The third dimension: new developments in cell culture models for colorectal research

Cellular models are important tools in various research areas related to colorectal biology and associated diseases. Herein, we review the most widely used cell lines and the different techniques to grow them, either as cell monolayer, polarized two-dimensional epithelia on membrane filters, or as three-dimensional spheres in scaffoldfree or matrix-supported culture conditions. Moreover, recent developments, such as gut-on-chip devices or the ex vivo growth of biopsy-derived organoids, are also discussed. We provide an overview on the potential applications but also on the limitations for each of these techniques, while evaluating their contribution to provide more reliable cellular models for research, diagnostic testing, or pharmacological validation related to colon physiology and pathophysiology.

Evaluating green infrastructure in urban environments using a multi-taxa and functional diversity approach

Forested areas within cities host a large number of species, responsible for many ecosystem services in urban areas. The biodiversity in these areas is influenced by human disturbances such as atmospheric pollution and urban heat island effect. To ameliorate the effects of these factors, an increase in urban green areas is often considered sufficient. However, this approach assumes that all types of green cover have the same importance for species. Our aim was to show that not all forested green areas are equal in importance for species, but that based on a multi-taxa and functional diversity approach it is possible to value green infrastructure in urban environments. After evaluating the diversity of lichens, butterflies and other-arthropods, birds and mammals in 31 Mediterranean urban forests in south-west Europe (Almada, Portugal), bird and lichen functional groups responsive to urbanization were found. A community shift (tolerant species replacing sensitive ones) along the urbanization gradient was found, and this must be considered when using these groups as indicators of the effect of urbanization. Bird and lichen functional groups were then analyzed together with the characteristics of the forests and their surroundings. Our results showed that, contrary to previous assumptions, vegetation density and more importantly the amount of urban areas around the forest (matrix), are more important for biodiversity than forest quantity alone. This indicated that not all types of forested green areas have the same importance for biodiversity. An index of forest functional diversity was then calculated for all sampled forests of the area. This could help decision-makers to improve the management of urban green infrastructures with the goal of increasing functionality and ultimately ecosystem services in urban areas.

Colletotrichum species in Australia

Forty-four species of Colletotrichum are confirmed as present in Australia based on DNA sequencing analyses. Many of these species were identified directly as a result of two workshops organised by the Subcommittee on Plant Health Diagnostics in Australia in 2015 that covered morphological and molecular approaches to identification of Colletotrichum. There are several other species of Colletotrichum reported from Australia that remain to be substantiated by DNA sequence-based methods. This body of work aims to provide a basis from which to critically examine a number of isolates of Colletotrichum deposited in Australian culture collections.

Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities.

GuiaTreeKey, a multi-access electronic key to identify tree genera in French Guiana

International audience

Anticancer and antibacterial secondary metabolites from the endophytic fungus Penicillium sp. CAM64 against multi-drug resistant Gram-negative bacteria.

Background: The emergence of multiple-drug resistance bacteria has become a major threat and thus calls for an urgent need to search for new effective and safe anti-bacterial agents. Objectives: This study aims to evaluate the anticancer and antibacterial activities of secondary metabolites from Penicillium sp. , an endophytic fungus associated with leaves of Garcinia nobilis . Methods: The culture filtrate from the fermentation of Penicillium sp. was extracted and analyzed by liquid chromatography– mass spectrometry, and the major metabolites were isolated and identified by spectroscopic analyses and by comparison with published data. The antibacterial activity of the compounds was assessed by broth microdilution method while the anticancer activity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: The fractionation of the crude extract afforded penialidin A-C (1-3), citromycetin (4), p-hydroxyphenylglyoxalaldoxime (5) and brefelfin A (6). All of the compounds tested here showed antibacterial activity (MIC = 0.50 – 128 μg/mL) against Gramnegative multi-drug resistance bacteria, Vibrio cholerae (causative agent of dreadful disease cholera) and Shigella flexneri (causative agent of shigellosis), as well as the significant anticancer activity (LC50 = 0.88 – 9.21 μg/mL) against HeLa cells. Conclusion: The results obtained indicate that compounds 1-6 showed good antibacterial and anticancer activities with no toxicity to human red blood cells and normal Vero cells.

