Rapid prototyping and tooling in the manufacture of mould elements for large parts

In the last two decades the advances in rapid prototyping and tooling (RPT) technologies made possible the use of materials alternative to steel in some mould components. They also have allowed novel fabrication strategies and new ways of thinking on the production of injection moulds for short series of plas-tics parts. These technologies induced a new generation of toolmaking techniques. In the scope of the devel-opment project Hybridmould 21 the pros and cons of some RPT technologies were studied to fabricate moulding blocks and inserts for hybrid injection moulds for large plastics parts. Essentially, the studies aimed at finding the best solution for quick production and compliance with specific product requirements, such as complex features, complex cooling layout and large size (around 1 m2) in short production series. This paper discusses the main advantages and disadvantages associated to a number of different RPT technologies for the manufacture of moulding element.Program QREN - ‘Concessão de Incentivos Financeiros no âmbito do Sistema de Incentivos à Investigação e Desenvolvimento Tecnológico’ (contract 2010/013307) – Project Hybridmould 2

Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion

The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1-3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4-6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9-12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mpsl, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.Lisboa Regional Operational Programme (Lisboa 2020) through the European Regional Development Fund (FEDER); Fundacao para a Ciencia e a Tecnologia (FCT; Portugal); FCT [SFRH/BPD/87482/2012, SFRH /BD/52438/2013, PD/BD/52428/2013, PD/00117/2012, CRM: 0027030, PTDC/BEX-BID/0395/2014, UID/BIM/04773/2013 CBMR 1334, IF/00851/2012/CP0185/CT0004]; Association for International Cancer Research [AICR 10-0553]; EMBO Installation Grant [IG2778]; European Research Council Starting Grant [ERC-2014-STG-638917]; [PPBI-POCI-01-0145-FEDER-022122]; [LISBOA-01-0145-FEDER-022170

Temporal and Spatial Control of Germ-Plasm RNAs

SummaryIn many species, germ cells form in a specialized germ plasm, which contains localized maternal RNAs [1–5]. In the absence of active transcription in early germ cells, these maternal RNAs encode germ-cell components with critical functions in germ-cell specification, migration, and development [6, 7]. For several RNAs, localization has been correlated with release from translational repression, suggesting an important regulatory function linked to localization [3, 4, 8, 9]. To address the role of RNA localization and translational control more systematically, we assembled a comprehensive set of RNAs that are localized to polar granules, the characteristic germ-plasm organelles. We find that the 3′-untranslated regions (UTRs) of all RNAs tested control RNA localization and instruct distinct temporal patterns of translation of the localized RNAs. We demonstrate necessity for translational timing by swapping the 3′UTR of polar granule component (pgc), which controls translation in germ cells, with that of nanos, which is translated earlier. Translational activation of pgc is concurrent with extension of its poly(A) tail length but appears largely independent of the Drosophila CPEB homolog ORB. Our results demonstrate a role for 3′UTR mediated translational regulation in fine-tuning the temporal expression of localized RNA, and this may provide a paradigm for other RNAs that are found enriched at distinct cellular locations such as the leading edge of fibroblasts or the neuronal synapse

A dual-function SNF2 protein drives chromatid resolution and nascent transcripts removal in mitosis

Mitotic chromatin is largely assumed incompatible with transcription due to changes in the transcription machinery and chromosome architecture. However, the mechanisms of mitotic transcriptional inactivation and their interplay with chromosome assembly remain largely unknown. By monitoring ongoing transcription in Drosophila early embryos, we reveal that eviction of nascent mRNAs from mitotic chromatin occurs after substantial chromosome compaction and is not promoted by condensin I. Instead, we show that the timely removal of transcripts from mitotic chromatin is driven by the SNF2 helicase-like protein Lodestar (Lds), identified here as a modulator of sister chromatid cohesion defects. In addition to the eviction of nascent transcripts, we uncover that Lds cooperates with Topoisomerase 2 to ensure efficient sister chromatid resolution and mitotic fidelity. We conclude that the removal of nascent transcripts upon mitotic entry is not a passive consequence of cell cycle progression and/or chromosome compaction but occurs via dedicated mechanisms with functional parallelisms to sister chromatid resolution.info:eu-repo/semantics/publishedVersio

Body composition, strength static and isokinetic, and bone health: comparative study between active adults and amateur soccer players

The Centro de Investigação do Desporto e Actividade Física (Research Unit for the Study of Sport and Physical Activity, uid/dtp/04213/2019) is being funded by Fundação para a Ciência e a Tecnologia. AOW is supported by São Paulo Research Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) process: 2017/27234-2.Objective: To compare tissue composition, total and regional bone mineral content and bone mineral density, static hand grip and knee joint isokinetic strength between amateur soccer players and Control Group. Methods: Cross-sectional study. Air displacement plethysmography was used to estimate body volume and, in turn, density. Body composition, bone mineral content and bone mineral density were assessed for the whole body and at standardized regions using dual energy X-ray absorptiometry. Static grip strength was assessed with an adjustable dynamometer, and peak torque derived from isokinetic strength dynamometer (concentric muscular knee actions at 60°/s). Magnitude of the differences between groups was examined using d-Cohen. Results: Compared to healthy active adults, soccer players showed larger values of whole body bone mineral content (+651g; d=1.60; p<0.01). In addition, differences between groups were large for whole body bone mineral density (d=1.20 to 1.90; p<0.01): lumbar spine, i.e. L1-L4 (+19.4%), upper limbs (+8.6%) and lower limbs (+16.8%). Soccer players attained larger mean values in strength test given by static hand grip protocol (+5.6kg, d=0.99; p<0.01). Conclusion: Soccer adequately regulates body composition and is associated better bone health parameters (bone mineral content and density at whole-body and at particular sites exposed to mechanical loadings).Objetivo: Comparar a composição de tecidos, o conteúdo mineral ósseo e a densidade mineral óssea totais e por regiões do corpo, a força de preensão manual estática, e força isocinética da articulação do joelho, entre um grupo de jogadores de futebol amadores e um Grupo Controle. Métodos: Estudo transversal utilizando pletismografia de ar deslocado para estimar o volume corporal, para subsequente cálculo da densidade corporal. A composição de tecidos, o conteúdo mineral ósseo e a densidade mineral óssea foram avaliados para o corpo todo e regiões padronizadas através da absorciometria de raios-X de dupla energia. A força de preensão manual estática foi avaliada por um dinamômetro ajustável. Os momentos máximos de força das ações musculares concêntricas para os extensores e flexores do joelho foram avaliados pela dinamometria isocinética (60°/s). Foi calculado o valor d-Cohen para apreciar a magnitude do efeito das diferenças entre grupos. Resultados: Os futebolistas apresentaram níveis superiores de conteúdo mineral ósseo em comparação com os adultos ativos do Grupo Controle (+651g; d=1,60; p<0,01) e obtiveram valores superiores de densidade mineral óssea (d: 1,20 a 1,90; p<0,01) para a coluna lombar, L1-L4 (+19,4%), membros superiores (+8,6%) e membros inferiores (+16,8%). Para a força de preensão (estática) a diferença foi moderada (d=0,99; p<0,01) com valores mais elevados apresentados pelo futebolistas (+5,6kg; d=0,99; p<0,01). Conclusão: A prática de futebol promove uma regulação adequada da composição corporal (tecidos magro e gordo) e ganhos na densidade mineral óssea, mais acentuada em partes do corpo com maior exposição aos impactos mecânicos da atividade motora.info:eu-repo/semantics/publishedVersio

Downregulation of RKIP Is Associated with Poor Outcome and Malignant Progression in Gliomas

Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of beta-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4. C8 and 02) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a beta-strand connected by a short loop to an alpha-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as,a potential model organism for MADD. (C) 2012 Elsevier B.V. All rights reserved.Fundacao para a Ciencia e Tecnologia (FCT/MCTES, Portugal) [PTDC/SAU-GMG/70033/2006, PTDC/QUI-BIQ/113027/2009, PTDC/BIA-BCM/111822/2009, PTDC/SAU-BID/111796/2009, SFRH/BPD/41609/2007, SFRH/BPD/74475/2010, SFRH/BPD/34763/2007]; CLIMB UK; [PEst-OE/EQB/LA0004/2011]info:eu-repo/semantics/publishedVersio

Zeolite structures loading with an anticancer compound as drug delivery systems

The authors are thankful to Dr. A. S. Azevedo for collecting the powder diffraction data.Two different structures of zeolites, faujasite (FAU) and Linde type A (LTA), were studied to investigate their suitability for drug delivery systems (DDS). The zeolites in the sodium form (NaY and NaA) were used as hosts for encapsulation of α-cyano-4- hydroxycinnamic acid (CHC). CHC, an experimental anticancer drug, was encapsulated in both zeolites by diffusion in liquid phase. These new drug delivery systems, CHC@zeolite, were characterized by spectroscopic techniques (FTIR, 1H NMR, 13C and 27Al solidstate MAS NMR, and UV−vis), chemical analysis, powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). The effect of the zeolites and CHC@zeolite drug deliveries on HCT-15 human colon carcinoma cell line viability was evaluated. Both zeolites alone revealed no toxicity to HCT-15 cancer cells. Importantly, CHC@zeolite exhibit an inhibition of cell viability up to 585-fold, when compared to the non-encapsulated drug. These results indicate the potential of the zeolites for drug loading and delivery into cancer cells to induce cell deathO.M. and R.A. are recipients of fellowships (SFRH/BD/36463/2007, SFRH/BI/51118/2010) from Fundação para a Ciência e a Tecnologia (FCT, Portugal). This work was supported by the FCT projects refs PEst-C/ QUI/UI0686/2011, PEst-C/CTM/LA0011/2011, and PTDC/ SAU-FCF/104347/2008, under the scope of “Programa Operacional Temático Factores de Competitividade” (COMPETE) of “Quadro Comunitário de Apoio III” and cofinanced by Fundo Comunitário Europeu FEDER, and the Centre of Chemistry and Life and Health Sciences Research Institute (University of Minho, Portugal)

NatF Contributes to an Evolutionary Shift in Protein N-Terminal Acetylation and Is Important for Normal Chromosome Segregation

N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe