provided expertise to develop 18 F nutrient uptake assays. F.X. and M.N.T injected and handled mice for 18 F nutrient uptake assays, and performed and provided expertise for PET imaging and autoradiography. T.H. and W.D.M. performed and provided expertise for intrarenal Renca experiments. R.W.J. and V.T.M generated and provided expertise for PyMT GEMM tumors. R.E.B and C.S.W. generated and provided expertise for AOM/DSS CRC tumors. B.I.R. R.T.O. and M.H.W. generated the pTZeo-EL-thy1.1 transposon construct and engineered MC38 cells using this transposon system. B.I.R, M.Z.M, and A.S. performed in vivo 2NBDG studies. J.E.B. provided expertise in characterizing TAM. A.R.P provided expertise in flow sorting for mRNA transcript analysis. B.I.R. and M.Z.M performed extracellular flux and mRNA transcript experiments. F.M.M. and E.F.M performed and provided expertise in cell staining for light microscopy. E.F.M performed light microscopy and pathologic examination of MC38 tumors. A.A (VU) conducted transcriptomic analysis. B.I.R and M.Z.M. analyzed all data generated in this study. J.C.R. and W.K.R. obtained funding for this study.Data Availability Statement (DAS) All data will be made available upon reasonable request to JCR/WKR. Tumor mRNA transcript data that support the findings of this study have been deposited in Gene Expression Omnibus (GEO) under accession GSE165223. These data are also found in Supplementary Information Table 4. Code Availability Statement (CAS)The code used to support tumor mRNA transcript analysis has been previously published (see methods references) and will be made available upon request to JCR/WKR.
Purpose of Review-Metabolic reprogramming is essential for the rapid proliferation of cancer cells and is thus recognized as a hallmark of cancer. In this review, we will discuss the etiologies and effects of metabolic reprogramming in colorectal cancer. Recent Findings-Changes in cellular metabolism may precede the acquisition of driver mutations ultimately leading to colonocyte transformation. Oncogenic mutations and loss of tumor suppressor genes further reprogram CRC cells to upregulate glycolysis, glutaminolysis, onecarbon metabolism, and fatty acid synthesis. These metabolic changes are not uniform throughout tumors, as subpopulations of tumor cells may rely on different pathways to adapt to nutrient availability in the local tumor microenvironment. Finally, metabolic cross-communication between stromal cells, immune cells, and the gut microbiota enable CRC growth, invasion, and metastasis. Summary-Altered cellular metabolism occurs in CRC at multiple levels, including in the cells that make up the bulk of CRC tumors, cancer stem cells, the tumor microenvironment, and hostmicrobiome interactions. This knowledge may inform the development of improved screening and therapeutics for CRC.
Thrombocytopenia affects up to 35% of all patients admitted to the neonatal intensive care unit (NICU). The causes of thrombocytopenia in neonates are very diverse, and include immune and nonimmune disorders. Most cases of thrombocytopenia encountered in the NICU are non-immune, and these will constitute the focus of this review. Specifically, we will first discuss the biological differences between neonatal and adult megakaryocytopoiesis, which contribute to explain the vulnerability of neonates to develop thrombocytopenia. Next, we will review new diagnostic tools that have allowed for a better evaluation of platelet production in neonates, without having to obtain a bone marrow sample. Finally, we will summarize our current understanding of the mechanisms underlying the thrombocytopenia in several common neonatal conditions, such as chronic intrauterine hypoxia, sepsis and necrotizing enterocolitis (NEC), and viral infections. A better understanding of the mechanisms underlying these varieties of thrombocytopenia is critical to develop disease-specific treatment protocols, and to begin to entertain the possibility of using novel thrombopoietic growth factors to treat selected neonates with severe thrombocytopenia. Platelet production in neonatesPlatelet production, or thrombopoiesis, is a complex process that can be schematically represented as consisting of four main steps. The first step is the production of the thrombopoietic stimulus, which drives the generation of megakaryocytes and ultimately platelets. Although a number of cytokines (i.e. IL-3, IL-6, IL-11, GM-CSF) and chemokines (i.e. SDF and FGF-4) contribute to this process, thrombopoietin (Tpo) is now widely recognized as the most potent known stimulator of platelet production. Thrombopoietin mostly acts by promoting the proliferation of megakaryocyte progenitors (the cells that multiply and give rise to megakaryocytes), and the maturation of the megakaryocytes. 1-3 Megakaryocyte maturation is a process characterized by a progressive increase in nuclear ploidy and cytoplasmic maturity that leads to the generation of large polyploid (8N-64N) megakaryocytes. Through a still poorly understood process, these mature megakaryocytes then generate and release new platelets into the circulation. 4-6 Address correspondence to: Martha Sola-Visner, MD, Assistant Professor of Pediatrics, Children's Hospital Boston, Division of Newborn Medicine, 300 Longwood Av., Enders Research Building, Rm. 961, Boston, MA 02115, Phone: 617-919-4845, Fax: 617-730-0260, Email: E-mail: Martha.Sola-Visner@childrens.harvard.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal di...
Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5–10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2–4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials.
ABSTRACT:We serially evaluated the effects of sepsis and/or necrotizing enterocolitis (NEC) on neonatal thrombopoiesis, using a panel of tests that included platelet counts, thrombopoietin concentrations (Tpo), circulating megakaryocyte progenitor concentrations (CMPs), and reticulated platelets (RPs). Variables analyzed included sepsis type, time after onset of sepsis, platelet counts, and gestational (GA) and postconceptional ages (PCA). Twenty neonates were enrolled. Ten had Gram-negative, six had Gram-positive, and four had presumed sepsis. Four neonates had NEC stage II or higher, and six developed thrombocytopenia. Overall, septic neonates had significantly elevated Tpo concentrations and circulating megakaryocyte progenitors. The highest Tpo levels were associated with Gramnegative or presumed sepsis. RP percentages were increased only in neonates with low platelet counts, while RP counts (RP% ϫ platelet count) were elevated in neonates with high platelet counts. Our findings suggest that septic neonates up-regulate Tpo production, leading to increased megakaryocytopoiesis and platelet release, although the degree of upregulation is moderate. The changes in RP% and RP count most likely reflect increased thrombopoiesis with variable degrees of platelet consumption. In addition, our findings suggest that different factors, likely including level of illness and/or specific platelet or bacterial products, can down-regulate the magnitude of the thrombopoietic response.
Objective:To characterize the underlying genetic defect in a family with 3 siblings affected by a severe, yet viable, congenital disorder.Methods:Extensive genetic and metabolic investigations were performed, and the affected children were imaged at different ages. Whole-genome genotyping and whole-exome sequencing were undertaken. A single large region (>8 Mb) of homozygosity in chromosome 4 (chr4:100,268,553–108,609,628) was identified that was shared only in affected siblings. Inspection of genetic variability within this region led to the identification of a novel mutation. Sanger sequencing confirmed segregation of the mutation with disease.Results:All affected siblings share homozygosity for a novel 4-bp deletion in the gene TBCK (NM_033115:c.614_617del:p.205_206del).Conclusions:This finding provides the genetic cause of a severe inherited disease in a family and extends the number of mutations and phenotypes associated with this recently identified disease gene.
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