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PDF file, 87KB, Differentially Expressed Genes by miR-708 Status in Never-Smoker Lung Adenocarcinoma.

Supplementary Tables 1-3 from Polymorphisms in Telomere Maintenance Genes and Risk of Lung Cancer

PDF file, 58KB, Potential Target Genes of miR-708.

This study was conducted to comprehensively evaluate the associations between polymorphisms in telomere maintenance genes (TERT, TRF1, TNKS1, TRF2, RAP1, and POT1) and lung cancer risk. We captured 35 polymorphisms in the genes and determined their frequencies in 27 healthy Koreans. Ten haplotype-tagging polymorphisms were examined in a case-control study that consisted of 720 lung cancer patients and 720 healthy controls. The TERT rs2735940 g.C > T and rs2736098 g.G > A, and TNKS1 rs6985140 g.A > G were significantly associated with the risk of lung cancer. In the haplotype analysis, the TERT rs2735940T/rs2736098A haplotype (ht4) was associated with a significantly increased risk of lung cancer compared with the rs2735940C/rs2736098G haplotype (adjusted odds ratio, 1.26; 95% confidence interval, 1.07-1.50; P = 0.008). When the TERT ht4 and TNKS1 rs6985140G as risk alleles, the risk of lung cancer increased in a dose-dependent manner as the number of risk alleles increased (Ptrend < 0.001). Subjects with two to four risk alleles were at a significantly increased risk of lung cancer (adjusted odds ratio, 1.67; 95% confidence interval, 1.23-2.27; P = 0.001) compared with subjects with zero risk allele. These findings suggest that genetic variants in the TERT and TNKS1 genes contribute to genetic susceptibility to lung cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(10):2773–81)

Purpose: miRNA plays an important role in human disease and cancer. We seek to investigate the expression status, clinical relevance, and functional role of miRNA in non–small cell lung cancer.Experimental Design: We conducted miRNA expression profiling in matched lung adenocarcinoma and uninvolved lung using 56 pairs of fresh-frozen (FF) and 47 pairs of formalin-fixed, paraffin-embedded (FFPE) samples from never smokers. The most differentially expressed miRNA genes were evaluated by Cox analysis and log-rank test. Among the best candidate, miR-708 was further examined for differential expression in two independent cohorts. Functional significance of miR-708 expression in lung cancer was examined by identifying its candidate mRNA target and through manipulating its expression levels in cultured cells.Results: Among the 20 miRNAs most differentially expressed between tested tumor and normal samples, high expression level of miR-708 in the tumors was most strongly associated with an increased risk of death after adjustments for all clinically significant factors including age, sex, and tumor stage (FF cohort: HR, 1.90; 95% CI, 1.08–3.35; P = 0.025 and FFPE cohort: HR, 1.93; 95% CI, 1.02–3.63; P = 0.042). The transcript for TMEM88 gene has a miR-708 binding site in its 3′ UTR and was significantly reduced in tumors high of miR-708. Forced miR-708 expression reduced TMEM88 transcript levels and increased the rate of cell proliferation, invasion, and migration in culture.Conclusions: miRNA-708 acts as an oncogene contributing to tumor growth and disease progression by directly downregulating TMEM88, a negative regulator of the Wnt signaling pathway in lung cancer. Clin Cancer Res; 18(13); 3658–67. ©2012 AACR.

PDF file, 154KB, TMEM88 is a putative target of miR-708 by MicroCosm database. The target site of miR-708 is located in the 3'UTR, 286 to 309 bases from downstream of the stop codon sequence of TMEM88. Asterisks indicate the binding site.

PDF file, 147KB, Validation of miR-708 Overexpression by Real-Time qPCR in Lung Squamous cell carcinoma. Non-paired mostly smoker Squamous cell carcinomas and normal FFPE tissues from the National Cancer Institute cohort were examined. Sample numbers (n) and tissue types are indicated on each graph. P values are as shown by unpaired 2-tailed t test.

