Clonal evolution through genetic bottlenecks and telomere attrition: Potential threats to in vitro data reproducibility

Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost‐efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.     

The association between mindfulness and emotional distress in adults with diabetes: Could mindfulness serve as a buffer? Results from Diabetes MILES: The Netherlands

People with diabetes have a higher risk of emotional distress (anxiety, depression) than non-diabetic or healthy controls. Therefore, identification of factors that can decrease emotional distress is relevant. The aim of the present study was to examine (1) the association between facets of mindfulness and emotional distress; and (2) whether mindfulness might moderate the association between potential adverse conditions (stressful life events and comorbidity) and emotional distress. Analyses were conducted using cross-sectional data (Management and Impact for Long-term Empowerment and Success—Netherlands): 666 participants with diabetes (type 1 or type 2) completed measures of mindfulness (Five Facet Mindfulness Questionnaire-Short Form; FFMQ-SF), depressive symptoms (Patient Health Questionnaire; PHQ-9), and anxiety symptoms (General Anxiety Disorder assessment; GAD-7). Hierarchical multiple regression analyses showed significant associations between mindfulness facets (acting with awareness, non-judging, and non-reacting) and symptoms of anxiety and depression (β = ?0.20 to ?0.33, all p < 0.001). These mindfulness facets appeared to have a moderating effect on the association between stressful life events and depression and anxiety (all p < 0.01). However, the association between co-morbidity and emotional distress was largely not moderated by mindfulness. In conclusion, mindfulness is negatively related to both depression and anxiety symptoms in people with diabetes and shows promise as a potentially protective characteristic against the influence of stressful events on emotional well-being.     

Variants in BAK1, SPRY4, and GAB2 are associated with pediatric germ cell tumors: A report from the children's oncology group

Germ cell tumors (GCT) are a rare form of childhood cancer that originate from the primordial germ cell. Recent genome‐wide association studies (GWAS) have identified susceptibility alleles for adult testicular GCT (TGCT). We test whether these SNPs are associated with GCT in pediatric and adolescent populations. This case‐parent triad study includes individuals with GCT diagnosed between ages 0 and 19. We evaluated 26 SNPs from GWAS of adult TGCT and estimated main effects for pediatric GCT within complete trios (N = 366) using the transmission disequilibrium test. We used Estimation of Maternal, Imprinting and interaction effects using Multinomial modelling to evaluate maternal effects in non‐Hispanic white trios and dyads (N = 244). We accounted for multiple comparisons using a Bonferroni correction. A variant in SPRY4 (rs4624820) was associated with reduced risk of GCT (OR [95% CI]: 0.70 [0.57, 0.86]). A variant in BAK1 (rs210138) was positively associated with GCT (OR [95% CI]: 1.70 [1.32, 2.18]), with a strong estimated effect for testis tumors (OR [95% CI]: 3.31 [1.89, 5.79]). Finally, a SNP in GAB2 (rs948662) was associated with increased risk for GCT (OR [95% CI]: 1.56 [1.20, 2.03]). Nominal associations (P < 0.05) were noted for eight additional loci. A maternal effect was observed for KITLG SNP rs4474514 (OR [95% CI]: 1.66 [1.21, 2.28]) and a paternal parent‐of‐origin effect was observed for rs7221274 (P = 0.00007), near TEX14, RAD51C, and PPM1E. We observed associations between SNPs in SPRY4, BAK1, and GAB2 and GCTs. This analysis suggests there may be common genetic risk factors for GCT in all age groups.     

Collective cell migration of the nephric duct requires FGF signaling

Background : During the course of development, the vertebrate nephric duct (ND) extends and migrates from the place of its initial formation, adjacent to the anterior somites, until it inserts into the bladder or cloaca in the posterior region of the embryo. The molecular mechanisms that guide ND migration are poorly understood. Results : A novel Gata3‐enhancer‐Gfp‐based chick embryo live imaging system was developed that permits documentation of ND migration at the individual cell level for the first time. FGF Receptors and FGF response genes are expressed in the ND, and FGF ligands are expressed in surrounding tissues. FGF receptor inhibition blocked nephric duct migration. Individual inhibitors of the Erk, p38, or Jnk pathways did not affect duct migration, but inhibition of all three pathways together did inhibit migration of the duct. A localized source of FGF8 placed adjacent to the nephric duct did not affect the duct migration path. Conclusions : FGF signaling acts as a “motor” that is required for duct migration, but other signals are needed to determine the directionality of the duct migration pathway. Developmental Dynamics 244:157–167, 2015. © 2014 Wiley Periodicals, Inc.     

Whole‐exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole‐exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis‐related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well‐established cancer gene lysine (K)‐specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome‐sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D‐mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development. © 2015 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.     

Toll-like receptor 9 modulates macrophage antifungal effector function during innate recognition of Candida albicans and Saccharomyces cerevisiae

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens. Macrophages sample the phagosomal content and orchestrate the innate immune response. Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA and is activated by fungal DNA. Here we demonstrate that specific triggering of TLR9 recruitment to the macrophage phagosomal membrane is a conserved feature of fungi of distinct phylogenetic origins, including Candida albicans, Saccharomyces cerevisiae, Malassezia furfur, and Cryptococcus neoformans. The capacity to trigger phagosomal TLR9 recruitment was not affected by a loss of fungal viability or cell wall integrity. TLR9 deficiency has been linked to increased resistance to murine candidiasis and to restriction of fungal growth in vivo. Macrophages lacking TLR9 demonstrate a comparable capacity for phagocytosis and normal phagosomal maturation compared to wild-type macrophages. We now show that TLR9 deficiency increases macrophage tumor necrosis factor alpha (TNF-α) production in response to C. albicans and S. cerevisiae, independent of yeast viability. The increase in TNF-α production was reversible by functional complementation of the TLR9 gene, confirming that TLR9 was responsible for negative modulation of the cytokine response. Consistently, TLR9 deficiency enhanced the macrophage effector response by increasing macrophage nitric oxide production. Moreover, microbicidal activity against C. albicans and S. cerevisiae was more efficient in TLR9 knockout (TLR9KO) macrophages than in wild-type macrophages. In conclusion, our data demonstrate that TLR9 is compartmentalized selectively to fungal phagosomes and negatively modulates macrophage antifungal effector functions. Our data support a model in which orchestration of antifungal innate immunity involves a complex interplay of fungal ligand combinations, host cell machinery rearrangements, and TLR cooperation and antagonism.     

Codon optimization of human factor VIII cDNAs leads to high-level expression

Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.