Metabolic engineering of Basfia succiniciproducens for the production of carbon-three compounds

The recent rise of bio-succinic acid as industrial platform chemical has brought the rumen bacterium Basfia succiniciproducens, a natural producer of this organic acid, into the focus of research. In order to upgrade this industrial producer into a cell factory of broader use, the potential of B. succiniproducens to produce carbon-three compounds was explored. A new genome engineering approach and the identification of suitable promoters for gene expression was targeted. For production of the C3 chemical alanine from glucose and xylose, a bioprocess was developed. Genetic engineering for β-alanine and 3-hydroxypropionate (3-HP) production was examined. A blue-white selection screening system for genome engineering was established, using a lacZ deficient strain. Suitable promoters for heterologous expression of different genes of interest were identified. Using B. succiniciproducens ALA-1, hosting a genomic copy of an alanine dehydrogenase from Geobacillus stearothermophilus, alanine production was improved to 29 g L-1 by establishing a fed-batch strategy under anaerobic conditions. Via equipment of B. succiniciproducens with an episomal copy of an aspartate 1-decarboxylase from Corynebacterium glutamicum or Vibrio natriegens, β-alanine synthesis was realized (450 mg L-1). The expression of a β-alanine pyruvate transaminase from Pseudomonas putida and a malonate semialdehyde reductase from Escherichia coli enabled production of 3-HP (100 mg L- 1) from glucose.Die Nutzung von Bio-Bernsteinsäure als industrielle Plattformchemikalie hat das Pansen-Bakterium Basfia succiniciproducens, einen natürlichen Produzenten dieser organischen Säure, in den Fokus der Forschung gebracht. Um diesen Produzentenstamm für weitere Anwendungen auszubauen, wurde das Potential von B. succiniciproducens zur Produktion von C3 Chemikalien erforscht. Eine neue genombasierte Methode zur genetischen Modifikation sowie geeignete Promotoren für die Genexpression wurden angestrebt. Für die Produktion der C3 Chemikalie Alanin aus Glucose und Xylose wurde ein Bioprozess entwickelt. Genetische Arbeiten zur Produktion von β-Alanin und 3-Hydroxypropionat (3-HP) wurden durchgeführt. Ein blau-weiß Screening System für genomische Modifikationen wurde zunächst in einem lacZ defizienten Stamm etabliert. Auch geeignete Promotoren für die heterologe Expression verschiedenster Ziel-Gene wurden identifiziert. Der Stamm B. succiniciproducens ALA-1, der eine genomische Kopie einer Alanin Dehydrogenase aus Geobacillus stearothermophilus besitzt, produzierte 29 g L-1 Alanin in einem anaeroben Fed-Batch Prozess. Durch Einbringen von episomalen Kopien einer Aspartat 1-Decarboxylase aus Corynebacterium glutamicum oder Vibrio natriegens wurde die β-Alanin (450 mg L-1) Synthese realisiert. Die Expression einer β-Alanin Pyruvat Transaminase aus Pseudomonas putida und einer Malonate semialdehyd Reduktase aus Escherichia coli ermöglichte die Produktion von 3-HP (100 mg L-1) aus Glucose

c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia

Background: Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels. Methods and Results: In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter. Conclusions: Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability

European LeukemiaNet laboratory recommendations for the diagnosis and management of chronic myeloid leukemia

From the laboratory perspective, effective management of patients with chronic myeloid leukemia (CML) requires accurate diagnosis, assessment of prognostic markers, sequential assessment of levels of residual disease and investigation of possible reasons for resistance, relapse or progression. Our scientific and clinical knowledge underpinning these requirements continues to evolve, as do laboratory methods and technologies. The European LeukemiaNet convened an expert panel to critically consider the current status of genetic laboratory approaches to help diagnose and manage CML patients. Our recommendations focus on current best practice and highlight the strengths and pitfalls of commonly used laboratory tests

Splenomegaly, elevated alkaline phosphatase and mutations in the SRSF2/ASXL1/RUNX1 gene panel are strong adverse prognostic markers in patients with systemic mastocytosis

We evaluated the impact of clinical and molecular characteristics on overall survival (OS) in 108 patients with indolent (n=41) and advanced SM (advSM, n=67). Organomegaly was measured by magnetic resonance imaging (MRI)-based volumetry of liver and spleen. In multivariate analysis of all patients, an increased spleen volume greater than or equal to450?ml (hazard ratio [HR], 5.2; 95% confidence interval [CI], [2.1–13.0]; P=0.003) and an elevated alkaline phosphatase (AP; HR 5.0 [1.1–22.2]; P=0.02) were associated with adverse OS. The 3-year OS was 100, 77, and 39%, respectively (P<0.0001), for patients with 0 (low-risk, n=37), 1 (intermediate-risk, n=32) or 2 (high-risk, n=39) parameters. For advSM patients with fully available clinical and molecular data (n=60), univariate analysis identified splenomegaly greater than or equal to1200?ml, elevated AP and mutations in the SRSF2/ASXL1/RUNX1 (S/A/R) gene panel as significant prognostic markers. In multivariate analysis, mutations in S/A/R (HR, 3.2 [1.1–9.6]; P=0.01) and elevated AP (HR 2.6 [1.0–7.1]; P=0.03) remained predictive adverse prognostic markers for OS. The 3-year OS was 76% and 38%, respectively (P=0.0003), for patients with 0-1 (intermediate-risk, n=28) or 2 (high-risk, n=32) parameters. We conclude that splenomegaly, elevated AP and mutations in the S/A/R gene panel are independent of the WHO classification and provide the most relevant prognostic information in SM patient

Does the Constitution Provide More Ballot Access Protection for Presidential Elections Than for U.S. House Elections?

Both the U.S. Constitution and The Federalist Papers suggest that voters ought to have more freedom to vote for the candidate of their choice for the U.S. House of Representatives than they do for the President or the U.S. Senate. Yet, strangely, for the last thirty-three years, the U.S. Supreme Court and lower courts have ruled that the Constitution gives voters more freedom to vote for the candidate of their choice in presidential elections than in congressional elections. Also, state legislatures, which have been writing ballot access laws since 1888, have passed laws that make it easier for minor-party and independent candidates to get on the ballot for President than for the U.S. House. As a result, voters in virtually every state invariably have far more choices on their general election ballots for the President than they do for the House. This Article argues that the right of a voter to vote for someone other than a Democrat or a Republican for the House is just as important as a voter’s right to do so for President, and that courts should grant more ballot access protection to minor-party and independent candidates for the House

Evidence for recombinant GRP78, CALR, PDIA3 and GPI as mediators of genetic instability in human CD34+ cells

Soluble factors released from irradiated human mesenchymal stromal cells (MSC) may induce genetic instability in human CD34+ cells, potentially mediating hematologic disorders. Recently, we identified four key proteins in the secretome of X-ray-irradiated MSC, among them three endoplasmic reticulum proteins, the 78 kDa glucose-related protein (GRP78), calreticulin (CALR), and protein disulfide-isomerase A3 (PDIA3), as well as the glycolytic enzyme glucose-6-phosphate isomerase (GPI). Here, we demonstrate that exposition of CD34+ cells to recombinant GRP78, CALR, PDIA3 and GPI induces substantial genetic instability. Increased numbers of γH2AX foci (p < 0.0001), centrosome anomalies (p = 0.1000) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells upon incubation with these factors. Specifically, γH2AX foci were found to be induced 4–5-fold in response to any individual of the four factors, and centrosome anomalies by 3–4 fold compared to control medium, which contained none of the recombinant proteins. Aberrant metaphases, not seen in the context of control medium, were detected to a similar extent than centrosome anomalies across the four factors. Notably, the strongest effects were observed when all four factors were collectively provided. In summary, our data suggest that specific components of the secretome from irradiated MSC act as mediators of genetic instability in CD34+ cells, thereby possibly contributing to the pathogenesis of radiation-induced hematologic disorders beyond direct radiation-evoked DNA strand breaks