PDF file, 90KB, Endogenous expression level of miR-708 in various cell lines. Real-time quantitative polymerase chain reaction analyses were performed as described in the "Methods" section.

PDF file, 69KB, Clinical Information of the three lung adenocarcinoma Cohorts.

OBJECTIVE: To identify hallmark genes and biomolecular processes in aortitis using high-throughput gene expression profiling. Additionally, to provide a range of potentially new drug targets (genes) and therapeutics from a pharmacogenomic network analysis. METHODS: Bulk RNA-seq was performed on surgically resected ascending aortic tissues from inflammatory (giant cell arteritis [GCA] with or without polymyalgia rheumatica [PMR] = 8; clinically isolated aortitis [CIA] = 17) and non-inflammatory (n = 25) aneurysms undergoing surgical aortic repair. Differentially expressed genes (DEGs) between the two patient groups were identified while controlling for clinical covariates. A protein-protein interaction model, drug-gene target information, and the DEGs were used to construct a pharmacogenomic network for identifying promising drug targets and potentially new treatment strategies in aortitis. RESULTS: Overall, tissue gene expression patterns were the most associated with disease state than with any other clinical characteristic. We identified 159 and 93 genes that were significantly upregulated and downregulated, respectively, in inflammatory aortic aneurysms compared to non-inflammatory aortic aneurysms. We found that the upregulated genes were enriched in immune-related functions, whereas the downregulated genes were enriched in neuronal processes. Notably, gene expression profiles of inflammatory aortic aneurysms from patients with GCA were no different than those from patients with CIA. Finally, our pharmacogenomic network analysis identified genes that could potentially be targeted by immunosuppressive drugs currently approved for other inflammatory diseases. CONCLUSION: We performed the first global transcriptomics analysis in inflammatory aortic aneurysms from surgically resected aortic tissues. We identified signature genes and biomolecular processes, while finding that CIA may be a limited presentation of GCA. Moreover, our computational network analysis revealed potential novel strategies for pharmacologic interventions, and suggests future biomarker discovery directions for the precise diagnosis and treatment of aortitis.

Background There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 μg of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated with DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. Results Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3′ region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R > 0.9) than RNAse-H enzymatic depletion (R > 0.85). Conclusion In this study, our results showed that 1 μg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.

BACKGROUND Prestorage leukoreduction has the advantage over poststorage leukoreduction in reducing leukocyte-derived molecules in red blood cells (RBC) unit, which induce immunomodulation. Our institution newly introduced prestorage leukoreduction, instead of conventional poststorage leukoreduction, for liver transplant recipients since March 2012. In this study, we aimed to evaluate the risk of posttransplant hepatocellular carcinoma (HCC) recurrence after the conversion of poststorage leukoreduction into prestorage leukoreduction for transfused allogeneic RBCs. METHODS Among 220 patients who underwent living-donor liver transplantation for HCC, 83 of 113 who received only poststorage-leukoreduced RBCs were matched with 83 of 107 who received only prestorage-leukoreduced RBCs using 1:1 propensity score matching based on factors like tumor biology. The primary outcome was overall HCC recurrence. Survival analysis was performed with death as a competing risk event. RESULTS In the matched cohort, recurrence probability at 1, 2, and 5 years posttransplant was 9.6%, 15.6%, and 18.1% in prestorage group and 15.6%, 21.6%, and 33.7% in poststorage group (hazard ratio [HR], 0.52; 0.28-0.97; P = 0.040). Multivariable analysis confirmed a significance of prestorage leukoreduction (HR, 0.29; 0.15-0.59; P < 0.001). Overall death risk was also lower with prestorage leukoreduction (HR, 0.51; 0.26-0.99; P = 0.049). In subgroup analysis for the unmatched cohort, recurrence risk was significantly lower in prestorage group within the patients who underwent surgery 2 years (HR, 0.24; 0.10-0.61; P = 0.002), 1 year (HR, 0.16; 0.03-0.92; P = 0.040), and 6 months (HR, 0.13; 0.02-0.85; P = 0.034), respectively, before and after the conversion to prestorage leukoreduction. CONCLUSIONS Our findings suggest a potential benefit of prestorage leukoreduction in reducing the risk of HCC recurrence in liver transplant recipients who received allogeneic RBCs during the perioperative period.

Background Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3′ mRNA-seq method sequences the 3′ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3′ mRNA-Seq using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation. Results The total mapped reads of QuantSeq 3′ mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3′ mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3′ mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67). Conclusion In this study, QuantSeq 3′ mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3′ mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.

Single cell RNA sequencing is a technology that provides the capability of analyzing the transcriptome of a single cell from a population. So far, single cell RNA sequencing has been focused mostly on human cells due to the larger starting amount of RNA template for subsequent amplification. One of the major challenges of applying single cell RNA sequencing to microbial cells is to amplify the femtograms of the RNA template to obtain sufficient material for downstream sequencing with minimal contamination. To achieve this goal, efforts have been focused on multiround RNA amplification, but would introduce additional contamination and bias. In this work, we for the first time coupled a microfluidic platform with multiple displacement amplification technology to perform single cell whole transcriptome amplification and sequencing of Porphyromonas somerae, a microbe of interest in endometrial cancer, as a proof-of-concept demonstration of using single cell RNA sequencing tool to unveil gene expression heterogeneity in single microbial cells. Our results show that the bacterial single-cell gene expression regulation is distinct across different cells, supporting widespread heterogeneity.

Nanosized Ni, NiCo-Y₂O₃ powders were successfully synthesized at low temperature via a simple polymer solution route. As an organic carrier, polyvinyl alcohol (PVA) afforded an atom-scale homogeneous precursor gel, which in turn gave fully crystallized nanosized Ni, NiCo-Y₂O₃ powder upon calcination at a low temperature under an Ar-4% H₂ atmosphere. The PVA content, calcination temperature, heating time, and reduction conditions affected the microstructure and crystallization behavior of the as-synthesized powders. The PVA content also influenced the synthesis behavior and microstructure of the final powder. The particle size increased with an increase in the calcination temperature and decrease in the PVA content. At a PVA-metal ion ratio of 4:1, the measured particle size was about 20 nm. The results of TEM mapping on the NiCo-Y (0.6 wt%) powders revealed a well-dispersed Y₂O₃ phase in the NiCo crystalline matrix.

Gene fusions involving the anaplastic lymphoma kinase ( ALK) gene occurs in approximately 3-5% of lung adenocarcinomas. The most common fusion partner is EML4 but several other fusion partners to the ALK kinase domain have been identified. Herein we report a lung adenocarcinoma case harboring a fusion between ALK and Striatin ( STRN ), with extended responses to targeted therapy. Using CT-guided needle biopsy approach, we successfully generated a patient-derived xenograft model from this tumor and molecularly characterized the STRN-ALK gene fusion.

Background Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown. Methods Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma. Results Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients. Conclusion Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM. Trial registration NCT03384108 and NCT03119883

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate β-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in β-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat β-cells. Conversely, TBK1 overexpression decreased sensitivity of β-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of β-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of β-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived β-cells and human islets. TBK1 expression was increased in β-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and β-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional β-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a β-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional β-cells.

Problem Prenatal exposure to metabolic dysregulation arising from maternal obesity can have negative health consequences in post-natal life. To date, the specific effects of maternal obesity on fetal immunity at a cellular level have not been well characterized. Method of study Using cord blood mononuclear cells (CBMCs) and cord plasma (n = 9/group) isolated from infants born to women with a high body mass index (BMI>25kg/m2 ) compared to women with a normal BMI (18-25kg/m2 ), we evaluated differences in immune cell populations using single-cell mass cytometry (CyTOF). CBMCs were matched according to potentially confounding variables, such as maternal and gestational age, ethnicity, smoking status, and gravidity. Statistical results were adjusted for fetal sex. Data were analyzed by viSNE and FlowSOM softwares in Cytobank™ . Results In newborn CBMCs from women with high BMI, we observed changes in frequency and phenotype of immune cell populations, including significant increases in CD4+ T cells and decreases in myeloid cell populations. IL-12p40 and MDC concentrations were significantly elevated in the high BMI group compared to control. Conclusion This study demonstrates an association between maternal obesity and fetal immunity. Our results warrant following long-term immunologic outcomes and associated clinical risks in children born to women with a high pre-pregnancy BMI.

Objective The aim of this study is to evaluate the association between fresh red blood cell (RBC) transfusion and recipient survival after liver transplantation. Background Fresh RBC products contain many viable leukocytes. Allogeneic leukocytes are responsible for adverse transfusion reactions in the immunocompromised host. Methods Among 343 liver transplant recipients who underwent perioperative RBC transfusion, 91 of 226 who did not receive fresh RBCs were matched with 91 of 117 who received fresh RBCs with 1:1 matching ratio using the propensity score based on the amount of transfused blood products and others. Survival analysis was performed using the Cox model. Results All transfused 3230 RBCs were leukoreduced and irradiated. Before matching, recipients in fresh RBC group received 3 U (2-6 U) of fresh RBCs. After a median follow-up of 60 months, 60 of 343 recipients (17.5%) died. Survival probability at 1/2/5 years after transplantation was 94.7%/92.0%/85.8% for nonfresh RBC group and 82.9%/76.0%/72.0% for fresh RBC group [death hazard ratio (HR) = 2.37 (1.43-3.94), P = 0.001]. In multivariable analysis, fresh RBC transfusion was significantly associated with increased death risk [HR = 2.33 (1.35-4.01), P = 0.002]. After matching, recipients in fresh RBC group received 3 U (2-5 U) of fresh RBCs. After a median follow-up of 56 months, 35 of 182 recipients (19.2%) died. Survival probability at 1/2/5 years was 95.6%/93.2%/86.0% for nonfresh RBC group and 85.7%/78.0%/73.0% for fresh RBC group [HR = 2.23 (1.43-3.94), P = 0.028]. Multivariable analysis confirmed a significance of fresh RBC transfusion [HR = 3.20 (1.51-6.78), P = 0.002]. Conclusion Our findings suggest a potential negative impact of fresh RBC transfusion on the survival of patients undergoing liver transplantation.

Introduction: Multiple gene expression profile assays have successfully prognosticated the overall survival (OS) outcomes of patients with Newly Diagnosed Multiple Myeloma (NDMM) and are now available for use in clinical practice. The genes included in these profiles encompass several aspects of cancer biology broadly such as cell-cycle biology, energy metabolism, migration, adhesion, immune evasion etc. Since altered energy metabolism is one of the hallmarks of all cancer cells, our goal was to take a deep-dive into specific genes associated with intracellular central carbon energy metabolism (CCEM) pathways such as oxidative phosphorylation (OxPhos) as well as glycolysis and lactate fermentation (Glyco-Lac) in MM cells to determine their impact on the pathogenesis and prognosis of MM patients. Methods: Gene expression level data via RNA sequencing from the COMMPASS dataset (made available by the Multiple Myeloma Research Foundation (MMRF)) was utilized. The expression levels of all genes associated with the OxPhos (N = 118) and Glyco-Lac (N = 34) pathways were evaluated in CD138+ cells derived from patients with NDMM. Each gene was dichotomized into low and high expression groups -based on their expression level above and below the median fragments per kilobase of transcript per million (FPKM), respectively. The prognostic significance of dichotomizing each of the 152 genes in this NDMM cohort was evaluated by levels of their hazard ratios (HRs) for OS determined by the log-rank method. Genes from the OxPhos and Glyco-Lac pathways were selected based on their significant negative impact on OS, wide representation of their biologic activity throughout the pathway and high independence from each other. This resulted in the selection of 12 distinct genes of interest. A scoring system consisting of assigning a point for every gene where their FPKM was above the median yielded a minimum of 0 and a maximum of 12 for the set of genes in the OxPhos and Glyco-Lac pathways to create an energy metabolism score (EMS). Patients were then categorized into "low" (0-8) or "high" (9-12) EMS groups. In addition, patients were designated as having high-risk cytogenetics if they had any of the following: t(4;14), t(14;16), t(14;20), del17p, and Amp 1q. Survival analysis was performed using the Kaplan-Meier survival analysis and compared via the log-rank method. Finally, the distribution of the EMS score among patients within a spectrum of plasma cell disorders such as, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma and MM as well as normal plasma cells was assessed using a previously published transcriptome dataset (GSE6477). Results: A total of 766 patients were included in this study. The selected genes included in this scoring system and their biological relevance in the OxPhos and Glyco-Lac pathways are depicted in Figure A. The distribution of low and high EMS groups across ISS stage and cytogenetic risk are depicted in Figure B and C respectively. The median OS (mOS) and PFS for the high (N = 248) vs. low (N = 518) EMS groups was 55 vs. 94 months (HR: 2.41; (95%CI: 1.85-3.14); P MGUS -> SMM -> MM) (Figure H). Conclusions: The expression levels of specific genes associated with the activity of intracellular CCEM pathways such as glycolysis, lactate fermentation and oxidative phosphorylation in plasma cells provides evidence for the impact of cellular energy metabolism on the pathogenesis and survival outcomes of MM. This provides an opportunity for developing future studies aimed at understanding the energy metabolism pathways of plasma cell malignancies in greater detail so as to exploit them for diagnostic and therapeutic purposes in MM. Figure 1 Figure 1. Disclosures Kumar: Novartis: Research Funding; Amgen: Consultancy, Research Funding; Beigene: Consultancy; Carsgen: Research Funding; Merck: Research Funding; Antengene: Consultancy, Honoraria; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Roche-Genentech: Consultancy, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Bluebird Bio: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Dispenzieri: Alnylam: Research Funding; Sorrento Therapeutics: Consultancy; Oncopeptides: Consultancy; Pfizer: Research Funding; Takeda: Research Funding; Janssen: Consultancy, Research Funding. Kapoor: AbbVie: Research Funding; Takeda: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding; Ichnos Sciences: Research Funding; Regeneron Pharmaceuticals: Research Funding; Glaxo SmithKline: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding. Dingli: Apellis: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; GSK: Consultancy; Novartis: Research Funding; Alexion: Consultancy. Lin: Gamida Cell: Consultancy; Takeda: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Juno: Consultancy; Novartis: Consultancy; Legend: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Bluebird Bio: Consultancy, Research Funding; Sorrento: Consultancy; Merck: Research Funding; Vineti: Consultancy. Gertz: Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria; Aurora Biopharma: Other: Stock option; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring; Ionis Pharmaceuticals: Other: Advisory Board; Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy. Fonseca: Aduro: Consultancy; Kite: Consultancy; Juno: Consultancy; Merck: Consultancy; Takeda: Consultancy; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; AbbVie: Consultancy; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; GSK: Consultancy.

Aberrant expression of neuroendocrine markers is extremely rare in endothelial neoplasms, with only a single report describing three cases. Although originally classified as conventional angiosarcoma, further assessment of these tumors revealed a strikingly composite morphology composed of retiform and epithelioid elements reminiscent of composite hemangioendothelioma, a rare subtype of hemangioendothelioma. To further investigate these findings, available materials from 11 morphologically distinctive endothelial tumors showing neuroendocrine marker expression were retrieved from our archives. Immunohistochemistry for CD31, CD34, FLI-1, synaptophysin, chromogranin, D2-40, ERG, keratin (OSCAR), and CAMTA1 was performed. Total RNA from five cases were extracted and subjected to whole transcriptome sequencing. Clinical follow-up was obtained. These tumors were found to arise in five males and six females in patients from 9 to 55 years in age (median 47 years). They arose both in superficial (wrist, ankle, scalp, hip, and foot) and deep (periaortic tissues, C5 vertebra, pulmonary vein, and liver) locations. All contained elongated, retiform vascular channels lined by hyperchromatic 'hobnail' endothelial cells and a solid growth of uniform epithelioid cells reminiscent of epithelioid hemangioendothelioma. Hemangioma-like foci also lined by hobnail endothelial cells were frequently present. Mitotic activity was typically

