Two viruses, MCV1 and MCV2, which infect Marinitoga bacteria isolated from deep-sea hydrothermal vents: functional and genomic analysis.

Viruses represent a driving force in the evolution of microorganisms including those thriving in extreme environments. However, our knowledge of the viral diversity associated to microorganisms inhabiting the deep-sea hydrothermal vents remains limited. The phylum of Thermotogae, including thermophilic bacteria, is well represented in this environment. Only one virus was described in this phylum, MPV1 carried by Marinitoga piezophila. In this study, we report on the functional and genomic characterization of two new bacterioviruses that infect bacteria from the Marinitoga genus. Marinitoga camini virus 1 and 2 (MCV1 and MCV2) are temperate siphoviruses with a linear dsDNA genome of 53.4 kb and 50.5 kb respectively. Here, we present a comparative genomic analysis of the MCV1 and MCV2 viral genomes with that of MPV1. The results indicate that even if the host strains come from geographically distant sites, their genomes share numerous similarities. Interestingly, heavy metals did not induce viral production, instead the host of MCV1 produced membrane vesicles. This study highlights interaction of mobile genetic elements (MGE) with their hosts and the importance of including hosts-MGEs' relationships in ecological studies.

Estimation of metagenome size and structure in an experimental soil microbiota from low coverage next-generation sequence data.

AIMS: A major challenge in metagenome studies is to estimate the true size of all combined genomes. Here, we present a novel approach to estimate the size of all combined genomes for low coverage next-generation sequencing (NGS) data through empirically determined copy numbers of random DNA fragments. METHODS AND RESULTS: Size estimates were made based on analyses of two experimental soil micro-ecosystems - simulating soil with and without earthworms. Our analyses showed combined genome sizes of about log 11 nucleotides for each of the soil micro-ecosystems, as estimated from qPCR determined copy numbers of random DNA fragments. This corresponds to more than 20000 unique bacterial genomes in each sample. There seemed, however, to be a bacterial subpopulation in the earthworm soil, not being present in the nonearthworm soil. To describe the structure of the metagenomes, both total DNA and amplified 16S rRNA gene sequence libraries were generated with 454-sequencing. Bioinformatic analysis of 454 sequence libraries showed a large functional but low taxonomic overlap between the samples with and without earthworms. A neutrality test indicated that rare species have a competitive advantage over abundant species in both micro-ecosystems providing a potential explanation for the large metagenome sizes. CONCLUSIONS: We have shown that the soil metagenome is very large and that the large size is probably a consequence of top-down selection of the dominant bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Estimates of metagenome size from low coverage NGS data will be important for guiding future NGS set-ups.

Establishment of Clinical and Lab Algorithms for the Identification of Carriers of Mutations in CYP21A2 - A Study of 365 Children and Adolescents.

BACKGROUND: Mutations of CYP21A2 encoding 21-hydroxylase are the most frequent cause of congenital adrenal hyperplasia (CAH) and are associated either with elevated basal or ACTH-stimulated levels of 17-hydroxyprogesterone (17OHP) in blood. OBJECTIVE: The study objective was to identify the most suitable of 12 different test algorithms and appropriate cut-off levels for that test to recognize patients with non-classical congenital adrenal hyperplasia (NCCAH) and carriers of clinically relevant mutations in CYP21A2. METHOD AND PATIENTS: Between July 2006 and July 2015 ACTH-tests were conducted in 365 children and adolescents (Age 1-20 y) suspected to have NCCAH. As a reference, results from subsequent gene sequencing of CYP21A2 was used. Inclusion criteria that were used were premature pubarche with accelerated bone age, hyperandrogenism, hirsutism, or menstrual irregularities. Receiver operating characteristics (ROC) were plotted. Evaluated test algorithms were composed around 17OHP measurements by radioimmunoassays. The most suitable test was identified by the greatest area under the curve (AUC). RESULTS: Among the 12 tested algorithms, the sum of 30 min and 60 min stimulated 17OHP values (sum17OHPstim) showed the highest AUC of 0.774 for identifying heterozygous and bi-allelic mutations. A cut-off of 10.1 µg/l was advisable. Bi-allelic mutations only were best identified calculating the difference between 30 min and basal 17OHP values (Δ17OHP30). A cut-off of 9.4 µg/l was most effective. CONCLUSION: Alternatively to the above mentioned cut-offs the difference of 60 min after stimulation to basal 17OHP (Δ17OHP60) can be used for the benefit of a combined test to identify both heterozygotes and bi-allelic patients. There are minimal decreases in sensitivity and specificity compared to an approach that applies two tests. However, it denotes a simpler approach in the clinical routine.

Epigenetic activation of O-linked ß-N-acetylglucosamine transferase overrides the differentiation blockage in acute leukemia.

BACKGROUND: Overriding the differentiation blockage in acute myeloid leukemia (AML) is the most successful mode-of-action in leukemia therapy - now curing the vast majority of patients with acute promyelocytic leukemia (APL) using all-trans retinoic acid (ATRA)-based regimens. Similar approaches in other leukemia subtypes, such as IDH1/2-mutated AML, are under active investigation. We herein present successful release of the differentiation blockage upon treatment with the natural (-)-Δ9-Tetrahydrocannabinol isomer dronabinol in vitro and in vivo. METHODS: Cellular maturation and differentiation were followed in two patients employing whole genome methylation profiling, proteome analyses, NGS deep sequencing and multispectral imaging flow cytometry. For functional studies lentiviral OGT knock-down in vitro and ex vivo cell models were created to evaluate proliferative, apoptotic and differentiating effects of OGT in acute leukemia. FINDINGS: In here, we provide molecular evidence that dronbinol is capable to override the differentiation blockage of acute leukemia blasts at the state of the leukemia-initiating clone. We further identify the O-linked ß-N-acetyl glucosamine (O-GlcNAc) transferase (OGT) to be crucial in this process. OGT is a master regulator enzyme adding O-GlcNAc to serine or threonine residues in a multitude of target proteins. Aberrant O-GlcNAc modification is implicated in pathologies of metabolic, neurodegenerative and autoimme diseases as well as cancers. We provide evidence that dronabinol induces transcription of OGT via epigenetic hypomethylation of the transcription start site (TSS). A lentiviral OGT-knock out approach proves the central role of OGT exerting antileukemic efficacy via a dual-mechanism of action: High concentrations of dronabinol result in induction of apoptosis, whereas lower concentrations drive cellular maturation. Most intriguingly, overriding of the differentiation blockage of acute leukemia blasts is validated in vivo following two patients treated with dronabinol. INTERPRETATION: In conclusion, we provide evidence for overcoming the differentiation blockage in acute leukemia in subentities beyond promyelocytic and IDH1/2-mutated leukemia and thereby identify O-GlcNAcylation as a novel (drugable) field for future leukemia research. FUNDING: Unrestricted grant support by the IZKF Program of the Medical Faculty Tübingen (MMS) and Brigitte Schlieben-Lange Program as well as the Margarete von Wrangell Program of the Ministry of Science, Research and the Arts, Baden-Württemberg, Germany (KKS) and Athene Program of the excellence initiative University of Tübingen (KKS).

A cDNA clone from Arabidopsis thaliana encoding plastidic ferredoxin:sulfite reductase.

A cDNA with an open reading frame of 1929 bp (termed sir) was isolated from a lambda ZapII library of Arabidopsis thaliana leaf tissue. The polypeptide sequence deduced from the cDNA is homologous to the ferredoxin-dependent sulfite reductase (EC 1.8.7.1) from Synechococcus PCC7942 and distantly related to the hemoprotein subunit of Escherichia coli NADPH-dependent sulfite reductase (EC 1.8.1.2). A molecular mass of 71.98 kDa can be predicted for a ferredoxin sulfite reductase from A. thaliana. The polypeptide consists of 642 amino acids including a transit peptide of 66 residues (6.72 kDa) that is assumed to direct the protein into the plastid. For expression and enzymatic characterization of a putative A. thaliana ferredoxin sulfite reductase, the DNA of the transit peptide was deleted by a PCR method. The truncated cDNA clone was expressed as his-tag fusion protein. The modified gene product was enzymatically inactive but specific cross-reaction with polyclonal antibodies against ferredoxin sulfite reductase from Synechococcus is seen as confirmation of its identity as higher plant ferredoxin sulfite reductase.

Variegate porphyria in a 46-year-old patient taking sibutramine for weight loss.

