Three-dimensional cell culture conditions promoted the Mesenchymal-Amoeboid Transition in the Triple-Negative Breast Cancer cell line MDA-MB-231.

Introduction: Breast cancer (BC) is the leading cause of death among women, primarily due to its potential for metastasis. As BC progresses, the extracellular matrix (ECM) produces more type-I collagen, resulting in increased stiffness. This alteration influences cellular behaviors such as migration, invasion, and metastasis. Specifically, cancer cells undergo changes in gene expression that initially promote an epithelial-to-mesenchymal transition (EMT) and subsequently, a transition from a mesenchymal to an amoeboid (MAT) migration mode. In this way, cancer cells can migrate more easily through the stiffer microenvironment. Despite their importance, understanding MATs remains challenging due to the difficulty of replicating in vitro the conditions for cell migration that are observed in vivo. Methods: To address this challenge, we developed a three-dimensional (3D) growth system that replicates the different matrix properties observed during the progression of a breast tumor. We used this model to study the migration and invasion of the Triple-Negative BC (TNBC) cell line MDA-MB-231, which is particularly subject to metastasis. Results: Our results indicate that denser collagen matrices present a reduction in porosity, collagen fiber size, and collagen fiber orientation, which are associated with the transition of cells to a rounder morphology with bleb-like protrusions. We quantified how this transition is associated with a more persistent migration, an enhanced invasion capacity, and a reduced secretion of matrix metalloproteinases. Discussion: Our findings suggest that the proposed 3D growth conditions (especially those with high collagen concentrations) mimic key features of MATs, providing a new platform to study the physiology of migratory transitions and their role in BC progression.

Cell dispersion during biofilm formation by Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium minutisporum and Lomentospora prolificans.

Dispersion is an essential step in the lifecycle of biofilms, since it enables the dissemination of microbial cells and, consequently, the potential colonization of new sites. Filamentous fungi belonging to the Scedosporium/Lomentospora genera are opportunistic human pathogens able to form multidrug-resistant biofilms on surfaces of different chemical compositions, environments and nutritional conditions. Despite the rising understanding of how biofilms are formed by Scedosporium/Lomentospora species, the cell dispersal step has not yet been explored. In the present study, the cell dispersion was investigated during biofilm formation by S. apiospermum, S. minutisporum, S. aurantiacum and L. prolificans cells. The results revealed that conidia were the major type of dispersed cells, which were detected throughout biofilm development (from 24 to 72 h). Dispersion was not influenced by increased glucose concentration (the main source for energetic metabolism) neither the presence of voriconazole (the most common antifungal used to treat scedosporiosis); however, the presence of mucin (a component of mucous, present in the lungs of cystic fibrosis patients, who are usually affected by these filamentous fungi) triggered cell dispersion. Contrarily, a poor nutritional environment (e.g., phosphate-buffered saline) inhibited this step. Overall, our study reveals new insights into the biofilm development of Scedosporium/Lomentospora species.

Safety and Growth Optimization of Lactic Acid Bacteria Isolated From Feedlot Cattle for Probiotic Formula Design.

In order to eliminate the widespread use of antibiotics in livestock production, the research for alternatives has increased lately. This study examined the safety of 40 lactic acid bacteria (LAB) isolated from bovine feedlot environment and previously selected as potential probiotics. A high sensitivity prevalence to ampicillin (AMP, 100%), gentamicin (GEN, 96.3%), kanamycin (KAN, 96.3%), clindamycin (CLI, 85.2%), chloramphenicol (CHL, 92.6%) and streptomycin (STR, 88.9%) while moderate and high resistance against erythromycin (ERY, 48%) and tetracycline (TET, 79%) respectively, were determined. Feedlot enterococci and pediococci displayed high resistance to CLI, ERY, GEN and TET (73, 100, 54.5, and 73%, respectively). Among fifteen resistance genes investigated, seven were identified in lactobacilli; their presence not always was correlated with phenotypic resistance. STR resistance genes, aadA and ant(6) were observed in 7.4 and 3.7% of isolates, respectively; genes responsible for aminoglycosides resistance, such as bla (7.4%), and aph(3")-III (3.7%) were also recognized. In addition, resistance cat and tetS genes (3.7 and 7.4%, respectively) were harbored by feedlot lactobacilli strains. The presence of ermB gene in 22.3% of isolates, including two of the six strains phenotypically resistant to ERY, exhibited the highest prevalence among the assessed antibiotics. None of the feedlot lactobacilli harbored virulence factors genes, while positive PCR amplification for ace, agg, fsrA, and atpA genes was found for enterococci. With the objective of producing large cell biomass for probiotic delivery, growth media without peptone but containing glucose and skim milk powder (Mgl and Mlac) were selected as optimal. Lactobacillus acidophilus CRL2074, L. amylovorus CRL2115, L. mucosae CRL2069, and L. rhamnosus CRL2084 were strains selected as free of antibiotic resistance and virulence determinants, able to reach high cell numbers in non-expensive culture media and being compatible among them.

Conidial germination in Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans: influence of growth conditions and antifungal susceptibility profiles

In the present study, we have investigated some growth conditions capable of inducing the conidial germination in Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans. Germination in Sabouraud medium (pH 7.0, 37ºC, 5% CO2) showed to be a typically time-dependent event, reaching ~75% in S. minutisporum and > 90% in S. apiospermum, S. aurantiacum and L. prolificans after 4 h. Similar germination rate was observed when conidia were incubated under different media and pHs. Contrarily, temperature and CO2 tension modulated the germination. The isotropic conidial growth (swelling) and germ tube-like projection were evidenced by microscopy and cytometry. Morphometric parameters augmented in a time-dependent fashion, evidencing changes in size and granularity of fungal cells compared with dormant 0 h conidia. In parallel, a clear increase in the mitochondrial activity was measured during the transformation of conidia-into-germinated conidia. Susceptibility profiles to itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin varied regarding each morphotype and each fungal species. Overall, the minimal inhibitory concentrations for hyphae were higher than conidia and germinated conidia, except for caspofungin. Collectively, our study add new data about the conidia-into-hyphae transformation in Scedosporium and Lomentospora species, which is a relevant biological process of these molds directly connected to their antifungal resistance and pathogenicity mechanisms.

Estabelecimento e crescimento in vitro de plantas de grápia / In vitro establishment and growth of apuleia seedlings

O objetivo deste trabalho foi avaliar o estabelecimento e crescimento in vitro de plantas de grápia (Apuleia leiocarpa (Vogel) J. F. Macbr.) em diferentes condições de cultivo. Sementes de grápia tratadas com ácido sulfúrico (H2SO4) por 20min foram imersas em etanol a 70% por 30s e em hipoclorito de sódio (0; 2,5 ou 5,0% de cloro ativo) por 5, 10 ou 15min. Aos 30 dias de cultivo, as sementes foram avaliadas quanto às porcentagens de desinfestação e germinação. As plantas assépticas foram transferidas para os meios de cultura WPM, MS e ½MS e avaliadas quanto ao comprimento (cm) da parte aérea e total das raízes e ao número de segmentos nodais e folhas aos 15 dias. Sementes de grápia também foram semeadas em meio de cultura WPM, suplementado com 4, 5 ou 6g L-1 de agar combinado com 10, 20 ou 30g L-1 de sacarose, e mantidos sob duas condições de luminosidade: luz durante todo o período de estabelecimento e escuro durante sete dias após a semeadura. Foram avaliados a porcentagem de germinação das sementes, o comprimento (cm) da parte aérea e total das raízes e o número de segmentos nodais e folhas aos 15 dias. Concluiu-se que os tratamentos com ácido sulfúrico e etanol foram suficientes para o estabelecimento in vitro de plantas assépticas de grápia. Plantas assépticas podem ser cultivadas em meio de cultura WPM ou MS, suplementados com 10g L-1 de sacarose e 4g L-1 de agar.