The relationship between Primary Biliary Cholangitis (PBC), a chronic cholestatic autoimmune liver disease, and the peripheral immune system remains to be fully understood. Herein, we performed the first mass cytometry (CyTOF)-based, immunophenotyping analysis of the peripheral immune system in PBC at single-cell resolution. CyTOF was performed on peripheral blood mononuclear cells (PBMCs) from PBC patients (n=33) and age-/sex-matched healthy controls (n=33) to obtain immune cell abundance and marker expression profiles. Hiearchical clustering methods were applied to identify immune cell types and subsets significantly associated with PBC. Subsets of gamma-delta T cells (CD3+TCRgd+), CD8+ T cells (CD3+CD8+CD161+PD1+), and memory B cells (CD3-CD19+CD20+CD24+CD27+) were found to have lower abundance in PBC than in control. In contrast, higher abundance of subsets of monocytes and naïve B cells were observed in PBC compared to control. Furthermore, several naïve B cell (CD3-CD19+CD20+CD24-CD27-) subsets were significantly higher in PBC patients with cirrhosis (indicative of late-stage disease) than in those without cirrhosis. Alternatively, subsets of CD8+CD161+ T cells and memory B cells were lower in abundance in cirrhotic relative to non-cirrhotic PBC patients. Future immunophenotyping investigations could lead to better understanding of PBC pathogenesis and progression, and also to the discovery of novel biomarkers and treatment strategies.

Therapeutic benefits of deep brain stimulation (DBS), a neurosurgical treatment for certain movement disorders and other neurologic conditions, are well documented, but DBS mechanisms remain largely unexplained. DBS is thought to modulate pathological neural activity. However, although astrocytes, the most numerous cell type in the brain, play a significant role in neurotransmission, chemical homeostasis and synaptic plasticity, their role in DBS has not been fully examined. To investigate astrocytic function in DBS, we applied DBS-like high frequency electrical stimulation for 24 h to human astrocytes in vitro and analyzed single cell transcriptome mRNA profile. We found that DBS-like high frequency stimulation negatively impacts astrocyte metabolism and promotes the release of extracellular matrix (matricellular) proteins, including IGFBP3, GREM1, IGFBP5, THBS1, and PAPPA. Our results suggest that astrocytes are involved in the long-term modulation of extra cellular matrix environments and that they may influence persistent cell-to-cell interaction and help maintain neuromodulation over time.

Formalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

Nitrogen-doped carbon nanofibers (N-CNFs) with different nitrogen contents were synthesized from vaporized acetonitrile as a simultaneous carbon and nitrogen source via the chemical vapor deposition method. N-CNF synthesis was conducted at 640 °C for 2 h, and the ratio of nitrogen and hydrogen mixture as reactant gas was adjusted to control the nitrogen content in the N-CNFs. The amount of hydrogen in the reactant gas was varied in the range from 0 to 40 mL min − 1 . The physical and electrochemical properties of the N-CNFs were characterized. The content of introduced nitrogen decreased with the ratio of supplied hydrogen, as opposed to the growth yield of the N-CNFs. Additionally, the nitrogen content and chemical states of the N-CNFs were examined and compared to reveal their correlations with the electrochemical behavior. The N-CNFs were found to have a noticeable electrochemical behavior such as capacitance and oxygen reduction reaction activity that was determined by the total nitrogen contents, especially those of pyridinic-nitrogen.

We used single cell RNA-Seq to examine molecular heterogeneity in multiple myeloma (MM) in 597 CD138 positive cells from bone marrow aspirates of 15 patients at different stages of disease progression. 790 genes were selected by coefficient of variation (CV) method and organized cells into four groups (L1–L4) using unsupervised clustering. Plasma cells from each patient clustered into at least two groups based on gene expression signature. The L1 group contained cells from all MGUS patients having the lowest expression of genes involved in the oxidative phosphorylation, Myc targets, and mTORC1 signaling pathways (p −14). In contrast, the expression level of these pathway genes increased progressively and were the highest in L4 group containing only cells from MM patients with t(4;14) translocations. A 44 genes signature of consistently overexpressed genes among the four groups was associated with poorer overall survival in MM patients (APEX trial, p p < 0.0001; HR, 2.00; 95% CI, 1.39–2.89). Our study, using single cell RNA-Seq, identified the most significantly affected molecular pathways during MM progression and provided a novel signature predictive of patient prognosis and treatment stratification.

Fuel cells have received worldwide attention as a next-generation renewable energy technology. However, catalyst cost and durability are the main issue hampering the commercialization of fuel cells. Many studies have focused on the physicochemical properties of the carbon support to improve the catalyst’s properties. Mesoporous carbons are suitable candidates because of their appropriate structural characteristics, including high surface area, large pore size, and regularly interconnected mesopores that permit efficient diffusion of the reactants and by-products. In this study, supports made from chestnut-like carbon consisting of platelet carbon nanofibers were fabricated by selective catalytic gasification of activated carbon. Pt/C catalysts were synthesized from these support structures using the impregnation method. Catalyst performance and characteristics were investigated by N2 adsorption/desorption isotherms, X-ray diffractions, and the rotating disk electrode technique for the oxygen reduction reaction.

Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

The von Hippel-Lindau tumor suppressor pVHL is an E3 ligase that targets hypoxia-inducible factors (HIFs). Mutation of VHL results in HIF up-regulation and contributes to processes related to tumor progression such as invasion, metastasis, and angiogenesis. However, very little is known with regard to post-transcriptional regulation of pVHL. Here we show that WD repeat and SOCS box-containing protein 1 (WSB1) is a negative regulator of pVHL through WSB1's E3 ligase activity. Mechanistically, WSB1 promotes pVHL ubiquitination and proteasomal degradation, thereby stabilizing HIF under both normoxic and hypoxic conditions. As a consequence, WSB1 up-regulates the expression of HIF-1α’s target genes and promotes cancer invasion and metastasis through its effect on pVHL. Consistent with this, WSB1 protein level negatively correlates with pVHL level and metastasis-free survival in clinical samples. This work reveals a new mechanism of pVHL's regulation by which cancer acquires invasiveness and metastatic tendency.

Although immune checkpoint inhibitors have resulted in durable clinical benefits in a subset of patients with advanced cancer, some patients who did not respond to initial anti-PD-1 therapy have been found to benefit from the addition of salvage chemotherapy. However, the mechanism responsible for the successful chemoimmunotherapy is not completely understood. Here we show that a subset of circulating CD8+ T cells expressing the chemokine receptor CX3CR1 are able to withstand the toxicity of chemotherapy and are increased in patients with metastatic melanoma who responded to chemoimmunotherapy (paclitaxel and carboplatin plus PD-1 blockade). These CX3CR1+CD8+ T cells have effector memory phenotypes and the ability to efflux chemotherapy drugs via the ABCB1 transporter. In line with clinical observation, our preclinical models identified an optimal sequencing of chemoimmunotherapy that resulted in an increase of CX3CR1+CD8+ T cells. Taken together, we found a subset of PD-1 therapy-responsive CD8+ T cells that were capable of withstanding chemotherapy and executing tumor rejection with their unique abilities of drug efflux (ABCB1), cytolytic activity (granzyme B and perforin), and migration to and retention (CX3CR1 and CD11a) at tumor sites. Future strategies to monitor and increase the frequency of CX3CR1+CD8+ T cells may help to design effective chemoimmunotherapy to overcome cancer resistance to immune checkpoint blockade therapy.

