Restricted mobility of Dnmt1 in preimplantation embryos: implications for epigenetic reprogramming

BACKGROUND: Mouse preimplantation development is characterized by both active and passive genomic demethylation. A short isoform of the prevalent maintenance DNA methyltransferase (Dnmt1S) is found in the cytoplasm of preimplantation embryos and transiently enters the nucleus only at the 8-cell stage. RESULTS: Using GFP fusions we show that both the long and short isoforms of Dnmt1 localize to the nucleus of somatic cells and the cytoplasm of preimplantation embryos and that these subcellular localization properties are independent of phosphorylation. Importantly, photobleaching techniques and salt extraction revealed that Dnmt1S has a very restricted mobility in the cytoplasm, while it is highly mobile in the nucleus of preimplantation embryos. CONCLUSION: The restricted mobility of Dnmt1S limits its access to DNA and likely contributes to passive demethylation and epigenetic reprogramming during preimplantationdevelopment

Deleterious Impact of a Novel CFH Splice Site Variant in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a heterogeneous disorder characterized by microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and acute kidney injury (AKI). In about 50% of cases, pathogenic variants in genes involved in the innate immune response including complement factors complement factor H (CFH), CFI, CFB, C3, and membrane co-factor protein (MCP/CD46) put patients at risk for uncontrolled activation of the alternative complement pathway. As aHUS is characterized by incomplete penetrance and presence of additional triggers for disease manifestation, genetic variant interpretation is challenging and streamlined functional variant evaluation is urgently needed. Here, we report the case of a 27-year-old female without previous medical and family history who presented with confusion, petechial bleeding, and anuric AKI. Kidney biopsy revealed glomerular thrombotic microangiopathy (TMA). Targeted next generation sequencing identified a paternally transmitted novel heterozygous splice site variant in the CFH gene [c.3134-2A>G; p.Asp1045_Thr1053del] which resulted in a partial in-frame deletion of exon 20 transcript as determined by cDNA analysis. On the protein level, the concomitant loss of 9 amino acids in the short consensus repeat (SCR) domains 17 and 18 of CFH includes a highly conserved cysteine residue, which is assumed to be essential for proper structural folding and protein function. Treatment with steroids, plasmapheresis, and the complement inhibitor eculizumab led to complete hematological and clinical remission after several months and stable renal function up to 6 years later. In conclusion, genetic investigation for pathogenic variants and evaluation of their functional impact, in particular in the case of splice site variants, is clinically relevant and enables not only better molecular understanding but helps to guide therapy with complement inhibitors

The underestimated burden of monogenic kidney disease in adults waitlisted for kidney transplantation

Purpose: Chronic kidney disease (CKD) is a major health-care burden. Increasing evidence suggests that a considerable proportion of patients are affected by a monogenic kidney disorder. Methods: In this study, the kidney transplantation waiting list at the Charité was screened for patients with undetermined cause of CKD. By next-generation sequencing (NGS) we targeted all 600 genes described and associated with kidney disease or allied disorders. Results: In total, 635 patients were investigated. Of these, 245 individuals had a known cause of CKD (38.5%) of which 119 had a proven genetic disease (e.g., ADPKD, Alport). The other 340 patients (53.5%) were classified as undetermined diagnosis, of whom 87 had kidney failure (KF) onset <40 years. To this latter group genetic testing was offered as well as to those patients (n = 29) with focal segmental glomerulosclerosis (FSGS) and all individuals (n = 21) suspicious for thrombotic microangiopathy (TMA) in kidney biopsy. We detected diagnostic variants in 26 of 126 patients (20.6%) of which 14 of 126 (11.1%) were pathogenic or likely pathogenic. In another 12 of 126 (9.5%) patients, variants of unknown significance (VUS) were detected. Conclusion: Our study demonstrates the diagnostic value of comprehensive genetic testing among patients with undetermined CKD

Alternative Splicing and Extensive RNA Editing of Human TPH2 Transcripts

Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre- mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour

A mammalianized synthetic nitroreductase gene for high-level expression

Background The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy. Methods We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays. Results In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect. Conclusion Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans

Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation

Non-neuronal, peripheral serotonin deficiency causes diabetes mellitus and identifies an intracellular role for serotonin in the regulation of insulin secretion

alternative splicing and RNA editing of human TPH2 transcripts

Serotonin (5-Hydroxytryptamin, 5-HT) ist ein monoaminerger Neurotransmitter, der an zahlreichen Aspekten der Verhaltenskontrolle beteiligt ist. Das serotonerge System nimmt seinen Ursprung in einer handvoll 5-HT synthetisierender Neuronen im Hirnstamm, die zusammen die Raphekerne B1 - B9 bilden. Seit über 40 Jahren ist bekannt, dass die Tryptophan-Hydroxylase (TPH) das geschwindigkeitsbestimmende Enzym in der 5-HT-Biosynthese ist. Erst kürzlich konnte ein zweites TPH-Gen (TPH2) identifiziert werden, das hauptsächlich im Gehirn exprimiert wird, während die Expression des bislang bekannten TPH-Gens (TPH1) vorwiegend in nicht neuronalen Geweben erfolgt. Dysfunktionen des serotonergen Systems im Gehirn konnten mit einer Vielzahl neuropsychiatrischer Erkrankungen, wie Depression, Schizophrenie und Suizidalverhalten in Zusammenhang gebracht werden und umfangreiche Beweise deuten auf TPH2 als Kandidatengen für 5-HT-verwandte psychiatrische Störungen. Zum besseren Verständnis der physiologischen Bedeutung von 5-HT im Gehirn und bei der Ätiologie psychiatrischer Erkrankungen wurde ein induzierbares Mausmodell etabliert. Das Modell basiert auf der spezifischen Expression der E. coli-Nitroreduktase (NTR) in den serotonergen Rapheneuronen und die damit verbundende Metabolisierung des inaktiven Protoxins CB1954 zu einem potenten Zytotoxin, wodurch die Ablation der 5-HT-Neuronen induziert und die zentralnervösen 5-HT-Spiegel konditionell gesenkt werden. Die transgenen NTR1 Mäuse zeigten jedoch trotz spezifischer NTR-Expression keinen Phänotyp nach Behandlung mit CB1954. Die Expression einer gfp markierten NTR in COS7-Zellen zeigte die Aggregation des Fusionsproteins und lies eine beeinträchtigte Translation durch Unterschiede im synonymen Codongebrauch zwischen Donor- und Wirtszelle vermuten. Eine synthetische NTR Version (ntro), deren Codongebrauch an die Präferenzen der Maus angepasst wurde, führte zu höheren Proteinausbeuten in verschiedenen Säugerzelllinien und sensibilisierte diese bereits bei einer zehnfach geringeren CB1954-Konzentration. Die Verbesserung des NTR/CB1954-Systems auf translationeller Ebene sollte sein Potential hinsichtlich der Untersuchung zellulärer Funktionen durch konditionale Zellablation in transgenen Tieren erhöhen und verspricht zusätzlich eine Anwendung in der humanen Krebstherapie durch GDEPT (gene-directed enzyme prodrug therapy). Zahlreiche Studien haben die positive Kopplung von Einzelstrangpolymorphismen (SNPs) im TPH2-Gen mit psychiatrischen Erkrankungen und ihren möglichen Einfluss auf die enzymatische Aktivität der TPH2 gezeigt. In der vorliegenden Arbeit ergab die Sequenzierung von TPH2-cDNAs von post mortem-Gehirnproben des Menschen, dass humane TPH2-Transkripte alternativ gespleißt werden, wobei sich die kinetischen Eigenschaften der kodierten Varianten TPH2A und TPH2B unterscheiden. Zudem werden die prä-mRNAs von TPH2a und TPH2b dynamisch editiert, durch spezifische sich gegenseitig ausschließende Editierungsmuster, die die enzymatische Aktivität der entsprechenden Proteine modulieren. Zusätzlich zur Dichotomie des serotonergen Systems, definiert durch die zwei geschwindigkeitsbestimmenden Enzyme TPH1 und TPH2, in peripheren bzw. neuronalen Geweben, ermöglicht die TPH2a/b-Editierung eine noch komplexere Feinregulation der zentralen 5-HT-Biosynthese. Letztendlich werden molekularbiologische Beweise präsentiert, die vermuten lassen, dass eine Dysregulation von alternativem Spleißen und RNA Editierung an der Ätiologie psychiatrischer Erkrankungen beteiligt ist. Eine wichtige Schlussfolgerung der hier gezeigten Ergebnisse ist die Tatsache, dass weder die aktuell verwendeten RNA- basierten Techniken noch immunhistochemische Proteinnachweismethoden eine Aussage über die TPH2-Aktivität in der psychiatrischen Forschung erlauben, wodurch die bisherigen Daten zu Störungen der TPH2-Expression bei psychiatrischen Erkrankungen sorgfältig nachgeprüft werden sollten.Serotonin (5-hydroxytryptamine, 5-HT) is a monoaminergic neurotransmitter involved in multiple facets of