Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system

Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1 μg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneous detection of the toxins. The IC₅₀'s using this method were 1.9 ± 0.1 μg L⁻¹ MC-LR, 1.3 ± 0.1 μg L⁻¹ CYN, 61 ± 4 μg L⁻¹ ANA-a, 5.4 ± 0.4 μg L⁻¹ STX and 4.9 ± 0.9 μg L⁻¹ DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC–IT-TOF-MS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC–IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts.

Herschel key program HERITAGE:A far-infrared source catalog for the Magellanic Clouds

Observations from the HERschel Inventory of the Agents of Galaxy Evolution (HERITAGE ) have been used to identify dusty populations of sources in the Large and Small Magellanic Clouds (LMC and SMC). We conducted the study using the HERITAGE catalogs of point sources available from the Herschel Science Center from both the Photodetector Array Camera and Spectrometer (PACS; 100 and 160 μm) and Spectral and Photometric Imaging Receiver (SPIRE; 250, 350, and 500 μm) cameras. These catalogs are matched to each other to create a Herschel band-merged catalog and then further matched to archival Spitzer IRAC and MIPS catalogs from the Spitzer Surveying the Agents of Galaxy Evolution (SAGE) and SAGE-SMC surveys to create single mid- to far-infrared (far-IR) point source catalogs that span the wavelength range from 3.6 to 500 μm. There are 35,322 unique sources in the LMC and 7503 in the SMC. To be bright in the FIR, a source must be very dusty, and so the sources in the HERITAGE catalogs represent the dustiest populations of sources. The brightest HERITAGE sources are dominated by young stellar objects (YSOs), and the dimmest by background galaxies. We identify the sources most likely to be background galaxies by first considering their morphology (distant galaxies are point-like at the resolution of Herschel) and then comparing the flux distribution to that of the Herschel Astrophysical Terahertz Large Area Survey (ATLAS ) survey of galaxies. We find a total of 9745 background galaxy candidates in the LMC HERITAGE images and 5111 in the SMC images, in agreement with the number predicted by extrapolating from the ATLAS flux distribution. The majority of the Magellanic Cloud-residing sources are either very young, embedded forming stars or dusty clumps of the interstellar medium. Using the presence of 24 μm emission as a tracer of star formation, we identify 3518 YSO candidates in the LMC and 663 in the SMC. There are far fewer far-IR bright YSOs in the SMC than the LMC due to both the SMC's smaller size and its lower dust content. The YSO candidate lists may be contaminated at low flux levels by background galaxies, and so we differentiate between sources with a high ("probable") and moderate ("possible ") likelihood of being a YSO. There are 2493/425 probable YSO candidates in the LMC/SMC. Approximately 73% of the Herschel YSO candidates are newly identified in the LMC, and 35% in the SMC. We further identify a small population of dusty objects in the late stages of stellar evolution including extreme and post-asymptotic giant branch, planetary nebulae, and supernova remnants. These populations are identified by matching the HERITAGE catalogs to lists of previously identified objects in the literature. Approximately half of the LMC sources and one quarter of the SMC sources are too faint to obtain accurate ample FIR photometry and are unclassified.

Sustainable binders for concrete: a structured approach from waste screening to binder composition development

Worldwide, the building sector requires the production of 4 billion tonnes of cement annually, consuming more than 40% of global energy. Alkali activated “cementless” binders have recently emerged as a novel eco-friendly construction material with a promising potential to replace ordinary Portland cement. These binders consist of a class of inorganic polymer formed mainly by the reaction between an alkaline solution and an aluminosilicate source. Precursor materials for this reaction can be found in secondary material streams from different industrial sectors, from energy to agro-alimentary. However, the suitability of these materials in developing the polymerisation reaction must be assessed through a detailed chemical and physical characterisation, ensuring the availability of required chemical species in the appropriate quantity and physical state. Furthermore, the binder composition needs to be defined in terms of proper alkali activation dosages, water content in the mix, and curing conditions. The mix design must satisfy mechanical requirements and compliance to desired engineering properties (workability, setting time) for ensuring the suitability of the binder in replacing Portland cement in concrete applications. This paper offers a structured approach for the development of secondary material-based binders, from their identification to mix design and production procedure development. Essential features of precursor material can be determined through chemical and physical characterisation methods and advanced microscope techniques. Important mixing parameters and binder properties requirements are examined and some examples of developed binders are reported.

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Molecular subtyping of primary prostate cancer reveals specific and shared target genes of different ETS rearrangements

This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

Nuclear trafficking and functions of endocytic proteins implicated in oncogenesis

A subset of proteins predominantly associated with early endosomes or implicated in clathrin-mediated endocytosis can shuttle between the cytoplasm and the nucleus. Although the endocytic functions of these proteins have been extensively studied, much less effort has been expended in exploring their nuclear roles. Membrane trafficking proteins can affect signalling and proliferation and this can be achieved either at a nuclear or endocytic level. Furthermore, some proteins, such as Huntingtin interacting protein 1, are known as cancer biomarkers. This review will highlight the limits of our understanding of their nuclear functions and the relevance of this to signalling and oncogenesis.

Clathrin is spindle-associated but not essential for mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.

METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.

CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.

Genomic Resources Notes Accepted 1 June 2015 - 31 July 2015

This article documents the public availability of (i) microbiomes in diet and gut of larvae from the dipteran Dilophus febrilis using massive parallel sequencing, (ii) SNP and SSR discovery and characterization in the transcriptome of the Atlantic mackerel (Scomber scombrus, L) and (iii) assembled transcriptome for an endangered, endemic Iberian cyprinid fish (Squalius pyrenaicus).

A promoção da autonomia dos alunos do ensino básico através das atividades experimentais de ciências

Relatório de Estágio apresentado à Escola Superior de Educação de Santarém para obtenção do Grau de Mestre em Ensino do 1.º e do 2.º Ciclo do Ensino Básico.Orientador:Professor Doutor Bento Cavadas

Expressão plástica : práticas e dinâmicas em contexto de ensino pré-escolar

As práticas educativas e os procedimentos pedagógicos, no plano do ensino pré-escolar, podem ser classificados mediante variadas propostas presentes na literatura e passíveis de serem observadas no terreno. Nesta perspetiva, e de modo introdutório ao tema, pergunto-me acerca dos modos pelos quais o agente educativo pode acompanhar e intervir pedagogicamente no âmbito da expressão plástica. Questão que surgiu por ter tido oportunidade de observar práticas de Expressão Plástica que parecem configurar num quadro marcado ora por um método de ensino muito condicionado pelas intenções do educador, ora por um método completamente aleatório, onde não se faz qualquer preparação prévia dando lugar ao que se entende por expressão livre. Face a este quadro questionei-me sobre qual deverá ser a minha atuação em termos de práticas e dinâmicas de expressão plástica no ensino pré-escolar. Neste relatório, reuni algumas respostas para a questão anterior, utilizando a revisão de literatura. Destaco, também, alguns dados empíricos recolhidos de maneira não sistemática pela observação direta e por entrevistas realizadas no âmbito desta investigação.

Ex vivo experimental model for percutaneous vertebroplasty: microCT utility

The aim of this study was to develop an ex vivo experimental animal model for percutaneous vertebroplasty, for further application in vivo to test novel bone injectable cements.

Development of a sheep vertebroplasty model for bioceramic materials assessment

Development of a sheep vertebroplasty model for bioceramic materials assessment Sheep has been widely used as an animal orthopaedic model. Although several studies report anatomic and biomechanical similarities as well as distinctions of ovine lumbar vertebrae when compared to human’s, only a few studies describe its actual use as a vertebroplasty model. Due to distinct anatomic features, sheep lumbar vertebrae pose a challenge when developing a minimally invasive procedure for vertebroplasty material testing, under conditions meant to be the most similar to clinical procedure. The present work describes the development of an appropriate surgical percutaneous vertebroplasty model in the lumbar spine of sheep, applicable in vivo, that minimizes the risk of post-surgical complications. This model was mechanically evaluated ex-vivo regarding its safety, and used to evaluate the injectability and radiopacity of two new bioceramic materials when compared to a commercial bioceramic bone substitute (Cerament™ SpineSupport). Microtomography techniques helped in the development of the model and results assessment. Under fluoroscopic guidance, a defect was created through a bilateral modified parapedicular access in the cranial hemivertebrae of 30 sheep lumbar vertebrae (L4, L5 and L6). The manually drilled defect had an average volume of 1209 ±226 mm3 and allowed the novel materials injection through a standardized injection cannula placed in one of the entrance points. Adequate defect filling was observed with all tested materials. No mechanical failure was observed under loads higher than the physiological.

Ex Vivo Model for Percutaneous Vertebroplasty

The testing of novel biomaterials for percutaneous vertebroplasty depends on suitable animal models. The aim of this study was to develop ex vivo a reproducible and feasible model of percutaneous vertebroplasty, for ulterior application in vivo. A large animal model was used (Merino sheep), due to its translational properties. Vertebroplasty was performed under tactile and fluoroscopic control, through a bilateral modified parapedicular access in lumbar vertebrae (n=12). Care was taken in order to avoid disruption of the vertebral foramen. The average defect volume was 1234±240 mm3. This mean volume ensures practical defects to test novel injectable biomaterials. 6 vertebrae were injected with a commercial cement (Cerament®, Bone Support, Sweden). Adequate defect filling was observed in all vertebrae. All vertebrae were assessed by microCT, prior to and post defect creation and after biomaterial injection. All vertebrae were mechanical tested. No mechanical failure was observed under loads higher than the physiological. Ultimately, this model is considered suitable for pre-clinical in vivo studies, mimicking clinical application.