The acute hepatic porphyrias can cause life-threatening attacks of neurovisceral symptoms that mimic other acute medical conditions. Variegate porphyria caused by mutations in the protoporphyrinogen oxidase (PPOX) gene is a latent disorder characterized by exacerbations induced by fasting, alcohol consumption or certain drugs. We describe the case of a 46-year-old female patient presenting with a first episode of symptomatic porphyria after 10 d of sibutramine treatment for weight loss. Genetic analysis showed a heterozygous R168H hot spot mutation in the PPOX gene. A putative effect of sibutramine on the hepatic haem biosynthetic pathway and reduced food intake have likely caused this exacerbation of a porphyria attack. Although this may be the first case report of this kind, the risk of acute porphyria should be considered in patients using pharmacotherapy for obesity.

[Familial Mediterranean fever. Rare manifestation without fever and with inconspicuous family case history]. / Familiäres Mittelmeerfieber. Seltene Manifestation ohne Fieber und mit unauffälliger Familienanamnese.

HISTORY AND ADMISSION FINDINGS: A 33-year-old man of Turkish descent had suffered from recurrent diffuse abdominal pain and shoulder pain since 13 years. Repeatedly performed investigations in the past had produced numerous diagnoses. The symptoms had been recurring quarterly to weekly, lasted three days on average and resolved spontaneously. He never had fever and the family history was unremarkable. DIAGNOSIS, TREATMENT, AND COURSE: Blood tests demonstrated increased parameters for systemic inflammation and mild normochromic normocytic anemia. In addition to splemomegaly the abdominal computed tomography revealed signs of sacroiliitis. There was no arthritis of the shoulder radiologically. Despite lack of familial history and fever genetic analysis of the Mediterranean fever gene (MEFV) revealed two heterozygous mutations in this MEFV gene for M694 and V726A. The patient was treated with colchicine and has now remained free of symptoms for meanwhile 10 months. There had been no comparable symptom-free period during the last 10 years. CONCLUSION: Sometimes the name "Familial Mediterranean Fever" (FMF) is misleading because this disease may, although rarely, occur without both, fever and familial history. Because of the increasing number of immigrants FMF should be considered in the initial differential diagnosis of patients of Mediterranean origin presenting with abdominal pain. Genetic analysis of the MEFV-gene as well as a therapeutic trial with colchicine, may help to detect FMF.

Reaction mechanism of thioredoxin: 3'-phospho-adenylylsulfate reductase investigated by site-directed mutagenesis.

Properties of purified recombinant adenosine 3'-phosphate 5'-phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are 23 microM and 10 microM, respectively; the enzyme has a Vmax of 94-99 mumol min-1 mg-1 and a molecular activity/catalytically active dimer of 95 s-1. Adenosine 3',5'-bisphosphate (PAdoP) inhibits competitively (Ki 4 microM) with respect to PAdoPS; adenosine 2',5'-bisphosphate and sulfite are not inhibitory. Alkylation by SH-group inhibitors irreversibly inactivates the enzyme. The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGI/LH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction of PAdoPS to sulfite was investigated by site-specific mutagenesis: cysH was mutated by single-strand-overlay extension PCR; the mutated genes were cloned in pBTac1 and expressed in E. coli RL 22 (delta cysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced Vmax by a factor of 4.5 x 10(3) (Vmax = 0.02-0.013 mumol min-1 mg-1) with marginal effects on Km for PAdoPS (19 microM) and reduced thioredoxin (14 microM). Mutation of Tyr209 drastically affected saturation with thioredoxin (Km 1.5 microM) and decreased Vmax (0.22-0.25 mumol min-1 mg-1) in addition to a small increase in Km for PAdoPS (31 microM). Chromophores as prosthetic groups were absent from recombinant PAdoPS reductase. Difference absorption spectra between reduced and oxidized forms of wild-type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (delta lambda max 292 nm) appears to be involved in the reduction. The data suggest a special ping-pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell-Chance type mechanism.

Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD.

While all known natural isolates of C. elegans contain multiple copies of the Tc1 transposon, which are active in the soma, Tc1 transposition is fully silenced in the germline of many strains. We mutagenized one such silenced strain and isolated mutants in which Tc1 had been activated in the germline ("mutators"). Interestingly, many other transposons of unrelated sequence had also become active. Most of these mutants are resistant to RNA interference (RNAi). We found one of the mutated genes, mut-7, to encode a protein with homology to RNaseD. This provides support for the notion that RNAi works by dsRNA-directed, enzymatic RNA degradation. We propose a model in which MUT-7, guided by transposon-derived dsRNA, represses transposition by degrading transposon-specific messengers, thus preventing transposase production and transposition.