behavioral control. The serotonergic projection system has its roots in a handful of selectively 5-HT-synthesizing neurons within the brainstem, which altogether constitute the raphe nuclei B1 - B9. It is known since more than four decades that tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the 5-HT biosynthesis. Recently, a second TPH gene (TPH2) was identified, which is mainly expressed in the brain, whereas the previously known TPH gene (TPH1) is predominantly expressed in non-neuronal tissues. Dysfunctions of the serotonergic system in the brain have been implicated in a variety of neuropsychatric disorders, like depression, schizophrenia and suicidal behaviour and overwhelming evidence points to TPH2 as a candidate gene for 5- HT-related psychiatric disorders. In order to better understand the physiological role of 5-HT in the brain and the etiology of psychiatric diseases a mouse model was established. The model is based on the specific expression of the E. coli nitroreductase (NTR), which is associated with the bioactivation of the inactive prodrug CB1954 to a powerful cytotoxin in the serotonergic raphe neurons. This leads to the ablation of the 5-HT neurons, thereby conditionally decreasing the 5-HT levels in the CNS. In spite of specific expression of NTR in the 5-HT neurons the transgenic NTR1 mice did not respond to CB1954. The expression of a gfp tagged NTR in COS7 cells showed the aggregation of the corresponding fusion protein, suggesting impaired translation by divergent synonymous codon usage between the donor and host cell. A synthetic NTR version (ntro), in which codon usage was adapted to mouse preferences, showed higher expression levels in different mammalian cell lines rendering them more sensitive to the prodrug CB1954 by one order of magnitude. The improvement of the NTR/CB1954 system at the translational level should increase its potential for the investigation of cellular functions by conditional targeted cell ablation in transgenic animals. Moreover, the ntro developed in this study is a promising candidate for human cancer treatment by GDEPT (gene-directed enzyme prodrug therapy). Numerous studies have proven positive linkage of single nucleotide polymorphisms (SNPs) in the TPH2 gene with psychiatric diseases and their possible influence on TPH2 enzymatic activity. By sequencing TPH2 cDNAs from human post mortem brain samples it could be shown that TPH2 exists in two alternative splice variants with distinct kinetic properties of the encoded TPH2A and TPH2B proteins. Further, the pre-mRNAs of TPH2a and TPH2b are dynamically edited in a mutually exclusive pattern, which modulates the enzymatic activity of the respective proteins. Thus, in addition to the dichotomy of the serotonergic system defined by the two rate limiting enzymes, TPH1 and TPH2, in peripheral and neuronal tissues, respectively, TPH2a/b editing allows an even more complex fine tuning of the central nervous 5-HT biosynthesis. Finally, molecular biological evidence is presented suggesting that a deregulated alternative splicing and RNA editing might be involved in the etiology of psychopathological diseases. An important implication of the results is the fact that neither currently used RNA-based techniques nor immunohistochemical protein detection methods allow estimating the enzymatic activity of TPH2 in psychiatry research. Thus, the here presented data should invite for careful reexamination of present reports on TPH2 expression disturbances in psychiatric disease

Spanloser Metallersatz: Endlosfaserverstärkte 3D-Druck-Filamente

Eine neu entwickelte Imprägniertechnologie ermöglicht die kostengünstige und flexible Herstellung von endlosfaserverstärkten, thermoplastischen 3D­Druck­Filamenten. In Kombination mit additiven Materialextrusionsverfahren gelingt eine wirtschaftliche Umsetzung von leichten und hochbelastbaren Bauteilen aus thermoplastischen Faserverbundwerkstoffen auch für Neueinsteiger und Kleinserien

A mammalianized synthetic nitroreductase gene for high-level expression

Abstract Background The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy. Methods We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays. Results In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect. Conclusion Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans.</p