The aim of this study was to evaluate the in vitro establishment and growth of apuleia (Apuleia leiocarpa (Vogel) J. F. Macbr.) seedlings under different culture conditions. Apuleia seeds were treated with sulfuric acid (H2SO4) for 20min and disinfected by the immersion in 70% of ethanol for 30s and sodium hypochlorite with 0, 2.5 and 5.0% of active chlorine for 5, 10 and 15min. The percentage of disinfection and germination were evaluated at 30 days. Aseptic seedlings were transferred to WPM, MS and ½ MS culture medium and evaluated for shoot and root (cm) growth and number of nodal segments and leaves at 15 days. Apuleia seeds were also sown in WPM medium supplemented with 4, 5 or 6g L-1 of agar combined with 10, 20 or 30g L-1 of sucrose. The cultures were kept under two luminescence conditions: light throughout the establishment period and dark during the first seven days. The percentage of germination, shoot and root (cm) growth and number of nodal segments and leaves were evaluated at 15 days. In conclusion, the sulfuric acid and ethanol treatments were enough for the in vitro production of apuleia aseptic seedlings. The aseptic seedlings can grow in both WPM and MS medium, supplemented with 10g L-1 of sucrose and 4g L-1 of agar.

Estabelecimento e crescimento in vitro de plantas de grápia / In vitro establishment and growth of apuleia seedlings

O objetivo deste trabalho foi avaliar o estabelecimento e crescimento in vitro de plantas de grápia (Apuleia leiocarpa (Vogel) J. F. Macbr.) em diferentes condições de cultivo. Sementes de grápia tratadas com ácido sulfúrico (H2SO4) por 20min foram imersas em etanol a 70% por 30s e em hipoclorito de sódio (0; 2,5 ou 5,0% de cloro ativo) por 5, 10 ou 15min. Aos 30 dias de cultivo, as sementes foram avaliadas quanto às porcentagens de desinfestação e germinação. As plantas assépticas foram transferidas para os meios de cultura WPM, MS e ½MS e avaliadas quanto ao comprimento (cm) da parte aérea e total das raízes e ao número de segmentos nodais e folhas aos 15 dias. Sementes de grápia também foram semeadas em meio de cultura WPM, suplementado com 4, 5 ou 6g L-1 de agar combinado com 10, 20 ou 30g L-1 de sacarose, e mantidos sob duas condições de luminosidade: luz durante todo o período de estabelecimento e escuro durante sete dias após a semeadura. Foram avaliados a porcentagem de germinação das sementes, o comprimento (cm) da parte aérea e total das raízes e o número de segmentos nodais e folhas aos 15 dias. Concluiu-se que os tratamentos com ácido sulfúrico e etanol foram suficientes para o estabelecimento in vitro de plantas assépticas de grápia. Plantas assépticas podem ser cultivadas em meio de cultura WPM ou MS, suplementados com 10g L-1 de sacarose e 4g L-1 de agar.(AU)

The aim of this study was to evaluate the in vitro establishment and growth of apuleia (Apuleia leiocarpa (Vogel) J. F. Macbr.) seedlings under different culture conditions. Apuleia seeds were treated with sulfuric acid (H2SO4) for 20min and disinfected by the immersion in 70% of ethanol for 30s and sodium hypochlorite with 0, 2.5 and 5.0% of active chlorine for 5, 10 and 15min. The percentage of disinfection and germination were evaluated at 30 days. Aseptic seedlings were transferred to WPM, MS and ½ MS culture medium and evaluated for shoot and root (cm) growth and number of nodal segments and leaves at 15 days. Apuleia seeds were also sown in WPM medium supplemented with 4, 5 or 6g L-1 of agar combined with 10, 20 or 30g L-1 of sucrose. The cultures were kept under two luminescence conditions: light throughout the establishment period and dark during the first seven days. The percentage of germination, shoot and root (cm) growth and number of nodal segments and leaves were evaluated at 15 days. In conclusion, the sulfuric acid and ethanol treatments were enough for the in vitro production of apuleia aseptic seedlings. The aseptic seedlings can grow in both WPM and MS medium, supplemented with 10g L-1 of sucrose and 4g L-1 of agar.(AU)

Stem anatomical characteristics of the climbing palm Desmoncus orthacanthos (Arecaceae) under two natural growth conditions in a tropical forest

Desmoncus orthacanthos is a Neotropical climbing palm that resembles rattan and therefore has similar potential applications. The genus Desmoncus (subfamily Arecoideae, subtribe Bactridinae) is distributed throughout the Americas, from veracruz, Mexico, to Brazil and Bolivia. The anatomical characteristics of its support tissue have not been thoroughly studied, although some observations from Central American artisans suggest that the stems collected from undisturbed sites possess better characteristics; these include a good capacity to withstand bending without breaking (i.e. higher fracture strength than plants from disturbed sites). Stem samples were collected from individuals from disturbed and undisturbed sites, at three points along the length of the stem (basal, medium and apical). Collections were made of one ramet from five individuals (n=5) at both sites. Each ramet was divided into three sections: basal, from soil surface to a height of 0.5 m; medium, from a height of 0.5 to 5.0 m; and apical, from a height 5.0 to 10.0 m. An anatomical analysis including vascular bundles, parenchyma elements and fibers was performed in the radial direction and also along the longitudinal direction of the stems. The amount of vascular bundles was greater for samples from undisturbed site stems; the amount of parenchyma cells differ between samples from both sites and the amount of fibers was greater for samples from disturbed site stems. The anatomical structural dimensions were smaller for samples from the undisturbed site stems. These findings partially confirm the artisans’ belief and supports the conclusion that microclimatic conditions affect plant anatomical structure. Rev. Biol. Trop. 56 (2): 937-949. Epub 2008 June 30.

Desmoncus orthacanthos es una palmera trepadora neotropical que puede, potencialmente, utilizarse en usos similares a los del ratán. El género Desmoncus (subfamilia Arecoideae, subtribu Bactridinae) se distribuye en América desde Veracruz, Mexico, hasta Brasil y Bolivia. Esta especie posee características anatómicas que no han sido ampliamente estudiadas pero observaciones hechas por artesanos centroamericanos sugieren que los tallos de sitios no perturbados por actividades humanas presentan mayor resistencia al doblado y menos tallos fracturados en el manejo. Las muestras de tallos fueron recolectadas de individuos que crecen en un sitio conservado y uno perturbado por actividades humanas, a tres alturas de la longitud del tallo (basal, media y apical). Se recolectó una rama de cada uno de cinco individuos (n=5) en ambos sitios. Cada rama fue dividida en tres secciones: basal, desde el nivel del suelo hasta los 0.5 m; medio, desde 0.5 a 5.0 m; y apical, desde 5.0 a 10.0 m. Se midió la cantidad por unidad de área y las dimensiones o tamaño (i.e. diámetro radial y diámetro perpendicular) de paquetes vasculares, células de parénquima y fibras. La cantidad de paquetes vasculares fue mayor en los tallos del sitio conservado, la cantidad de células de parénquima fue diferente entre sitios y la cantidad de fibras fue mayor en los tallos del sitio perturbado por actividades humanas. Estas características muestran cierta relación con los resultados mecánicos en otro estudio de los autores, los cuales confirman parcialmente las observaciones de los artesanos, lo que puede estar estrechamente relacionado con las características microclimáticas de los sitios de crecimiento.

Temperature and pH conditions for mycelial growth of Agaricus brasiliensis on axenic cultivation / Condições de temperatura e pH para o crescimento micelial de Agaricus brasiliensis em cultivo axênico

Few studies have been done to determine Agaricus brasiliensis Wasser et al. (A. blazei; A. subrufescens) basic mycelial growth characteristics on axenic cultivation. This study aimed to determine the optimal temperature and initial pH for mycelial growth of A. brasiliensis on malt extract agar medium to develop axenic cultivation techniques. Studied initial pH values for mycelial growth were adjusted to 3.0, 4.0, 5.0, 5.5, with HCl, 6.0, 7.0, 8.0, with NaOH, and again 7.0 and 8.0, with CaCO3. Studied temperatures for mycelial growth were 22 ºC, 25 ºC, 28 ºC, 31 ºC and 34 ºC. It was concluded that A. brasiliensis can grow in axenic cultivation at temperature range from 22 oC to 34 ºC, with optimal temperature range from 28 oC to 31 ºC and optimal temperature value of 30.5 ºC ± 0.3 ºC. It also grows in initial pH range from 4.0 to 7.0, adjusted with HCl or NaOH but not CaCO3, with optimal initial pH range from 5.5 to 6.0 and optimal initial pH value of 5.56 ± 0.05. Mycelial growth is inhibited with pH of 3.0 or lower, 8.0 or higher, or when CaCO3 is used to adjust pH in the substratum to 7.0 or higher.

