Inoculated cell density as a determinant factor of the growth dynamics and metastatic efficiency of a breast cancer murine model

Copyright: © 2016 Gregório et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.4T1 metastatic breast cancer model have been widely used to study stage IV human breast cancer. However, the frequent inoculation of a large number of cells, gives rise to fast growing tumors, as well as to a surprisingly low metastatic take rate. The present work aimed at establishing the conditions enabling high metastatic take rate of the triple-negative murine 4T1 syngeneic breast cancer model. An 87% 4T1 tumor incidence was observed when as few as 500 cancer cells were implanted. 4T1 cancer cells colonized primarily the lungs with 100% efficiency, and distant lesions were also commonly identified in the mesentery and pancreas. The drastic reduction of the number of inoculated cells resulted in increased tumor doubling times and decreased specific growth rates, following a Gompertzian tumor expansion. The established conditions for the 4T1 mouse model were further validated in a therapeutic study with peguilated liposomal doxorubicin, in clinical used in the setting of metastatic breast cancer. Inoculated cell density was proven to be a key methodological aspect towards the reproducible development of macrometastases in the 4T1 mouse model and a more reliable pre-clinical assessment of antimetastatic therapies.Ana Cristina Gregório is a student of the international PhD program in Experimental Biology and Biomedicine (PDBEB) from the Institute for Interdisciplinary Research, University of Coimbra and recipient of the fellowship SFRH/BD/51190/2010 from the Portuguese Foundation for Science and Technology (FCT). The work was supported by the grants PTDC/SAU-BMA/121028/2010 (FCT) and UID/NEU/04539/2013 (FEDER/COMPETE 2020/FCT).info:eu-repo/semantics/publishedVersio

Robust colour constancy in red-green dichromats

Colour discrimination has been widely studied in red-green (R-G) dichromats but the extent to which their colour constancy is affected remains unclear. This work estimated the extent of colour constancy for four normal trichromatic observers and seven R-G dichromats when viewing natural scenes under simulated daylight illuminants. Hyperspectral imaging data from natural scenes were used to generate the stimuli on a calibrated CRT display. In experiment 1, observers viewed a reference scene illuminated by daylight with a correlated colour temperature (CCT) of 6700K; observers then viewed sequentially two versions of the same scene, one illuminated by either a higher or lower CCT (condition 1, pure CCT change with constant luminance) or a higher or lower average luminance (condition 2, pure luminance change with a constant CCT). The observers' task was to identify the version of the scene that looked different from the reference scene. Thresholds for detecting a pure CCT change or a pure luminance change were estimated, and it was found that those for R-G dichromats were marginally higher than for normal trichromats regarding CCT. In experiment 2, observers viewed sequentially a reference scene and a comparison scene with a CCT change or a luminance change above threshold for each observer. The observers' task was to identify whether or not the change was an intensity change. No significant differences were found between the responses of normal trichromats and dichromats. These data suggest robust colour constancy mechanisms along daylight locus in R-G dichromacy.This work was supported by FEDER through the COMPETE Program and by the Portuguese Foundation for Science and Technology (FCT) in the framework of the project PTDC/MHC-PCN/4731/2012 and by Ministerio de Ciencia y Competitividad in the framework of the project PSI2012-37778. LA was supported by Beca Santander JPI2014. The publication of this article was funded by the Anglia Ruskin Open Access Fund.info:eu-repo/semantics/publishedVersio

On the formulation of pH-sensitive liposomes with long circulation times

Strategies used to enhance liposome-mediated drug delivery in vivo include the enhancement of stability and circulation time in the bloodstream, targeting to specific tissues or cells, and facilitation of intracytoplasmic delivery. pH-sensitive liposomes have been developed to mediate the introduction of highly hydrophilic molecules or macromolecules into the cytoplasm. These liposomes destabilize under acidic conditions found in the endocytotic pathway, and usually contain phosphatidylethanolamine (PE) and titratable stabilizing amphiphiles. Formulations without PE have also been developed. Encapsulated compounds are thought to be transported into the cytoplasm through destabilization of or fusion with the endosome membrane. Incorporation of a low mole percentage of poly(ethylene glycol) (PEG)-conjugated lipids into pH-sensitive liposomes confers prolonged circulation times to these liposomes, which are otherwise cleared rapidly. While the incorporation of PEG-lipids reduces the pH-dependent release of encapsulated fluorescent markers in vitro, it does not hinder the cytoplasmic delivery of the markers per cell-associated liposome. This suggests that intracellular delivery is not dictated simply by the destabilization of the liposomes. Antibodies or ligands to cell surface receptors can be coupled to pH-sensitive or sterically stabilized pH-sensitive liposomes for targeting. pH-sensitive liposomes have been used to deliver anticancer drugs, antibiotics, antisense oligonucleotides, ribozymes, plasmids, proteins and peptides to cells in culture or in vivo. © 2004 Elsevier B.V. All rights reserved

Targeting of sterically stabilised pH-sensitive liposomes to human T-leukaemia cells

The main aim of this work was to develop novel targeted sterically stabilised pH-sensitive liposomes tailored to promote efficient intracellular delivery of therapeutic molecules into human T-leukaemia cells. Our results indicate that the targeting moiety (thiolated transferrin) was successfully coupled to the distal reactive maleimide terminus of poly(ethylene glycol)-phospholipid conjugates incorporated in the liposomal bilayer. Results from atomic force microscopy studies, performed to characterise vesicle surface topology, indicated that, to a certain extent, thiolated transferrin has the ability to associate in a non-specific manner with the lipid membrane of pegylated liposomes. This is an issue not commonly reported in the literature but which is crucial to demonstrate the targeting proof of principle. Nevertheless, fluorimetric studies together with confocal microscopy clearly demonstrate that liposomes bearing covalently coupled transferrin associate more extensively to human T-leukaemia cells in vitro than non-targeted liposomes. Cell mechanistic studies indicate that targeted liposomes bind specifically to transferrin receptors and are internalised via receptor-dependent endocytotic pathway. In addition, the biophysical features exhibited by the developed liposomes, namely their ability to promote pH-triggered cytoplasmic delivery of loaded material, make them promising delivery systems for in vivo targeting of therapeutic molecules to tumours.http://www.sciencedirect.com/science/article/B6T6C-4DVT9WH-1/1/5592c4a7248e7be29f239e55046f842

Laccase production by free and immobilized fungal mycelium of Trametes versicolor

The production of laccase by free and immobilized mycelium of Trametes versicolor was evaluated. Fermentation experiments were carried out using a Trametes defined medium [1] supplemented with tween-80 (0.5%, w/v) and xylidine (30 μM) to stimulate and induce the secretion of extracellular enzyme. The support for the mycelium immobilization consisted in 0.3 cm3 cubes of synthetic fiber (Scotch Brite, 3M Spain, SA), which were added to the culture system at 1 g/100 mL of medium. Assays were performed in 250-mL Erlenmeyer flasks and in a 2 L stirred tank bioreactor. For the flasks fermentations, three 7 mm diameter plugs from the fungal monoculture plate were inoculated into 50 mL culture medium in the presence or not of the immobilization support. For the assays in bioreactor, the volume of inoculum necessary to obtain an initial cell concentration of 70 mg/L was transferred to the reactor containing 1 L of culture medium with or without the immobilization support. In both cases, the fungus was incubated at 28 °C and 180 rpm. During the experiments, samples were periodically withdrawn for laccase and glucose determinations. Synthetic fiber was used as immobilization support since this material was demonstrated to be of great potential for fungi immobilization [2]. Additionally, many studies have demonstrated that fermentation systems with immobilized cells are able to increase the process productivity. However, the laccase production by T. versicolor (present study) did not show this performance. In both systems (Erlenmeyer flasks and bioreactor), the highest laccase production was obtained when using free mycelium. Additionally, the maximum laccase production obtained in bioreactor was lower than the maximum found in Erlenmeyer-flasks, suggesting that the conditions used in the bioreactor should be optimized to increase the laccase production results. Due to the great importance of the laccases in the industrial sector, more studies will be performed aiming to find a strategy to maximize the production of this enzyme by T. versicolo

Laccase production by free and immobilized mycelia of Peniophora cinerea and Trametes versicolor: a comparative study

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production.S.C. Silverio acknowledges the financial support from FCT (Fundacao para a Ciencia e para a Tecnologia, Ph.D. grant SFRH/BD/43439/2008), Portugal; and S. Moreira acknowledges CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Brazil

An equation for deriving spatial variations in carbonate production rates from sediment deposition rates and dilution: Application to Santa Maria Island, Azores