Biometry and body chemical eomfosition of some Japanese and Eufopean fish gpecles in culture

Dissertação de mest. em Aquacultura, Unidade de Ciências e Tecnologias dos Recursos Aquáticos, Univ. do Algarve, 1997

Dynamics of some limnological characteristics in pacu (Piaractus mesopotamicus) culture tanks as function of handling

Visando obter subsídios para um estudo da qualidade da água em tanques de piscicultura, realizou-se um experimento de 166 dias com uma espécie nativa, pacu (Piaractus mesopotamicus). Nos tanques foram testados dois níveis diferentes de proteína na dieta (16% e 34% de proteína bruta) e três densidades de estocagem (0,25; 0,50 e 0,77 peixes/m²). Dos resultados obtidos foi observado que a interação entre a densidade de estocagem e a duração do experimento interferiram nas variáveis bicarbonato e alcalinidade e a interação entre a densidade de estocagem e a porcentagem de proteína interferiram nas concentrações de CO2 livre e total, condutividade e pH (P < 0,05). A temperatura da água nos tanques variou significativamente ao longo do período estudado (P < 0,05), diminuindo gradativamente do verão para o inverno. Não houve diferença significativa no tempo de residência da água nos tanques (P > 0,05) durante a duração do experimento. Os demais parâmetros não sofreram interferência dos tratamentos ao longo do período de estudo.

Population growth and development of two species of Cladocera, Moina micrura and Diaphanosoma birgei, in laboratory

O objetivo do presente trabalho foi testar a influência de quatro dietas alimentares sobre o crescimento populacional, desenvolvimento, comprimento total, peso seco e valor nutricional de duas espécies zooplanctônicas, Moina micrura and Diaphanosoma birgei, com os seguintes tratamentos alimentares: somente alga (A), alga + vitaminas (AV), alga + ração (AR) e alga + ração + vitaminas (ARV). O pico de crescimento para as duas espécies estudadas ocorreu mais rápido no tratamento AV. em geral, o tratamento AV para M. micrura mostrou melhores resultados para taxa intrínseca, fecundidade, desenvolvimento embrionário e pós-embrionário. Já a longevidade e número total de desovas apresentaram melhores resultados no tratamento AR (p < 0,05). Para D. birgei, os melhores resultados foram obtidos nos tratamentos contendo ração e vitamina (p < 0,05). A maior porcentagem de proteínas e lipídeos para os dois cladóceros ocorreu nos tratamentos contendo ração, já o carboidrato foi maior no tratamento contendo somente alga (p < 0,05). em geral, as dietas contendo ração e vitamina apresentaram os melhores resultados para o desenvolvimento dos cladóceros, com qualidade de água adequada para cultivo, podendo ser utilizadas em culturas com altas concentrações em laboratório.

Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil

Flavobacterium columnare é o agente etiológico da columnariose em peixes de água doce, ocasionando enfermidade na pele e nas brânquias, provocando freqüentemente um grande número de mortalidade. O objetivo deste estudo foi o isolamento e a caracterização de Flavobacterium columnare em peixes tropicais no Brasil. Piracanjuba (Brycon orbignyanus), pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum) e cascudo (Hypostomus plecostomus) foram examinados externamente com relação a sinais característicos de columnariose, como manchas acinzentadas na cabeça, região dorsal e pedúnculo caudal dos peixes. A amostragem compreendeu a coleta de 50 exemplares de peixes, representando as quatro diferentes espécies escolhidas para este estudo. Amostras para o isolamento foram obtidas através de raspado com swab estéril das lesões e do rim dos peixes clinicamente diagnosticados como acometidos por columnarios e imediatamente semeados em meios de culturas artificiais (líquido e sólido) próprios para o estudo de Flavobacterium segundo Carlson e Pacha (1968). No meio líquido, houve o desenvolvimento de microrganismos que observados em gota pendente apresentaram a forma de bacilos finos, longos, móveis por deslizamento. Através da coloração de Gram, apresentaram morfologia de bacilos finos, Gram negativos, agrupados em colunas. em meio sólido, as colônias eram pequenas, cinza-amareladas, com borda em forma de raiz. No total, foram obtidos quatro isolamentos: 01 cepa de Brycon orbignyanus; 01 cepa de Piaractus mesopotamicus; 01 cepa de Colossoma macropomum; e 01 cepa de Hypostomus plecostomus. A caracterização bioquímica das amostras, como absorção do vermelho Congo, produção de flexirrubina, produção de H2S e redução do nitrato, sugere que os isolamentos poderiam ser classificados como Flavobacterium columnare.

Multi-Etiological Nature of Tuberculosis-Like Lesions in Condemned Pigs at the Slaughterhouse.

Tuberculosis-like lesions (TBL) in pigs have been associated with microorganisms other than mycobacteria. In this work a histopathological and microbiological evaluation of TBL in pigs is shown. A total of 352 samples belonging to 171 pigs totally condemned at slaughterhouse due to generalized TBL were sampled and selected for analysis. Pyogranulomatous (56.2%) and granulomatous lesions (20.2%) were observed in all analysed organs. Most of the granulomas observed in both lymph nodes and lungs belonged to more advanced stages of development (stages III and IV) whereas in the liver and the spleen most of lesions belonged to intermediate stages (stages II and III). Different microorganisms were simultaneously detected from TBL in the 42.7% of the animals. Mycobacterium tuberculosis complex (MTC) (38%), coryneform bacteria (40.3%) and streptococci (28.1%) were the main groups of microorganisms detected after bacteriological analysis, with Trueperella pyogenes and Streptococcus suis as the most frequently isolated species. Mycobacteria belonging to MTC were the most frequently detected pathogens in granulomatous and pyogranulomatous lesions in submandibular lymph nodes (32.7%) and coryneform bacteria were the microorganisms more frequently isolated from lungs (25.9%) and spleen samples (37.2%). These results may provide new insights into the pathogenesis and diagnosis of this pathology. The importance of coryneform bacteria and streptococci in such processes must be evaluated in future studies.

First report of Ciguatoxins in two starfish species: Ophidiaster ophidianus and Marthasterias glacialis

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk.

A multi-model approach to evaluate the role of environmental variability and fishing pressure in sardine fisheries

Understanding the fluctuations in population abundance is a central question in fisheries. Sardine fisheries is of great importance to Portugal and is data-rich and of primary concern to fisheries managers. In Portugal, sub-stocks of Sardina pilchardus (sardine) are found in different regions: the Northwest (IXaCN), Southwest (IXaCS) and the South coast (IXaS-Algarve). Each of these sardine sub-stocks is affected differently by a unique set of climate and ocean conditions, mainly during larval development and recruitment, which will consequently affect sardine fisheries in the short term. Taking this hypothesis into consideration we examined the effects of hydrographic (river discharge), sea surface temperature, wind driven phenomena, upwelling, climatic (North Atlantic Oscillation) and fisheries variables (fishing effort) on S. pilchardus catch rates (landings per unit effort, LPUE, as a proxy for sardine biomass). A 20-year time series (1989-2009) was used, for the different subdivisions of the Portuguese coast (sardine sub-stocks). For the purpose of this analysis a multi-model approach was used, applying different time series models for data fitting (Dynamic Factor Analysis, Generalised Least Squares), forecasting (Autoregressive Integrated Moving Average), as well as Surplus Production stock assessment models. The different models were evaluated, compared and the most important variables explaining changes in LPUE were identified. The type of relationship between catch rates of sardine and environmental variables varied across regional scales due to region-specific recruitment responses. Seasonality plays an important role in sardine variability within the three study regions. In IXaCN autumn (season with minimum spawning activity, larvae and egg concentrations) SST, northerly wind and wind magnitude were negatively related with LPUE. In IXaCS none of the explanatory variables tested was clearly related with LPUE. In IXaS-Algarve (South Portugal) both spring (period when large abundances of larvae are found) northerly wind and wind magnitude were negatively related with LPUE, revealing that environmental effects match with the regional peak in spawning time. Overall, results suggest that management of small, short-lived pelagic species, such as sardine quotas/sustainable yields, should be adapted to a regional scale because of regional environmental variability.

