Integration of molecular typing results into tuberculosis surveillance in Germany-A pilot study.

An integrated molecular surveillance for tuberculosis (TB) improves the understanding of ongoing TB transmission by combining molecular typing and epidemiological data. However, the implementation of an integrated molecular surveillance for TB is complex and requires thoughtful consideration of feasibility, demand, public health benefits and legal issues. We aimed to pilot the integration of molecular typing results between 2008 and 2010 in the German Federal State of Baden-Württemberg (population 10.88 Million) as preparation for a nationwide implementation. Culture positive TB cases were typed by IS6110 DNA fingerprinting and results were integrated into routine notification data. Demographic and clinical characteristics of cases and clusters were described and new epidemiological links detected after integrating typing data were calculated. Furthermore, a cross-sectional survey was performed among local public health offices to evaluate their perception and experiences. Overall, typing results were available for 83% of notified culture positive TB cases, out of which 25% were clustered. Age <15 years (OR = 4.96, 95% CI: 1.69-14.55) and being born in Germany (OR = 2.01, 95% CI: 1.44-2.80) were associated with clustering. At cluster level, molecular typing information allowed the identification of previously unknown epidemiological links in 11% of the clusters. In 59% of the clusters it was not possible to identify any epidemiological link. Clusters extending over different counties were less likely to have epidemiological links identified among their cases (OR = 11.53, 95% CI: 3.48-98.23). The majority of local public health offices found molecular typing useful for their work. Our study illustrates the feasibility of integrating typing data into the German TB notification system and depicts its added public health value as complementary strategy in TB surveillance, especially to uncover transmission events among geographically separated TB patients. It also emphasizes that special efforts are required to strengthen the communication between local public health offices in different counties to enhance TB control

Generation of an induced pluripotent stem cell line (MHHi018-A) from a patient with Cystic Fibrosis carrying p.Asn1303Lys (N1303K) mutation

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene which encodes for a chloride ion channel regulating the balance of salt and water across secretory epithelia. Here we generated an iPSC line from a CF patient homozygous for the p.Asn1303Lys mutation, a Class II folding defect mutation. This iPSC line provides a useful resource for disease modeling and to investigate the pharmacological response to CFTR modulators in iPSC derived epithelia

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

Synonymous GATA2 mutations result in selective loss of mutated RNA and are common in patients with GATA2 deficiency

Deficiency of the transcription factor GATA2 is a highly penetrant genetic disorder predisposing to myelodysplastic syndromes (MDS) and immunodeficiency. It has been recognized as the most common cause underlying primary MDS in children. Triggered by the discovery of a recurrent synonymousGATA2variant, we systematically investigated 911 patients with phenotype of pediatric MDS or cellular deficiencies for the presence of synonymous alterations inGATA2. In total, we identified nine individuals with five heterozygous synonymous mutations: c.351C>G, p.T117T (N = 4); c.649C>T, p.L217L; c.981G>A, p.G327G; c.1023C>T, p.A341A; and c.1416G>A, p.P472P (N = 2). They accounted for 8.2% (9/110) of cases with GATA2 deficiency in our cohort and resulted in selective loss of mutant RNA. While for the hotspot mutation (c.351C>G) a splicing error leading to RNA and protein reduction was identified, severe, likely late stage RNA loss without splicing disruption was found for other mutations. Finally, the synonymous mutations did not alter protein function or stability. In summary, synonymousGATA2substitutions are a new common cause of GATA2 deficiency. These findings have broad implications for genetic counseling and pathogenic variant discovery in Mendelian disorders

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage

Cytoplasmic p21(WAF1/CIP1 )expression is correlated with HER-2/ neu in breast cancer and is an independent predictor of prognosis

BACKGROUND: HER-2 (c-erbB2/Neu) predicts the prognosis of and may influence treatment responses in breast cancer. HER-2 activity induces the cytoplasmic location of p21(WAFI/CIPI )in cell culture, accompanied by resistance to apoptosis. p21(WAFI/CIPI )is a cyclin-dependent kinase inhibitor activated by p53 to produce cell cycle arrest in association with nuclear localisation of p21(WAFI/CIPI). We previously showed that higher levels of cytoplasmic p21(WAFI/CIPI )in breast cancers predicted reduced survival at 5 years. The present study examined HER-2 and p21(WAFI/CIPI )expression in a series of breast cancers with up to 9 years of follow-up, to evaluate whether in vitro findings were related to clinical data and the effect on outcome. METHODS: The CB11 anti-HER2 monoclonal antibody and the DAKO Envision Plus system were used to evaluate HER-2 expression in 73 patients. p21(WAFI/CIPI )staining was performed as described previously using the mouse monoclonal antibody Ab-1 (Calbiochem, Cambridge, MA, USA). RESULTS: HER-2 was evaluable in 67 patients and was expressed in 19% of cases, predicting reduced overall survival (P = 0.02) and reduced relapse-free survival (P = 0.004; Cox regression model). HER-2-positive tumours showed proportionately higher cytoplasmic p21(WAFI/CIPI )staining using an intensity distribution score (median, 95) compared with HER-2-negative cancers (median, 47) (P = 0.005). There was a much weaker association between nuclear p21(WAFI/CIPI )and HER-2 expression (P = 0.05), suggesting an inverse relationship between nuclear p21(WAF1/CIP1 )and HER-2. CONCLUSION: This study highlights a new pathway by which HER-2 may modify cancer behaviour. HER-2 as a predictor of poor prognosis may partly relate to its ability to influence the relocalisation of p21(WAFI/CIPI )from the nucleus to the cytoplasm, resulting in a loss of p21(WAFI/CIPI)tumour suppressor functions. Cytoplasmic p21(WAFI/CIPI )may be a surrogate marker of functional HER-2 in vivo

Alternative splicing of barley clock genes in response to low temperature:evidence for alternative splicing conservation

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement

Perlecan Maintains microvessel integrity in vivo and modulates their formation in vitro

Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF165 rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function

Expression and activity profiles of DPP IV/CD26 and NEP/CD10 glycoproteins in the human renal cancer are tumor-type dependent

[Background] Cell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO).[Methods] Peptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining.Peer reviewe

The MLL recombinome of acute leukemias in 2017

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients