Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome.

Axonal protein synthesis and degradation are rapidly regulated by extrinsic signals during neural wiring, but the full landscape of proteomic changes remains unknown due to limitations in axon sampling and sensitivity. By combining pulsed stable isotope labeling of amino acids in cell culture with single-pot solid-phase-enhanced sample preparation, we characterized the nascent proteome of isolated retinal axons on an unparalleled rapid timescale (5 min). Our analysis detects 350 basally translated axonal proteins on average, including several linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) show distinct signatures with more than 100 different nascent protein species up- or downregulated within the first 5 min followed by further dynamic remodeling. Switching repulsion to attraction triggers opposite regulation of a subset of common nascent proteins. Our findings thus reveal the rapid remodeling of the axonal proteomic landscape by extrinsic cues and uncover a logic underlying attraction versus repulsion

Production of carboxylates from high rate activated sludge through fermentation

The aim of this work was to study the key parameters affecting fermentation of high rate activated A-sludge to carboxylates, including pH, temperature, inoculum, sludge composition and iron content. The maximum volatile fatty acids production was 141 mg C g1 VSSfed, at pH 7. Subsequently the potentialfor carboxylate and methane production for A-sludge from four different plants at pH 7 and 35 C were compared. Initial BOD of the sludge appeared to be key determining carboxylate yield from A-sludge. Whereas methanogenesis could be correlated linearly to the quantity of ferric used for coagulation, fermentation did not show a dependency on iron presence. This difference may enable a strategywhereby A-stage sludge is separated to achieve fermentation, and iron dosing for phosphate removal is only implemented at the B-stage

A simple cytofluorimetric score may optimize testing for biallelic CEBPA mutations in patients with acute myeloid leukemia

Acute myeloid leukemia with biallelic mutation of CEBPA (CEBPA-dm AML) is a distinct good prognosis entity recognized by WHO 2016 classification. However, testing for CEBPA mutation is challenging, due to the intrinsic characteristics of the mutation itself. Indeed, molecular analysis cannot be performed with NGS technique and requires Sanger sequencing. The association of recurrent mutations or translocations with specific immunophenotypic patterns has been already reported in other AML subtypes. The aim of this study was the development of a specific cytofluorimetric score (CEBPA-dm score), in order to distinguish patients who are unlikely to harbor the mutation. To this end, the correlation of CEBPA-dm score with the presence of the mutation was analyzed in 50 consecutive AML patients with normal karyotype and without NPM1 mutation (that is mutually exclusive with CEBPA mutation). One point each was assigned for expression of HLA DR, CD7, CD13, CD15, CD33, CD34 and one point for lack of expression of CD14. OS was not influenced by sex, age and CEBPA-dm score. Multivariate OS analysis showed that CEBPA-dm (p < 0.02) and FLT3-ITD (p < 0.01) were the strongest independent predictors of OS. With a high negative predictive value (100%), CEBPA-dm score < 6 was able to identify patients who are unlikely to have the mutation. Therefore, the application of this simple score might optimize the use of expensive and time-consuming diagnostic and prognostic assessment in the baseline work up of AML patients

On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons.

Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.This work was supported by Wellcome Trust Grants (085314/Z/08/Z and 203249/Z/16/Z) to C.E.H. and (100329/Z/12/Z) to W.A.H., European Research Council Advanced Grant (322817) to C.E.H., Champalimaud Vision Award to C.E.H. and by the Netherlands Organization for Scientific Research (NWO Rubicon 019.161LW.033) to M.K. CFK acknowledges funding from the UK Engineering and Physical Sciences Research Council, EPSRC (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z) and the UK Medical Research Council, MRC (grants MR/K015850/1 and MR/K02292X/1) and Infinitus (China) Ltd

Depletion of SIRT6 enzymatic activity increases acute myeloid leukemia cells' vulnerability to DNA-damaging agents.

Genomic instability plays a pathological role in various malignancies, including acute myeloid leukemia (AML), and thus represents a potential therapeutic target. Recent studies demonstrate that SIRT6, a NAD <sup>+</sup> -dependent nuclear deacetylase, functions as genome-guardian by preserving DNA integrity in different tumor cells. Here, we demonstrate that also CD34 <sup>+</sup> blasts from AML patients show ongoing DNA damage and SIRT6 overexpression. Indeed, we identified a poor-prognostic subset of patients, with widespread instability, which relies on SIRT6 to compensate for DNA-replication stress. As a result, SIRT6 depletion compromises the ability of leukemia cells to repair DNA double-strand breaks that, in turn, increases their sensitivity to daunorubicin and Ara-C, both in vitro and in vivo In contrast, low SIRT6 levels observed in normal CD34 <sup>+</sup> hematopoietic progenitors explain their weaker sensitivity to genotoxic stress. Intriguingly, we have identified DNA-PKcs and CtIP deacetylation as crucial for SIRT6-mediated DNA repair. Together, our data suggest that inactivation of SIRT6 in leukemia cells leads to disruption of DNA-repair mechanisms, genomic instability and aggressive AML. This synthetic lethal approach, enhancing DNA damage while concomitantly blocking repair responses, provides the rationale for the clinical evaluation of SIRT6 modulators in the treatment of leukemia

Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD+-independent HDACs are an established therapeutic target, the relevance of NAD+-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD+-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD+ levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD+-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited

Sampling bias inverts ecogeographical relationships in island reptiles

Aim: Species richness is one of the commonest measures of biodiversity, and is a basis for analyses at multiple scales. Data quality may affect estimations of species richness, but most broad-scale studies do not take sampling biases into account. We analysed reptile richness on islands that have received different sampling efforts, and assessed how inventory completeness affects the results of ecogeographical analyses. We also used simulations to evaluate under what circumstances insufficient sampling can bias the outcome of biodiversity analyses. Location: Mediterranean islands. Methods: We gathered data on reptile richness from 974 islands, assuming better sampling in islands with specific inventories. We used Moran's eigenvector mapping to analyse the factors that determine whether an island has been surveyed, and to identify the relationships between reptile richness, geographical parameters and anthropic parameters. We simulated islands, mimicking patterns of true data, and sampled them with varying effort. Simulated richness was analysed using the same approach used for real-world data. Results: The probability that islands were sampled for reptiles was higher in large, human-populated islands. The relationship between human impact and reptile richness was negative in well-surveyed islands, but positive in islands that had not been systematically surveyed, because densely populated and accessible islands receive better sampling. In simulations, analyses successfully retrieved the relationships between species richness and human presence only if the average species detection probability was 6575%. Poorer sampling resulted in biased regression results. Main conclusions: Human activities may strongly affect biodiversity, but human presence and accessibility improve sampling effort and thus the quality of biodiversity information. Therefore, regressing known species richness on parameters representing human presence may result in apparent positive relationships. These two facets of human presence (positive on biodiversity knowledge, negative on actual biodiversity) represent a major challenge for ecogeographical studies, as not taking them into account would bias analyses and underestimate human impact