CellProfiler plugins -- an easy image analysis platform integration for containers and Python tools

CellProfiler is a widely used software for creating reproducible, reusable image analysis workflows without needing to code. In addition to the >90 modules that make up the main CellProfiler program, CellProfiler has a plugins system that allows for creation of new modules which integrate with other Python tools or tools that are packaged in software containers. The CellProfiler-plugins repository contains a number of these CellProfiler modules, especially modules that are experimental and/or dependency-heavy. Here, we present an upgraded CellProfiler-plugins repository with examples of accessing containerized tools, improved documentation, and added citation/reference tools to facilitate the use and contribution of the community.Comment: 17 pages, 2 figures, 1 tabl

Interplay between solid lipid nanoparticles, TGF-B pathway and epithelial-mesenchymal transition in tumor and non-tumor cells from prostate epithelium

Orientador: Marcelo Bispo de JesusDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O estudo da nanotecnologia, e mais especificamente de nanopartículas têm crescido, principalmente de suas propriedades e efeitos colaterais. Dentre as nanopartículas, se destacam as nanopartículas lipídicas sólidas (SLN), produzidas com componentes biocompatíveis e biodegradáveis. Muito se estuda sobre os efeitos benéficos de nanopartículas na entrega específica de fármacos e genes às células, porém pouco se sabe sobre os efeitos em vias de sinalização intracelular. Nesse trabalho estudamos os efeitos de SLN catiônica em células não tumorais e tumorais do epitélio de próstata humano, relacionadas à via do TGF-beta e transição epitélio-mesenquimal (EMT). A via do TGF-beta está relacionada com o aumento da invasão e migração celular no câncer, estimulando a metástase através da ativação da EMT. Nossos resultados mostram que as SLN aumentam a migração de células de câncer de próstata PC-3, efeito não observado em células não tumorais de próstata PNT1A ¿ um resultado inesperado e inédito. Observamos que o aumento da migração depende da nanopartícula e não dos seus componentes, e é um efeito concentração-dependente. Ainda, proteínas relacionadas à EMT, como Zeb1, mostraram-se aumentadas após tratamento com a SLN, demonstrando a importância dessa via no efeito biológico. Além disso, o papel biológico, i.e., a transfecção, mediado por SLN é significativamente reduzido quando inibimos os receptores do TGF-beta ou a Smad3. Concluímos que a SLN é capaz de interferir na motilidade de células PC-3 através de sua ativação da via do TGF-beta e a EMT. Tal efeito, ao extrapolar para a clínica, poderia agravar o quadro de um paciente caso esses resultados reproduzam em ensaios in vivo. A compreensão dos mecanismos moleculares disparado por nanomateriais, bem como suas consequências para o funcionamento celular, é importante para o desenvolvimento de novos nanomateriais e para delimitar seu uso seguro e eficazAbstract: The study of nanotechnology, and more specifically of nanoparticles, has grown, mainly due to its properties and side effects. Among the nanoparticles, we highlight the Solid Lipid Nanoparticles (SLN), produced with biocompatible and biodegradable components. Much has been researched about the beneficial effects of nanoparticles on the specific delivery of drugs and genes to cells, but little is known about the effects on intracellular signaling pathways. In this work we studied the effects of cationic SLN in non-tumor and tumor cells of the human prostate epithelium, related to the TGF-beta pathway and epithelial-mesenchymal transition (EMT). The TGF-beta pathway is related to increased cell invasion and migration in cancer, stimulating metastasis through activation of EMT. Our results show that SLNs increase the migration of PC-3 prostate cancer cells, an effect not seen in non-tumor prostate cells PNT1A - an unexpected and unprecedented result. We observed that the increase in migration depends on the nanoparticle and not on its components, and is a concentration-dependent effect. Also, EMT-related proteins, such as Zeb1, were shown to be increased after treatment with SLN, demonstrating the importance of this pathway in the biological effect. In addition, the biological effect, i.e., transfection, mediated by SLN is significantly reduced when we inhibit TGF-beta receptors or Smad3. We conclude that SLN is capable of interfering in the motility of PC-3 cells through its activation of the TGF-beta pathway and EMT. Such an effect, when extrapolating to the clinic, could worsen a patient's condition if these results reproduce in in vivo trials. Understanding the molecular mechanisms triggered by nanomaterials, as well as their consequences for cellular functioning, is important for the development of new nanomaterials and to delimit their safe and effective useMestradoBioquimicaMestra em Biologia Funcional e Molecular88882.329744/2010-01CAPE

Production of β-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: Characterization and catalytic properties of the enzymatic extract

Background: β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Results: A high production of β-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The optimal activity of β-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5–10.5. The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 10% of ethanol. Conclusions: β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the utility of this enzyme in diverse industrial processes

Production and Catalytic Properties of Amylases from Lichtheimia ramosa

The present study compared the production and the catalytic properties of amylolytic enzymes obtained from the fungi Lichtheimia ramosa (mesophilic) and Thermoascus aurantiacus (thermophilic). The highest amylase production in both fungi was observed in wheat bran supplemented with nutrient solution (pH 4.0) after 96 hours of cultivation, reaching 417.2 U/g of dry substrate (or 41.72 U/mL) and 144.5 U/g of dry substrate (or 14.45 U/mL) for L. ramosa and T. aurantiacus, respectively. The enzymes showed higher catalytic activity at pH 6.0 at 60°C. The amylases produced by L. ramosa and T. aurantiacus were stable between pH 3.5–10.5 and pH 4.5–9.5, respectively. The amylase of L. ramosa was stable at 55°C after 1 hour of incubation, whereas that of T. aurantiacus maintained 60% of its original activity under the same conditions. Both enzymes were active in the presence of ethanol. The enzymes hydrolyzed starch from different sources, with the best results obtained with corn starch. The enzymatic complex produced by L. ramosa showed dextrinizing and saccharifying potential. The enzymatic extract produced by the fungus T. aurantiacus presented only saccharifying potential, releasing glucose monomers as the main hydrolysis product

Mechanistic insights into the intracellular release of doxorubicin from pH-sensitive liposomes

pH-sensitive liposomes are interesting carriers for drug-delivery, undertaking rapid bilayer destabilization in response to pH changes, allied to tumor accumulation, a desirable behavior in the treatment of cancer cells. Previously, we have shown that pH-sensitive liposomes accumulate in tumor tissues of mice, in which an acidic environment accelerates drug delivery. Ultimately, these formulations can be internalized by tumor cells and take the endosome-lysosomal route. However, the mechanism of doxorubicin release and intracellular traffic of pH-sensitive liposomes remains unclear. To investigate the molecular mechanisms underlying the intracellular release of doxorubicin from pH-sensitive liposomes, we followed HeLa cells viability, internalization, intracellular trafficking, and doxorubicin’s intracellular delivery mechanisms from pH-sensitive (SpHL-DOX) and non-pH-sensitive (nSpHL-DOX) formulations. We found that SpHL-DOX has faster internalization kinetics and intracellular release of doxorubicin, followed by strong nuclear accumulation compared to nSpHL-DOX. The increased nuclear accumulation led to the activation of cleaved caspase-3, which efficiently induced apoptosis. Remarkably, we found that chloroquine and E64d enhanced the cytotoxicity of SpHL-DOX. This knowledge is paramount to improve the efficiency of pH-sensitive liposomes or to be used as a rational strategy for developing new formulations to be applied in vivo