Poucos estudos foram desenvolvidos para determinar as condições básicas de crescimento micelial do fungo Agaricus brasiliensis Wasser et al. (A. blazei, A. subrufescens). O objetivo deste trabalho foi determinar a faixa ótima de temperatura e pH para o crescimento micelial, em agar-extrato-de-malte, de A. brasiliensis, visando o desenvolvimento de técnicas de cultivo axênica. Os valores de pH estudados foram 3,0, 4,0, 5,0 e 5,5, ajustados com HCl, 6,0, 7,0 e 8,0, ajustados com NaOH, e 7,0 e 8,0, ajustados com CaCO3. As temperaturas de crescimento estudadas foram 22 ºC; 25 ºC; 28 ºC; 31 ºC e 34 oC. Concluiu-se que A. brasiliensis cresce em uma faixa de temperatura ótima de 28 oC a 31 ºC, com valor ótimo de temperatura de 30,5 ºC ± 0,3 ºC. A faixa de pH inicial ótimo no substrato é de 5,5 a 6,0 e o valor de pH inicial ótimo é de 5,56 ± 0,05. O crescimento do micélio é inibido com pH de 3,0 ou inferior, 8,0 ou superior, ou quando CaCO3 é utilizado para ajuste do pH para 7,0 ou superior.

Temperature and pH conditions for mycelial growth of Agaricus brasiliensis on axenic cultivation / Condições de temperatura e pH para o crescimento micelial de Agaricus brasiliensis em cultivo axênico

Few studies have been done to determine Agaricus brasiliensis Wasser et al. (A. blazei; A. subrufescens) basic mycelial growth characteristics on axenic cultivation. This study aimed to determine the optimal temperature and initial pH for mycelial growth of A. brasiliensis on malt extract agar medium to develop axenic cultivation techniques. Studied initial pH values for mycelial growth were adjusted to 3.0, 4.0, 5.0, 5.5, with HCl, 6.0, 7.0, 8.0, with NaOH, and again 7.0 and 8.0, with CaCO3. Studied temperatures for mycelial growth were 22 ºC, 25 ºC, 28 ºC, 31 ºC and 34 ºC. It was concluded that A. brasiliensis can grow in axenic cultivation at temperature range from 22 oC to 34 ºC, with optimal temperature range from 28 oC to 31 ºC and optimal temperature value of 30.5 ºC ± 0.3 ºC. It also grows in initial pH range from 4.0 to 7.0, adjusted with HCl or NaOH but not CaCO3, with optimal initial pH range from 5.5 to 6.0 and optimal initial pH value of 5.56 ± 0.05. Mycelial growth is inhibited with pH of 3.0 or lower, 8.0 or higher, or when CaCO3 is used to adjust pH in the substratum to 7.0 or higher.

Poucos estudos foram desenvolvidos para determinar as condições básicas de crescimento micelial do fungo Agaricus brasiliensis Wasser et al. (A. blazei, A. subrufescens). O objetivo deste trabalho foi determinar a faixa ótima de temperatura e pH para o crescimento micelial, em agar-extrato-de-malte, de A. brasiliensis, visando o desenvolvimento de técnicas de cultivo axênica. Os valores de pH estudados foram 3,0, 4,0, 5,0 e 5,5, ajustados com HCl, 6,0, 7,0 e 8,0, ajustados com NaOH, e 7,0 e 8,0, ajustados com CaCO3. As temperaturas de crescimento estudadas foram 22 ºC; 25 ºC; 28 ºC; 31 ºC e 34 oC. Concluiu-se que A. brasiliensis cresce em uma faixa de temperatura ótima de 28 oC a 31 ºC, com valor ótimo de temperatura de 30,5 ºC ± 0,3 ºC. A faixa de pH inicial ótimo no substrato é de 5,5 a 6,0 e o valor de pH inicial ótimo é de 5,56 ± 0,05. O crescimento do micélio é inibido com pH de 3,0 ou inferior, 8,0 ou superior, ou quando CaCO3 é utilizado para ajuste do pH para 7,0 ou superior.