First report of Ciguatoxins in two starfish species: Ophidiaster ophidianus and Marthasterias glacialis

Ciguatera fish poisoning (CFP) is a syndrome caused by the ingestion of fish contaminated with Ciguatoxins (CTXs). These phycotoxins are produced mainly by dinoflagellates that belong to the genus Gambierdiscus that are transformed in more toxic forms in predatory fish guts, and are more present in the Indo-Pacific and Caribbean areas. It is estimated that CFP causes per year more than 10,000 intoxications worldwide. With the rise of water temperature and anthropogenic intervention, it is important to study the prevalence of CFP in more temperate waters. Through inter- and subtidal sampling, 22 species of organisms were collected, in Madeira and Azores archipelagos and in the northwestern Moroccan coast, during September of 2012 and June and July of 2013. A total of 94 samples of 22 different species of bivalves, gastropods, echinoderms and crustaceans where analyzed by Ultra Performance Liquid Chromatography-Mass Spectometry-Ion Trap-Time of Flight (UPLC-MS-IT-TOF) and Ultra Performance Chromatography- Mass Spectrometry (UPLC-MS). Our main aim was to detect new vectors and ascertain if there were some geographical differences. We detected for the first time putative CTXs in echinoderms, in two starfish species—M. glacialis and O. ophidianus. We detected differences regarding uptake values by organisms and geographical location. Toxin amounts were significant, showing the importance and the need for continuity of these studies to gain more knowledge about the prevalence of these toxins, in order to better access human health risk. In addition, we suggest monitoring of these toxins should be extended to other vectors, starfish being a good alternative for protecting and accessing human health risk.

Effects of culture conditions on larval growth and survival of stalked barnacles (Pollicipes pollicipes).

Pollicipes pollicipes (Crustacea: Scalpelliformes) is a highly prized food in Portugal and Spain and con- sequently a species of considerable interest to aqua- culture. Surprisingly, however, larval culture conditions for this barnacle have not been opti- mized. This study investigated the effects of temper- ature, diet, photoperiod and salinity on the growth and survival of P. pollicipes larvae. Temperature had a significant effect on specific growth rate (2.6–5.9% total width per day, from 11 to 24°C), reducing mean development time to the cyprid from 25 days at 11 °C to 10 days at 24°C, although this was accompanied by a significant increase in mortality to over 90% above 22°C. Mid- range temperatures (15–20°C) maximized total survival (19–31% respectively). Algal diets of Tetra- selmis suecica, T. suecica/Skeletonema marinoi and S. marinoi/Isochrysis galbana did not affect specific growth rate significantly, but survival (on average 39% in 15 days) and the proportion of high-quality healthy cyprids was significantly higher on the lat- ter two diets (11–15% of initial number of larvae). Photoperiod did not significantly affect the survival, although specific growth rate was significantly higher at 24:0 and 16:8 L:D. Salinity (20– 40 g L 1 range) did not affect growth and survival significantly. The best growth and survival were accomplished using rearing temperatures of 15–20°C, daily feeding with T. suecica/S. marinoi or I. galbana/S. marinoi and a photoperiod of 24:0 L:D.