risk ratio (RR) and cumulative incidence for outcome misclassification was developed from a published methodology and used to demonstrate the impact of correction on point estimates. RESULTS: Eight (19%) of 43 papers selected reported estimates of outcome ascertainment accuracy with positive predictive value (PPV) the most commonly reported measure (7 of 8 studies). Three studies (7%) corrected for the bias, 1 by exposure strata, and 5 (12%) restricted analyses to confirmed cases. The app (app http://apps.p-95.com/ISPE/) uses values of PPV and sensitivity (or a range of possible values) in each exposure strata and returns corrected point estimates and confidence intervals. The app demonstrates that small differences between comparison groups in PPV or sensitivity can introduce bias even when accuracy estimates are high. CONCLUSIONS: Outcome misclassification is not usually corrected in pharmacoepidemiology database studies although correction methods using routinely measured indices are available. Error indices are needed for each comparison group to correct RR estimates for these errors. The app should encourage understanding of this bias and increase adjustment.

Sequesterpene Lactones Isolated from a Brazilian Cerrado Plant (Eremanthus spp.) as Anti-Proliferative Compounds, Characterized by Functional and Proteomic Analysis, are Candidates for New Therapeutics in Glioblastoma.

Gliomas are responsible for more than 60% of all primary brain tumors. Glioblastoma multiforme (GBM), a grade IV tumor (WHO), is one of the most frequent and malignant gliomas. Despite two decades of advances in the discovery of new markers for GBM, the chemotherapy of choice falls to temozolomide after surgery and radiotherapy, which are not enough to increase the survival of patients to more than 15 months. It is urgent to discover new anti-glioma compounds. Many compounds derived from natural products have been used in the development of anti-tumor drugs. In this work, we have screened six low molecular weight sesquiterpene lactones, isolated from Eremanthus spp., and studied their function as anti-proliferative agents against GBM strains. We demonstrated that two of them, goyazensolide and lychnofolide, were effective in reducing cell viability, preventing the formation of anchorage-dependent colony and were able to pass through a mimetic blood-brain barrier making them candidates for glioma therapy, being more potent than temozolomide, according to in vitro assays for the cell lines tested. Proteomic analysis revealed a number of altered proteins involved in glycolytic metabolism and cellular catabolism.

Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells.

Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-ß, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.

Estimating the Burden of Medically Attended Norovirus Gastroenteritis: Modeling Linked Primary Care and Hospitalization Datasets.

Background: Norovirus is the leading cause of community-acquired and nosocomial acute gastroenteritis. Routine testing for norovirus is seldom undertaken, and diagnosis is mainly based on presenting symptoms. This makes understanding the burden of medically attended norovirus-attributable gastroenteritis (MA-NGE) and targeting care and prevention strategies challenging. Methods: We used linked population-based healthcare datasets (Clinical Practice Research Datalink General Practice OnLine Database linked with Hospital Episode Statistics Admitted Patient Care) to model the incidence of MA-NGE associated with primary care consultations or hospitalizations according to age groups in England in the period July 2007-June 2013. Results: Mean annual incidence rates of MA-NGE were 4.9/1000 person-years and 0.7/1000 person-years for episodes involving primary care or hospitalizations, respectively. Incidence rates were highest in children aged <5 years: 34.0 consultations/1000 person-years and 3.3 hospitalizations/1000 person-years. Medically attended norovirus-attributable gastroenteritis hospitalization rates were second highest in adults aged >65 years (1.7/1000 person-years). Conclusions: In this particular study, the burden of MA-NGE estimated from healthcare datasets was higher than previously estimated in small cohort studies in England. Routinely collected primary care and hospitalization datasets are useful resources to estimate and monitor the burden of MA-NGE in a population over time.

Identifying rare diseases using electronic medical records: the example of allergic bronchopulmonary aspergillosis.

PURPOSE: The purpose of the study is to evaluate whether primary care electronic medical records (EMRs) from patients with severe asthma can be used to identify allergic bronchopulmonary aspergillosis (ABPA) cases. METHODS: This cross-sectional feasibility study was conducted in adults with active and severe asthma registered with the Clinical Practice Research Datalink. A set of keywords flagged terms potentially indicative of ABPA in free-text comments of patients' EMRs to produce a grid on the basis of keywords' hit or miss. The grid was examined for occurrence and concurrence of keywords to discern patterns of concurrence potentially indicative of an underlying diagnosis of ABPA. RESULTS: The analyses included 3 653 169 free-text items from 21 054 patients. In total, 52 patients (0.25%) had at least one mention of 'ABPA' in their medical record; 67% of these patients also had a mention of 'aspergillus/aspergillosis', 54% of 'bronchiectasis', 42% of 'itraconazole' and 62% of 'IgE'. The term 'aspergillus/aspergillosis' occurred with a proportion of 1.84% (N = 387); 9% of these patients also had a mention of 'ABPA', and the remaining 91% were potential additional cases of ABPA. From the observed concurrence of keywords, we were able to devise a potential algorithm to identify cases with varying degrees of specificity. CONCLUSIONS: This study suggests that analysis of free text within asthmatic patients' EMRs may be used to identify potential cases of ABPA. This could be an efficient approach to identify rare conditions and to quantify their potential burden. © 2017 The Authors. Pharmacoepidemiology & Drug Safety Published by John Wiley & Sons Ltd.

DSP30 enhances the immunosuppressive properties of mesenchymal stromal cells and protects their suppressive potential from lipopolysaccharide effects: A potential role of adenosine.

Multipotent mesenchymal stromal cells (MSC) are imbued with an immunosuppressive phenotype that extends to several immune system cells. In this study, we evaluated how distinct Toll-like receptor (TLR) agonists impact immunosuppressive properties of bone marrow (BM)-MSC and explored the potential mechanisms involved. We show that TLR4 stimulation by lipopolysaccharide (LPS) restricted the ability of MSC to suppress the proliferation of T lymphocytes, increasing the gene expression of interleukin (IL)-1ß and IL-6. In contrast, stimulation of TLR9 by DSP30 induced proliferation and the suppressive potential of BM-MSC, coinciding with reducing tumor necrosis factor (TNF)-α expression, increased expression of transforming growth factor (TGF)-ß1, increased percentages of BM-MSC double positive for the ectonucleotidases CD39+CD73+ and adenosine levels. Importantly, following simultaneous stimulation with LPS and DSP30, BM-MSC's ability to suppress T lymphocyte proliferation was comparable with that of non-stimulated BM-MSC levels. Moreover, stimulation of BM-MSC with LPS reduced significantly the gene expression levels, on co-cultured T lymphocyte, of IL-10 and interferon (IFN)γ, a cytokine with potential to enhance the immunosuppression mediated by MSC and ameliorate the clinical outcome of patients with graft-versus-host disease (GVHD). Altogether, our findings reiterate the harmful effects of LPS on MSC immunosuppression, besides indicating that DSP30 could provide a protective effect against LPS circulating in the blood of GVHD patients who receive BM-MSC infusions, ensuring a more predictable immunosuppressive effect. The novel effects and potential mechanisms following the stimulation of BM-MSC by DSP30 might impact their clinical use, by allowing the derivation of optimal "licensing" protocols for obtaining therapeutically efficient MSC.

Importance of feasibility assessments before implementing non-interventional pharmacoepidemiologic studies of vaccines: lessons learned and recommendations for future studies.

PURPOSE: Investigational and marketed vaccines are increasingly evaluated, and manufacturers are required to put in place mechanisms to monitor long-term benefit-risk profiles. However, generating such evidence in real-world settings remains challenging, especially when rare adverse events are assessed. Planning of an appropriate study design is key to conducting a valid study. The aim of this paper is to illustrate how feasibility assessments support the generation of robust pharmacoepidemiological data. METHODS: Following an initiative launched by the International Society for Pharmacoepidemiology in May 2014, a working group including members of the private and public sectors, was formed to assess the value of conducting feasibility assessments as a necessary step before embarking on larger-scale post-licensure studies. Based on five real-life examples of feasibility assessments, lessons learned and recommendations were issued by the working group to support scientific reasoning and decision making when designing pharmacoepidemiologic vaccine studies. RESULTS: The working group developed a toolbox to provide a pragmatic approach to conducting feasibility assessments. The toolbox contains two main components: the scientific feasibility and the operational feasibility. Both components comprise a series of specific questions aimed at overcoming methodological and operational challenges. CONCLUSIONS: A feasibility assessment should be formalized as a necessary step prior to the actual start of any pharmacoepidemiologic study. It should remain a technical evaluation and not a hypothesis testing. The feasibility assessment report may facilitate communication with regulatory agencies toward improving the quality of study protocols and supporting the endorsement of study objectives and methods addressing regulatory commitments. © 2016 The Authors. Pharmacoepidemiology and Drug Safety published by John Wiley & Sons Ltd.

PROTEINS OF THE INTEGUMENTARY SYSTEM OF THE HONEYBEE, Apis mellifera.

The integument of insects and other arthropods is composed of an inner basal lamina coated by the epidermis, which secretes the bulk of the outer integument layer, the cuticle. The genome sequencing of several insect species has allowed predicting classes of proteins integrating the cuticle. However, only a small proportion of them, as well as other proteins in the integumentary system, have been validated. Using two-dimensional gel electrophoresis coupled with mass spectrometry, we identified 45 different proteins in a total of 112 selected gel spots derived from thoracic integument samples of developing honeybee workers, including 14 cuticular proteins (AmelCPR 3, AmelCPR 12, AmelCPR 16, AmelCPR 27, apidermin 2, apidermin 3, endocuticle structural glycoprotein SgAbd-8-like, LOC100577363, LOC408365, LOC413679, LOC725454, LOC100576916, LOC725838, and peritrophin 3-C analogous). Gene ontology functional analysis revealed that the higher proportions of the identified proteins have molecular functions related to catalytic and structural molecule activities, are involved in metabolic biological processes, and pertain to the protein class of structural or cytoskeletal proteins and hydrolases. It is noteworthy that 26.7% of the identified proteins, including five cuticular proteins, were revealed as protein species resulting from allelic isoforms or derived from posttranslational modifications. Also, 66.7% of the identified cuticular proteins were expressed in more than one developmental phase, thus indicating that they are part of the larval, pupal, and adult cuticle. Our data provide experimental support for predicted honeybee gene products and new information on proteins expressed in the developing integument.

Linker for activation of T-cell family member2 (LAT2) a lipid raft adaptor protein for AKT signaling, is an early mediator of alkylphospholipid anti-leukemic activity.

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 µm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 µm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five µm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

Proteomic analysis of exosomes secreted during the epithelial-mesenchymal transition and potential biomarkers of mesenchymal high-grade serous ovarian carcinoma.

BACKGROUND: The epithelial-mesenchymal transition (EMT) promotes cell signaling and morphology alterations, contributing to cancer progression. Exosomes, extracellular vesicles containing proteins involved in cell-cell communication, have emerged as a potential source of biomarkers for several diseases. METHODS: Our aim was to assess the proteome content of exosomes secreted after EMT-induction to identify potential biomarkers for ovarian cancer classification. EMT was induced in the ovarian cancer cell line CAOV3 by treating it with EGF (10 ng/mL) for 96 h following 24 h of serum deprivation. Subsequently, exosomes were isolated from the supernatant using selective centrifugation after decellularization, and their characteristics were determined. The proteins present in the exosomes were extracted, identified, and quantified using Label-Free-Quantification (LFQ) via Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). To identify potential biomarkers, the obtained proteomic data was integrated with the TGGA database for mRNA expression using principal component analysis and a conditional inference tree. RESULTS: The exosomes derived from CAOV3 cells exhibited similar diameter and morphology, measuring approximately 150 nm, regardless of whether they were subjected to EMT stimulation or not. The proteomic analysis of proteins from CAOV3-derived exosomes revealed significant differential regulation of 157 proteins, with 100 showing upregulation and 57 downregulation upon EMT induction. Further comparison of the upregulated proteins with the TCGA transcriptomic data identified PLAU, LAMB1, COL6A1, and TGFB1 as potential biomarkers of the mesenchymal HGSOC subtype. CONCLUSIONS: The induction of EMT, the isolation of exosomes, and the subsequent proteomic analysis highlight potential biomarkers for an aggressive ovarian cancer subtype. Further investigation into the role of these proteins is warranted to enhance our understanding of ovarian cancer outcomes.

Analysis of ovarian cancer cell secretome during epithelial to mesenchymal transition reveals a protein signature associated with advanced stages of ovarian tumors.

Ovarian cancer (OvCA) is the most lethal neoplasia among gynecologic malignancies and faces high rates of new cases particularly in South America. In special, the High Grade Serous Ovarian Carcinoma (HGSC) presents very poor prognosis with deaths caused mainly by metastasis. Among several mechanisms involved in metastasis, the Epithelial to Mesenchymal Transition (EMT) molecular reprogramming represents a model for latest stages of cancer progression. EMT promotes important cellular changes in cellular adhesion and cell-cell communication, which particularly depends on the paracrine signaling from neighbor cells. Considering the importance of cellular communication during EMT and metastasis, here we analyzed the changes in the secretome of the ovarian cancer cell line Caov-3 induced to EMT by Epidermal Growth Factor (EGF). Using a combination of GEL-LC-MS/MS and stable isotopic metabolic labelling (SILAC), we identified up-regulated candidates during EMT as a starting point to identify relevant proteins for HGSC. Based on public databases, our candidate proteins were validated and prioritized for further analysis. Importantly, several of the protein candidates were associated with cellular vesicles, which are important to the cell-cell communication and metastasis. Furthermore, the association of candidate proteins with gene expression data uncovered a subset of proteins correlated with the mesenchymal subtype of ovarian cancer. Based on this relevant molecular signature for aggressive ovarian cancer, supported by protein and gene expression data, we developed a targeted proteomic method to evaluate individual OvCA clinical samples. The quantitative information obtained for 33 peptides, representative of 18 proteins, was able to segregate HGSC from other tumor types. Our study highlighted the richness of the secretome and EMT to reveal relevant proteins for HGSC, which could be used in further studies and larger patient cohorts as a potential stratification signature for ovarian cancer tumor that could guide clinical conduct for patient treatment.

Hepatitis B virus infection and the risk of liver disease progression in type 2 diabetic patients with potential nonalcoholic fatty liver disease: a retrospective, observational, cohort study in the United Kingdom Clinical Practice Research Datalink.

OBJECTIVE: Assess the risk of progression to cirrhosis and hepatocellular carcinoma (HCC) due to hepatitis B virus (HBV)-infection in patients with nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). METHODS: Retrospective cohort study in the UK Clinical Practice Research Datalink with three cohorts: subjects with T2DM and HBV infection (T2DM+HBV cohort; N = 297), with T2DM without HBV-infection (T2DM cohort; N = 261 865), and with HBV-infection without T2DM (HBV cohort; N = 3630). Primary analyses were performed on the three cohorts and secondary analyses on subcohorts including patients with NAFLD diagnosis code (N = 6599). Case/outcome definitions were formulated with International Classification of Diseases/Read codes/laboratory results and classified using validated algorithms. Adjusted incidence rate ratios (IRR) were estimated with a Poisson regression model. RESULTS: When comparing the T2DM+HBV and T2DM cohorts, adjusted IRRs were 14.06 (95% confidence interval: 4.47-44.19) for cirrhosis and 2.83 (1.06-7.55) for HCC. When comparing the T2DM+HBV and HBV cohorts, adjusted IRRs were 0.68 (0.21-2.27) for cirrhosis and 1.39 (0.46-4.20) for HCC. No cirrhosis cases were identified in T2DM+NAFLD+HBV patients; IRs were 16.92/10 000 person-years (12.97-21.69) and 85.24/10 000 person-years (10.32-307.91) in the T2DM+NAFLD and NAFLD+HBV cohorts. CONCLUSION: HBV-infection increased significantly the risk for cirrhosis among T2DM patients, however, not beyond the expected incremental risk among infected non-T2DM subjects. Our approach to evaluate the role of T2DM/NAFLD and HBV-infection in liver disease progression could be applied to other settings with higher HBV prevalence.

Proteomics analysis reveals the role of ubiquitin specific protease (USP47) in Epithelial to Mesenchymal Transition (EMT) induced by TGFß2 in breast cells.

Epithelial to Mesenchymal Transition (EMT) is a normal cellular process that is also triggered during cancer progression and metastasis. EMT induces cellular and microenviromental changes, resulting in loss of epithelial features and acquisition of mesenchymal phenotypes. The growth factor TGFß and the transcription factor SNAIL1 (SNAIL) have been described as inducers of EMT. Here, we carried out an EMT model with non-tumorigenic cell line MCF-10A induced with the TGFß2 specific isoform of TGF protein family. The model was validated by molecular, morphological and functional experiments and showed correlation with the up-regulation of SNAIL. In order to identify additional regulators of EMT in this non-tumorigenic model, we explored quantitative proteomics, which revealed the Ubiquitin carboxyl-terminal hydrolase 47 (USP47) as one of the top up-regulated proteins. USP47 has a known role in cell growth and genome integrity, but not previously correlated to EMT. After validating USP47 alterations using MRM and antibody-based assays, we demonstrated that the chemical inhibition of USP47 with the inhibitor P5091 reduced expression of EMT markers and reverted morphological changes in MCF-10A cells undergoing EMT. These results support the involvement of USP47 in our EMT model as well as potential applications of deubiquitinases as therapeutic targets for cancer progression management. BIOLOGICAL SIGNIFICANCE: Metastasis is responsible for most cancer-associated mortality. Additionally, metastasis requires special attention, as the cellular transformations make treatment at this stage very difficult or occasionally impossible. Early steps in cancer metastasis involve the ability to detach from the solid tumor mass and invade the surrounding stromal tissues through cohesive migration, or a mesenchymal or amoeboid invasion. One of the first steps for metastatic cascade is denominated epithelial to mesenchymal transition (EMT), which can be triggered by different factors. Here, our efforts were directed to better understand this process and identify new pathways that contributes for acquisition of EMT, mainly focused on post translational modifications related to ubiquitin proteasome system. Our model of EMT induction by TGFß2 mimics early stage of metastatic cancer in epithelial breast cells and a proteomic study carried out for such model demonstrates that the deubiquitinase enzyme USP47 acts in SNAIL stabilization, one of the most important transcription factors for EMT phenotype acquisition and consequent metastasis. In addition, the inhibiton of USP47 with P5091, reverted the EMT phenotype. Together the knowledge of such processes of cancer progression and regulation can help in designing new strategies for combined therapies for control of cancer in early stages.

NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia.

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17-11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

Herpes zoster related healthcare burden and costs in immunocompromised (IC) and IC-free populations in England: an observational retrospective database analysis.

OBJECTIVE: Individuals with immunocompromised (IC) conditions are at a higher risk of developing herpes zoster (HZ) than IC-free individuals. This study assessed the healthcare resource utilisation (HCRU) burden and costs, of HZ in IC and IC-free individuals ≥18 years of age (YOA). METHODS: We conducted an observational retrospective study in a cohort of IC (n=621 588) and IC-free (n=621 588) individuals, matched by age, gender and General Practitioner practice region, contributing to the Clinical Practice Research Datalink database from 2000 to 2012 and linked to the Hospital Episode Statistics inpatient data. HCRU (ie, primary and secondary care consultations, hospital inpatient stays and treatment prescriptions) was analysed from 7 days before to: (1) 30, (2) 365 days after the HZ diagnosis date for individuals with (1) HZ only (no postherpetic neuralgia (PHN)) and (2) individuals with HZ and PHN only. Healthcare costs were computed by multiplying the number of units of resources used by the unit costs, summed across all HCRU categories to obtain a total cost per subject. Values were expressed in 2014 UK pound sterling (£) and presented for HZ cases overall, stratified by age (ie, 18-49, 50-59, 60-69, 70-79 and ≥80 YOA) and IC status. RESULTS: The percentage of HZ cases requiring hospitalisation was higher in IC individuals (2.7% vs 0.4% in IC and IC-free individuals aged 18-49 YOA, respectively and 9.5% vs 7.5% in IC and IC-free individuals aged ≥80 YOA, respectively). Similarly, HZ-related mean treatment costs per subject were higher in IC individuals (£189 vs £104 in IC and IC-free individuals aged 18-49 YOA, respectively and £557 vs £401 in IC and IC-free individuals aged ≥80 YOA, respectively). Costs varied considerably by IC condition. CONCLUSIONS: Individuals with IC conditions, have a high burden of HZ, associated with an increased risk of HZ and high HZ-related healthcare costs.

Evaluation of a risk score to predict future Clostridium difficile disease using UK primary care and hospital data in Clinical Practice Research Datalink.

We evaluated the applicability of a Clostridium difficile infection (CDI) risk index developed for patients at hospital discharge to identify persons at high-risk of CDI in a primary care population. This retrospective observational study used data from the UK Clinical Practice Research Datalink, linked with Hospital Episodes Statistics. The risk index was based on the following patient characteristics: age, previous hospitalizations, days in hospital, and prior antibiotics use. Individual risk scores were calculated by summing points assigned to pre-defined categories for each characteristic. We assessed the association of risk factors with CDI by multivariate logistic regression. The estimated CDI incidence rate was 4/10,000 and 2/10,000 person-years in 2008 and 2012, respectively. On an index with a maximal risk of 19, a cut-off for high risk of ≥7 had sensitivity, specificity and positive predictive values of 80%, 87% and 12%, respectively. A high-risk person had a ~ 35% higher risk of CDI than a low-risk person. Multivariate risk factor analysis indicated a need to reconsider the relative risk scores. The CDI risk index can be applied to the UK primary care population and help identify study populations for vaccine development studies. Reassessing the relative weights assigned to risk factors could improve the index performance in this setting.

A proteomics outlook towards the elucidation of epithelial-mesenchymal transition molecular events.

The main cause of death in cancer is the spread, or metastasis, of cancer cells to distant organs with consequent tumor formation. Additionally, metastasis is a process that demands special attention, as the cellular transformations make cancer at this stage very difficult or occasionally even impossible to be cured. The main process that converts epithelial tumor cells to mesenchymal-like metastatic cells is the Epithelial to Mesenchymal Transition (EMT). This process allows stationary and polarized epithelial cells, which are connected laterally to several types of junctions as well as the basement membrane, to undergo multiple biochemical changes that enable disruption of cell-cell adherence and apical-basal polarity. Moreover, the cells undergo important reprogramming to remodel the cytoskeleton and acquire mesenchymal characteristics such as enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and a large increase in the production of ECM components. As expected, the alterations of the protein complement are extensive and complex, and thus exploring this by proteomic approaches is of particular interest. Here we review the overall findings of proteome modifications during EMT, mainly focusing on molecular signatures observed in multiple proteomic studies as well as coordinated pathways, cellular processes and their clinical relevance for altered proteins. As a result, an interesting set of proteins is highlighted as potential targets to be further investigated in the context of EMT, metastasis and cancer progression.

Proteomic Identification and Time-Course Monitoring of Secreted Proteins During Expansion of Human Mesenchymal Stem/Stromal in Stirred-Tank Bioreactor.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) is widely recognized for the treatment of several diseases, including acute graft-vs.-host disease (GVHD), hematological malignancies, cardiovascular, bone, and cartilage diseases. More recently, this therapeutic efficacy has been attributed to the bioactive molecules that these cells secrete (secretome), now being referred as medicinal signaling cells. This fact raises the opportunity of therapeutically using MSC-derived soluble factors rather than cells themselves, enabling their translation into the clinic. Indeed, many clinical trials are now studying the effects of MSC-secretome in the context of cell-free therapy. MSC secretome profile varies between donors, source, and culture conditions, making their therapeutic use very challenging. Therefore, identifying these soluble proteins and evaluating their production in a reproducible and scalable manner is even more relevant. In this work, we analyzed the global profile of proteins secreted by umbilical cord matrix (UCM) derived-MSC in static conditions by using mass spectrometry, enabling the identification of thousands of proteins. Afterwards, relevant proteins were chosen and monitored in the supernatant of a fully-controllable, closed and scalable system (bioreactor) by using multiple reaction monitoring (MRM) mass spectrometric technique in a time-dependent manner. The results showed that the majority of interesting proteins were enriched through time in culture, with the last day of culture being the ideal time for supernatant collection. The use of this regenerative "soup," which is frequently discarded, could represent a step toward a safe, robust and reproducible cell-free product to be used in the medical therapeutic field. The future use of chemically defined culture-media will certainly facilitate secretome production according to Good Manufacturing Practice (GMP) standards.