Knowledge of how carbonates are produced on shelves is needed for working out how these “carbonate factories” generate stratigraphy by providing particles for potential export or local deposition. Production rates can be derived straightforwardly in low-energy environments from one-dimensional analysis (age–depth variations) but rates are less easily derived for high-energy hydrodynamical environments where particles are transported away from their sites of production. This particularly affects knowledge of spatial variations in production rates, needed for working out controlling influences of light, hydrodynamics, and nutrient availability. We show here that, if a non-carbonate component of the sediment, such as terrigenous particles arising from coastal and subaerial erosion, is conserved and thus acts as a tracer, rates of carbonate production can in principle be derived from carbonate content data, if sediment transport fluxes can also be constrained. In the equation developed here, the spatial rate of change of carbonate content is caused by dilution of the terrigenous component by the newly produced carbonate and depends on the sediment transport flux. We investigate this idea using data from Santa Maria Island, Azores, an inactive volcanic island in a temperate environment. Geochemical, X-ray diffraction (XRD), and X-ray fluorescence (XRF) data of surface–sediment grab samples indicate nearly simple mixing trends between two components (volcanic rock and marine carbonate), as needed for our simple dilution-based equation to apply. High-resolution boomer seismic data reveal thicker (> 1 m) deposits in the mid- to outer shelf of the island, which we interpret as having been emplaced during the Holocene. These effectively provide time-averaged depositional fluxes and, assuming conservation of mass, can be used to constrain transport fluxes. The derived equation is used to predict the observed deposit thicknesses into the mid-shelf alongside coincident increasing carbonate percentages. The thicknesses are replicated only if carbonate production rates increase with depth and distance away from the coastline into the mid-shelf, quantifying the variation of production of such a nearshore environment for the first time. We speculate that mollusks dominating the production have a preference for sand that is less frequently or strongly agitated by waves, although nutrient availability from occasional upwelling may also regulate growth to create this trend.Peer reviewe

Componentes químicos associados à resistência de Lycopersicon hirsutum f. typicum a Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Este trabalho foi conduzido em casa de vegetação no Campus da Universidade Federal de Viçosa, Minas Gerais, Brasil de maio a setembro de 1997 e objetivou estudar a resistência do acesso LA 1777 de Lycopersicon hirsutum f. typicum a Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) e os prováveis compostos químicos envolvidos na resistência. Utilizaram-se as cultivares de Santa Clara (tomate para consumo in natura) e IPA-5 (tomate industrial) de Lycopersicon esculentum como padrões de suscetibilidade à praga. Avaliaram-se os números de minas pequenas (comprimento 0,5 cm); sobrevivência, estádio de desenvolvimento do inseto, peso de pupas, proporção sexual, fertilidade de adultos e a taxa de eclosão de larvas. Foram identificados picos nas correntes de íons do extrato hexânico das folhas e as prováveis substâncias associadas a estes utilizando-se cromatógrafo a gás acoplado a espectrômetro de massa. L. hirsutum f. typicum apresentou resistência à traça do tomateiro em relação a L. esculentum, o que refletiu em menor número de minas grandes/folha e maiores duração da fase larval, mortalidade de larvas e número de minas pequenas de T. absoluta. Foram identificados 14 picos nas correntes de íons, com tempos de 10,43 (pico 1) a 26,03 min. (pico 14). Os picos 8, 9, 10 e 12 estiveram relacionados com a resistência de L. hirsutum f. typicum a T. absoluta. O pico 14 ocorreu apenas na cultivar Santa Clara e esteve associado ao aumento da suscetibilidade do tomateiro a T. absoluta. Os prováveis compostos associados ao pico oito foram os sesquiterpenos 2,5,5-trimetil-1,3,6-heptatrieno, (+) canfeno, farneseno ou santalol. As prováveis substâncias associadas ao pico nove foram os sesquiterpenos α-bergamoteno, farneseno, β-sinesal e farnesol. Não foram encontradas substâncias prováveis associadas aos picos 10, 12 e 14.This work was carried out in a greenhouse at the Universidade Federal de Viçosa "Campus", Minas Gerais, Brazil, between May and September 1997 and its objective was study the resistance of the accession LA 1777 of Lycopersicon hirsutum f. typicum to Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and the compounds possibly involved in the resistance. The varieties Santa Clara (tomato for in natura consumption) and IPA-5 (industrial tomato) of Lycopersicon esculentum were used as pest susceptibility standards. The number of small and large mines ( 0.5 cm long, respectively); survival; development stage; pupa weight; sexual proportion; fertility, and rate of larvae eclosion were assessed. Peaks of the ion current of hexane extract of tomato leaves and the possible compounds associated with them were identified using a gas chromatograph coupled to a mass spectrometer. L. hirsutum f. typicum showed resistance to the tomato leafminer in comparison with L. esculentum, what was reflected by the smaller number of large mines/ leaf and greater length of larval phase, mortality of larvae and number of small mines of T. absoluta. Fourteen peaks were identified in the total ion current with retention times ranging from 10.43 (peak 1) to 26.03 min (peak 14). The peaks 8, 9, 10 and 12 were associated with T. absoluta resistance in L. hirsutum f. typicum. The peak 14 occurred only in the variety Santa Clara with an increased tomato susceptibility to T. absoluta. The probable compounds associated with peak 8 were the sesquiterpenes 2,5,5-trimethyl-1,3,6-heptatriene, (+) camphene, farnesene or santalol. The probable substances associated with peak 9 were the sesquiterpenes α-bergamotene, farnesene, β-sinesal, or farnesol. No substance associated with peaks 10, 12 and 14 were found