This study aimed to evaluate the effect of oral iron supplementation in frequent donors in Korea, based solely on donation history.The hemoglobin (Hb) level, ferritin level, soluble transferrin receptor (sTfR), total iron binding capacity (TIBC), and transferrin saturation of frequent donors at high risk for iron deficiency were compared to those of first donors. The frequent donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects.A total of 53 male and 57 female frequent donors were recruited. After 4-week iron supplementation, among the men, the prevalence of a: low Hb level (13.0 g/dL) decreased from 25% to 2%; low ferritin level (15.0 ng/mL) decreased from 58% to 4%; iron deficient erythropoiesis (IDE) (log(sTfR/ferritin) ≥ 2.07) decreased from 77% to 33%. Among the women, the percentage of a: low Hb level (12.0 g/dL) decreased from 44% to 9%; low ferritin level decreased from 79% to 11%; IDE decreased from 95% to 47%. In total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. No serious adverse events were reported.Ferritin level, a reliable indicator of iron status, increased and IDE decreased significantly after four-week iron supplementation in the female, but not in the male, donor group, compared to those of control donors. Four-week oral iron supplement was not enough to restore iron storage level in the male donor group.

Interior ballistics are completed in tens of milliseconds, as are all gun-firing phenomena. Thus, some data cannot be measured directly through experimentation. Therefore, such complex gun-firing phenomena are traditionally clarified by numerical analysis. In the twophase flow of interior ballistics, interphase drag has a strong effect on propellant particle movement. This drag is a momentum sink in the gas phase and a corresponding source of momentum for the solid phase. Previous studies have calculated the drag force on the propellant particles using Ergun’s empirical equation, which was developed for a dense bed and relates the drag to the pressure drop through porous media. However, the particulate bed is fluidized in the course of the cycle of interior ballistics, thus indicating that the flow field is transient with regions of high Reynolds number beyond the range of experimental data. The Ergun equation is examined through a compensation study and calibrated based on the Reynolds number using the numerical method. Moreover, the influence of different drag models on flow behavior and propellant movement in interior ballistics is analyzed.

Background Adenosine signaling has been implicated in several neurological and psychiatric disorders. Previously, we found that astrocytic excitatory amino acid transporter 2 (EAAT2) and aquaporin 4 (AQP4) are downregulated in the striatum of mice lacking type 1 equilibrative nucleoside transporter (ENT1). Methods To further investigate the gene expression profile in the striatum, we preformed Illumina Mouse Whole Genome BeadChip microarray analysis of the caudate–putamen (CPu) and nucleus accumbens (NAc) in ENT1 null mice. Gene expression was validated by RT-PCR, Western blot, and immunofluorescence. Using transgenic mice expressing enhanced green fluorescence protein (EGFP) under the control of the glial fibrillary acidic protein (GFAP) promoter, we examined EGFP expression in an ENT1 null background. Results Glial fibrillary acidic protein was identified as a top candidate gene that was reduced in ENT1 null mice compared to wild-type littermates. Furthermore, EGFP expression was significantly reduced in GFAP-EGFP transgenic mice in an ENT1 null background in both the CPu and NAc. Finally, pharmacological inhibition or siRNA knockdown of ENT1 in cultured astrocytes also reduced GFAP mRNA levels. Conclusions Overall, our findings demonstrate that ENT1 regulates GFAP expression and possibly astrocyte function.

Tungsten carbides (WC) nanoparticles on platelet type-carbon nanofibers (p-CNFs) catalysts have been synthesized for alkaline direct ethanol fuel cells (ADEFC). Physical properties of WC/CNFs samples with various WC contents are analyzed by transmission electron microscope (TEM), thermal gravimetric analysis (TGA) and nitrogen isotherm (BET). The WC/CNFs catalysts showed an improved kinetics for the ethanol oxidation than p-CNFs did. It indicates that the significant increase in the catalytic activity for ethanol oxidation on WC/CNFs than p-CNFs did due to the synergistic structural effect between WC nanoparticles and the p-CNFs supports. WC/CNFs also showed good performances in ADEFC single cells. The maximum current density of P4W3 and P4W4 was 9.0 and 4.4 mA cm −2 , respectively. These catalysts can be used as the ethanol oxidation in direct ethanol fuel cells in alkaline media.