Directed Evolution of Protein-Based Neurotransmitter Sensors for MRI

The production of contrast agents sensitive to neuronal signaling events is a rate-limiting step in the development of molecular-level functional magnetic resonance imaging (molecular fMRI) approaches for studying the brain. High-throughput generation and evaluation of potential probes are possible using techniques for macromolecular engineering of protein-based contrast agents. In an initial exploration of this strategy, we used the method of directed evolution to identify mutants of a bacterial heme protein that allowed detection of the neurotransmitter dopamine in vitro and in living animals. The directed evolution method involves successive cycles of mutagenesis and screening that could be generalized to produce contrast agents sensitive to a variety of molecular targets in the nervous system

Mobilized Peripheral Blood Stem Cells Versus Unstimulated Bone Marrow As a Graft Source for T-Cell-Replete Haploidentical Donor Transplantation Using Post-Transplant Cyclophosphamide.

Purpose T-cell-replete HLA-haploidentical donor hematopoietic transplantation using post-transplant cyclophosphamide was originally described using bone marrow (BM). With increasing use of mobilized peripheral blood (PB), we compared transplant outcomes after PB and BM transplants. Patients and Methods A total of 681 patients with hematologic malignancy who underwent transplantation in the United States between 2009 and 2014 received BM (n = 481) or PB (n = 190) grafts. Cox regression models were built to examine differences in transplant outcomes by graft type, adjusting for patient, disease, and transplant characteristics. Results Hematopoietic recovery was similar after transplantation of BM and PB (28-day neutrophil recovery, 88% v 93%, P = .07; 100-day platelet recovery, 88% v 85%, P = .33). Risks of grade 2 to 4 acute (hazard ratio [HR], 0.45; P \u3c .001) and chronic (HR, 0.35; P \u3c .001) graft-versus-host disease were lower with transplantation of BM compared with PB. There were no significant differences in overall survival by graft type (HR, 0.99; P = .98), with rates of 54% and 57% at 2 years after transplantation of BM and PB, respectively. There were no differences in nonrelapse mortality risks (HR, 0.92; P = .74) but relapse risks were higher after transplantation of BM (HR, 1.49; P = .009). Additional exploration confirmed that the higher relapse risks after transplantation of BM were limited to patients with leukemia (HR, 1.73; P = .002) and not lymphoma (HR, 0.87; P = .64). Conclusion PB and BM grafts are suitable for haploidentical transplantation with the post-transplant cyclophosphamide approach but with differing patterns of treatment failure. Although, to our knowledge, this is the most comprehensive comparison, these findings must be validated in a randomized prospective comparison with adequate follow-up

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by 13C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively

Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine

The development of molecular probes that allow in vivo imaging of neural signaling processes with high temporal and spatial resolution remains challenging. Here we applied directed evolution techniques to create magnetic resonance imaging (MRI) contrast agents sensitive to the neurotransmitter dopamine. The sensors were derived from the heme domain of the bacterial cytochrome P450-BM3 (BM3h). Ligand binding to a site near BM3h's paramagnetic heme iron led to a drop in MRI signal enhancement and a shift in optical absorbance. Using an absorbance-based screen, we evolved the specificity of BM3h away from its natural ligand and toward dopamine, producing sensors with dissociation constants for dopamine of 3.3–8.9 μM. These molecules were used to image depolarization-triggered neurotransmitter release from PC12 cells and in the brains of live animals. Our results demonstrate the feasibility of molecular-level functional MRI using neural activity–dependent sensors, and our protein engineering approach can be generalized to create probes for other targets.Charles A. Dana Foundation. Brain and Immuno-ImagingRaymond and Beverley Sackler FoundationNational Institutes of Health (U.S.) (grant R01-DA28299)National Institutes of Health (U.S.) (grant DP2-OD2441)National Institutes of Health (U.S.) (grant R01-GM068664)Jacobs Institute for Molecular Engineering for Medicine. Jacobs Institute for Molecular Engineering for MedicineNational Institutes of Health (U.S.) (grant R01-DE013023

Is Corporate Social Responsibility an Agency Problem? Evidence from CEO Turnovers

We empirically examine two competing claims: first, if a firm’s Corporate Social Responsibility (CSR) activity is driven by its CEO’s private rent extraction (i.e. an agency problem), firms with higher CSR ratings are poorly governed and their managers are less likely to be dismissed for poor financial performance. In contrast, if CSR reflects owners’ preferences, CEOs of firms with higher CSR ratings are more likely to be removed in light of poor financial performance. We find that CEO turnover-financial performance sensitivity increases in firm CSR scores during the last years of both the outgoing CEO as well as his predecessor. Further, firm CSR ratings do not change following CEO turnover suggesting that CSR ratings are a firm characteristic. Our findings are consistent with the view that CSR is driven by shareholder preferences

Clonal interference of signaling mutations worsens prognosis in core-binding factor acute myeloid leukemia

Mutations in receptor tyrosine kinase/RAS signaling pathway genes are frequent in core-binding factor (CBF) acute myeloid leukemias (AMLs), but their prognostic relevance is debated. A subset of CBF AML patients harbors several signaling gene mutations. Genotyping of colonies and of relapse samples indicates that these arise in independent clones, thus defining a process of clonal interference (or parallel evolution). Clonal interference is pervasive in cancers, but the mechanisms underlying this process remain unclear, and its prognostic impact remains unknown. We analyzed a cohort of 445 adult and pediatric patients with CBF AML treated with intensive chemotherapy and with deep sequencing of 6 signaling genes (, , , , , ). A total of 152 (34%), 167 (38%), and 126 (28%) patients harbored no, a single, and multiple signaling clones (clonal interference), respectively. Clonal interference of signaling mutations was associated with older age ( = .004) and inv(16) subtype ( = .025) but not with white blood cell count or mutations in chromatin or cohesin genes. The median allele frequency of signaling mutations was 31% in patients with a single clone or clonal interference ( = .14). The repertoire of , , and / variants differed between groups. Clonal interference did not affect complete remission rate or minimal residual disease after 1-2 courses, but it did convey inferior event-free survival ( < 10), whereas the presence of a single signaling clone did not ( = .44). This inferior outcome was independent of clinical parameters and of the presence of specific signaling clones. Our results suggest that specific clonal architectures can herald distinct prognoses in AML

Scoring System Prognostic of Outcome in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation for Myelodysplastic Syndrome

To develop a system prognostic of outcome in those undergoing allogeneic hematopoietic cell transplantation (allo HCT) for myelodysplastic syndrome (MDS)

Dietary practices in isovaleric acidemia:A European survey

Background: In Europe, dietary management of isovaleric acidemia (IVA) may vary widely. There is limited collective information about dietetic management. Aim: To describe European practice regarding the dietary management of IVA, prior to the availability of the E-IMD IVA guidelines (E-IMD 2014). Methods: A cross-sectional questionnaire was sent to all European dietitians who were either members of the Society for the Study of Inborn Errors of Metabolism Dietitians Group (SSIEM-DG) or whom had responded to previous questionnaires on dietetic practice (n=53). The questionnaire comprised 27 questions about the dietary management of IVA. Results: Information on 140 patients with IVA from 39 centres was reported. 133 patients (38 centres) were given a protein restricted diet. Leucine-free amino acid supplements (LFAA) were routinely used to supplement protein intake in 58% of centres. The median total protein intake prescribed achieved the WHO/FAO/UNU [2007] safe levels of protein intake in all age groups. Centres that prescribed LFAA had lower natural protein intakes in most age groups except 1 to 10 y. In contrast, when centres were not using LFAA, the median natural protein intake met WHO/FAO/UNU [2007] safe levels of protein intake in all age groups. Enteral tube feeding was rarely prescribed. Conclusions: This survey demonstrates wide differences in dietary practice in the management of IVA across European centres. It provides unique dietary data collectively representing European practices in IVA which can be used as a foundation to compare dietary management changes as a consequence of the first E-IMD IVA guidelines availability. (C) 2017 The Authors. Published by Elsevier Inc

CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex

The gene encoding the transcription factor C/EBP alpha is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause ablation of full-length C/EBP alpha without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBP alpha p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBP alpha p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBP alpha p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations