Human amniotic membrane (hAM) has recently drawn attention as an upcoming anti-cancer therapy. Regarding the strategies which have already investigated, little is known about hAM protein extracts (hAMPE) effect on cancer. So, this work aims to study the effect of hAMPE in metabolic activity of several human cancer cell lines. hAMPE were mechanically obtained, thus avoiding the effect of detergents and other reagents commonly used in protein extraction under the cell lines studied. After quantification of proteins in hAMPE, their effect on the metabolic activity of 21 human cancer cell lines was assessed by 3-(4,5-dimethylthia-zolyl-2)2,5-diphenyltetrazolium bromide (MTT) assay. Our results indicate that there is an inhibition of metabolic activity until 25 and 50% in two and seven cell lines, respectively. Five cell lines proved to be very sensitive to hAMPE, being its metabolic activity more than 50% inhibited. Our results show that hAMPE can inhibit the metabolic activity of some human cancer cell lines. However, research about this cell line-dependent response to hAMPE becomes indispensable.

With the ability to improve a patient’s oral health and functionality, dental implants have become the primary treatment option for patients with total or partial tooth loss. Over the past decade, several global dental implant companies, such as ZSystems ®, have invested in the commercialisation and development of solutions for zirconia implants. To promote osseointegration, companies have developed a surface treatment based on sandblasting and acid etching (SBAE), which produces a surface roughness of 1 to 3 μm. Laser surface texturing can replace the commercial method as an eco-process that avoids solid and acid residues. However, diseases such as diabetes, osteoporosis, obesity and hepatitis can hamper bone regeneration around dental implants. A functional connection between the surface of the dental implant and the surrounding host tissues - soft (marginal and attached gingiva) and hard (alveolar bone) - is facilitated by angiogenesis and osseointegration. The use of bioactive glass has emerged as an approach to further enhance osseointegration, as it is known for its ability to form a hydroxyapatite-like layer when in contact with physiological fluids, thereby stimulating bone bonding and regeneration. The addition of angiogenic agents to the implant surface also reduces the risk of implant failure in relation to the progression of peri-implantitis. Phytotherapeutics such as acemannan, the major polysaccharide in aloe vera, have demonstrated angiogenic potential by inducing the expression of vascular endothelial growth factor. In this work, 3 mol% yttria-stabilised zirconia samples were subjected to three different textures: sandblasting and acid etching, as the commercial approach; random laser texture, to replace SBAE procedures; and micropit texture, with periodic deep spots. The samples were then dip-coated in 58S bioactive glass solution and thermally treated to compare a conventional method with laser technology. A second acemannan coating was added to investigate its potential as an angiogenic agent. The aim was to understand how different textures and coatings affect cell viability and adhesion, and consequently improve osseointegration. The results showed that the sandblasted and acid-etched surface exhibited traces of monoclinic phase transformation in contrast to the random laser surface. The laser thermally treated surfaces produced a smoother surface, whereas the conventional treatment showed a more heterogeneous and granular surface. Cell metabolic activity results identified the random texture with laser-treated 58S mesoporous bioactive glass as the most promising condition.

The effect of tributyltin (TBT) on growth and metabolic activity of three estuarine bacteria with different TBT resistance profiles was investigated in an organic-rich culture medium (TSB) and in phosphate buffered saline (PBS) buffer. Exposure to TBT was assessed by determining its effect on growth (OD(600 nm) measurement), bacterial productivity (leucine incorporation), viability (CFU counts), aggregation and cell size (from Live/Dead analysis), ATP and NADH concentrations. TBT exposure resulted in decrease of bacterial density, cell size, and metabolic activity. In addition, cell aggregates were observed in the TBT-treated cultures. TBT strongly affected bacterial cell metabolism and seemed to exert an effect on its equilibrium, interfering with cell activity. Also, TBT toxicity was lower when cells were grown in TSB than in PBS, suggesting that a nutrient-rich growth medium can protect cells from TBT toxicity. This study contributes to our understanding of the TBT-resistant cell behavior reflected in its physiology and metabolic activity. This information is of utmost importance for further studies of TBT bioremediation.

Construction of 16S rDNA libraries was undertaken to examine the structure of free-living bacterial communities in the estuarine system Ria de Aveiro, Portugal. Samples were collected in April 2002, from two sites representing marine (station N1) and brackish (station I6) water zones. Clones were characterized by RFLP patterns and sequence analysis of representative clones revealed that both libraries were clearly dominated by α-proteobacteria, followed by γ-proteobacteria and β-proteobacteria. Clones affiliated with the δ-proteobacteria, Verrucomicrobia, Bacteroidetes and Actinobacteria were exclusive of station I6 and sequences related to the Firmicutes were only found in station N1. Sequences retrieved are included in only a few major bacterial divisions and in general, shared a high degree of homology with sequences deposited in nucleotide databases, and recovered from aquatic environments of diverse geographic regions. Differences between the two sites may reflect adaptation to different environmental conditions, especially salinity. The pattern of prokaryotic diversity is comparable to other coastal and estuarine environments previously studied.

Cell viability and metabolic activity are ubiquitous parameters used in biochemistry, molecular biology, and biotechnological studies. Virtually all toxicology and pharmacological projects include at some point the evaluation of cell viability and/or metabolic activity. Among the methods used to address cell metabolic activity, resazurin reduction is probably the most common. At variance with resazurin, resorufin is intrinsically fluorescent, which simplifies its detection. Resazurin conversion to resorufin in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. UV–Vis absorbance is an alternative technique but is not as sensitive. In contrast to its wide empirical “black box” use, the chemical and cell biology fundamentals of the resazurin assay are underexplored. Resorufin is further converted to other species, which jeopardizes the linearity of the assays, and the interference of extracellular processes has to be accounted for when quantitative bioassays are aimed at. In this work, we revisit the fundamentals of metabolic activity assays based on the reduction of resazurin. Deviation to linearity both in calibration and kinetics, as well as the existence of competing reactions for resazurin and resorufin and their impact on the outcome of the assay, are addressed. In brief, fluorometric ratio assays using low resazurin concentrations obtained from data collected at short time intervals are proposed to ensure reliable conclusions.

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary e ects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. E ects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using di erent fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH e ect on e ux pumps was also studied in an E. coli strain with an over-expression of several e ux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these e ects displayed slight di erences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block effux pump activity in a dose-dependent fashion in the E. coli strain.

Nannochloropsis oculata is well-recognized as a potential microalgal source of valuable compounds such as polyunsaturated fatty acids, particularly, eicosapentaenoic acid (EPA). The content and profile of these lipids is highly dependent on the growth conditions and can, therefore, be tailored through modulation of the growth parameters, specifically, temperature. Moreover, biological activities are composition dependent. In the present work, lipid extracts obtained from N. oculata, grown under constant temperature and under modulated temperature stress (to increase EPA content; Str) were characterized by GC-FID and several bioactivities were evaluated, namely, antioxidant (L-ORACFL), cytotoxic (MTT), adipolytic, anti-hepatic lipid accumulation (steatosis), and anti-inflammatory properties. Both extracts exhibited antioxidant activity (c.a. 49 µmol Troloxequivalent/mgextract) and the absence of toxicity (up to 800 µg/mL) toward colon and hepatic cells, adipocytes, and macrophages. They also induced adipolysis and the inhibition of triglycerides hepatic accumulation, with a higher impact from Str. In addition, anti-inflammatory activity was observed in the lipopolysaccharide-induced inflammation of macrophages in the presence of either extract, since lower levels of pro-inflammatory interleukin-6 and interferon-β were obtained, specifically by Str. The results presented herein revealed that modulated temperature stress may enhance the health effects of N. oculata lipid extracts, which may be safely utilized to formulate novel food products.

Flow cytometry is often used for viability and vitality assessment in bacteria and yeasts. However, its application to the study of fungal spore development is uncommon, probably due to the difficulties in successfully staining these cells. In the current study, we used flow cytometry for the first time to assess the effects of a disinfection treatment on the survival, growth and metabolic activity of fungal spores (Penicillium chrysogenum, Aspergillus nidulans and Aspergillus niger) submitted to gamma radiation (0e15 kGy). The Forward and Side-Scatter parameters of the cytometer were used to assess the differences in size and complexity of particles. Furthermore, two fluorescent dyes were used: Propidium Iodide to assess the membrane integrity and spore viability, in a culture-independent procedure; and Dihydroethidium to measure the changes in metabolic activity of irradiated spores in their first 10 h of growth in a liquid culture medium. Our results support that flow cytometry is a valuable tool in assessing different biological parameters and biocide effects, as it allowed accurate determination of the viability, growth and metabolic activity of gamma-irradiated spores. The fluorescence of Propidium Iodide was 5e7 more intense in unviable spores. The Dihydroethidium fluorescence increase was associated with faster growth. Control and low radiation doses allowed the germination and growth of spores, while higher doses led to growth inhibition and lower fluorescence.

This work aimed to study the influence of gamma radiation on the growth and production of some active substances of Arthrospira platensis. Biomass production was significantly inhibited (p ≤ 0.05) by 21 and 34%, with respect to the control at 2.0 and 2.5 kGy, respectively. Chlorophyll-a content showed 11% reduction at 2.5 kGy compared to the control. As a result of growth and Chl-a inhibition, chlorophyll productivity recorded a continuous significant decrease below the control in the cells exposed to 1, 1.5, 2 and 2.5 kGy by 8, 12, 15 and 25%, respectively after 15 days of incubation. In addition, phycobillins productivity showed significant decrease by 10 and 36% below the control at 2 and 2.5 kGy of gamma radiation, respectively. Protein production decreased significantly by 24% at 1.5 kGy; low doses of gamma irradiation (0.5, 1.0 and 1.5 kGy) induced carbohydrate production by 106, 246 and 146%, respectively. Lipid content increased significantly over the control at 0.5 kGy of gamma irradiation by 22%, which was decreased at higher doses. Interestingly, carotenoid productivity showed significant increase at all used gamma doses up to 155% over the control.

Oxalate film formation is a pathology that often occurs in mural paintings and may result from the concomitant action of microorganisms and environmental conditions. Low Choir of the Convent of Nossa Senhora da Saudação (Portugal) has mural paintings with an extraordinary beauty, which over time have been suffered polychromy degradation and biofilm formation, presenting an ideal case study to investigate the role and impact of microorganisms in the biodeterioration process. Bacterial populations, filamentous fungi belonging to the genera Cladosporium, Penicillium, Nectria and yeast strain of the genera Rhodotorula were isolated from these wall paintings. The penetration of fungal hyphae in the microstructure of mortars, observed by scanning electron microscopy, seems to be responsible for cracking and detachments in some areas of the painting. The study revealed that the veils on the surface of the paintings are essentially oxalates and that these biofilms are caused by metabolic activity of bacterial communities. Furthermore, the colour alteration of green areas due to microorganisms was detected by Raman microscopy, in real samples and under in vitro conditions, being the result of the metabolic activity of microorganisms present on the paintings, which promote calcium oxalates formation over the malachite paint layers.