CALU-3 lung cells three-dimensionally assembled onto CellFate® matrix present angiotensin-converting enzyme-2 activity

Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO₂ humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air–liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs.     

Activities of enzymes that hydrolyze adenine nucleotides in lymphocytes from patients with rheumatoid arthritis

The purpose of this study was to investigate the activities of ecto‐nucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5; CD39) and adenosine deaminase (E‐ADA; EC 3.5.4.4) in lymphocytes from patients with rheumatoid arthritis (RA). Thirty patients diagnosed with RA through American College of Rheumatology criteria as well as 30 healthy patients were selected. Peripheral blood lymphocytes were isolated, and E‐NTPDase and E‐ADA activities were assayed. The results demonstrated an increased E‐NTPDase activity (both ATP and ADP as substrates) and a decreased E‐ADA activity in RA patients. These data suggest an organic effort to preserve the adenosine level, which is known to have anti‐inflammatory and analgesic properties, working as a potent suppressor of immune response. Copyright © 2012 John Wiley & Sons, Ltd.     

Habitat size determine algae biomass in tank-bromeliads

The primary goal of this study was to evaluate the relative importance of habitat physical properties, bottom-up and top-down factors, and their interaction on algae biomass in tank-bromeliads. We sampled algae biomass (chlorophyll-a concentration), micro-metazoan density, mosquito abundance, and several environmental variables, including nutrient concentrations and characteristics of the habitat physical structure, in a survey of 64 tank-bromeliads of four different species (Aechmea nudicaulis, Aechmea lingulata, Neoregelia cruenta, and Vriesea neoglutinosa). We analyzed the complete and individual bromeliad species datasets using an information-theoretic model selection approach (Akaike’s information criterion). Bromeliad species, maximum water volume, and bromeliad diameter comprised the best model for determining chlorophyll-a concentrations for the complete dataset. The maximum water volume also comprised the best model to explain chlorophyll-a concentrations in three of four bromeliad species datasets. Interactions between consumers and nutrient concentration were included in the subsequent models, but they were not statistically significant. Taken together, these results demonstrate that the impact of habitat size on the associated autotrophic biomass occurs possibly via changes in community susceptibility to disturbances, particularly drought. We can conclude that habitat size is more important than resource availability or herbivory on phytotelm autotrophic biomass regulation in these natural microcosms.     

Volatile affairs in microbial interactions

Microorganisms are important factors in shaping our environment. One key characteristic that has been neglected for a long time is the ability of microorganisms to release chemically diverse volatile compounds. At present, it is clear that the blend of volatiles released by microorganisms can be very complex and often includes many unknown compounds for which the chemical structures remain to be elucidated. The biggest challenge now is to unravel the biological and ecological functions of these microbial volatiles. There is increasing evidence that microbial volatiles can act as infochemicals in interactions among microbes and between microbes and their eukaryotic hosts. Here, we review and discuss recent advances in understanding the natural roles of volatiles in microbe–microbe interactions. Specific emphasis will be given to the antimicrobial activities of microbial volatiles and their effects on bacterial quorum sensing, motility, gene expression and antibiotic resistance.     

Galactofuranose attenuates cellular adhesion of Aspergillus fumigatus

Galactofuranose (Gal f ) is a major molecule found in cell wall polysaccharides, secreted glycoproteins, membrane lipophosphoglycans and sphingolipids of Aspergillus fumigatus . The initial step in the Galf synthetic pathway is the re-arrangement of UDP-galactopyranose to UDP-Galf through the action of UDP-galactopyranose mutase. A mutant lacking the Af UGM1 gene encoding the UDP-galactopyranose mutase has been constructed. In the mutant, though there is a moderate reduction in the mycelial growth associated with an increased branching, it remains as pathogenic and as resistant to cell wall inhibitors and phagocytes as the wild-type parental strain. The major phenotype seen is a modification of the cell wall surface that results in an increase in adhesion of the mutants to different inert surfaces (glass and plastic) and epithelial respiratory cells. The adhesive phenotype is due to the unmasking of the mannan consecutive to the removal of galactofuran by the ugm1 mutation. Removal of the mannan layer from the mutant surface by a mannosidase treatment abolishes mycelial adhesion to surfaces.     

Desensitization to ANG II in guinea pig ileum depends on membrane repolarization: role of maxi-K+ channel

Desensitization of ANG II tonic contractile response of theguinea pig ileum is related to membrane repolarization determined byCa²⁺-activatedK⁺(maxi-K⁺) channelopening. ANG II-stimulated depolarized myocytes presented sustainedactivation of maxi-K⁺ channels,characterized by reduction from 415 to 12 ms of the closed timeconstant. ANG II desensitization was prevented by 100 nM iberiotoxin,being reversible within 30 min. Depolarization by KCl, higher than 4 mM, impaired desensitization, suggesting that the membrane potentialmust attain a threshold to counteract the repolarization induced bymaxi-K⁺ channel opening. Once thisvalue is attained, there is no time dependency because thedesensitization process was shut off by addition of KCl along the timecourse of the tonic response. In contrast, the sustained ACh toniccomponent was not altered by these maneuvers. We conclude thatdesensitization of the ANG II tonic component is foremost due to theopening of maxi-K⁺ channels,leading to membrane repolarization, thus closing the voltage-dependentCa²⁺ channels responsible for theCa²⁺ influx that sustains thetonic component in this muscle.

     

Genetic diversity of <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>strains isolated in northeastern Brazil as revealed by DNA sequencing,PCR-based analyses and molecular karyotyping

Background  

Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes.