Confocal imaging protocols for live/dead staining in three-dimensional carriers

In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideally applied to 3D tissue or scaffolds with thickness up to several millimetres, this application is surprisingly rare and scans are often done on slices with thickness <20 μm. Here, we present novel protocols for the staining of 3D tissue (e.g. intervertebral disc tissue) and scaffolds, such as fibrin gels or alginate beads.

Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms--which stain is suitable?

BACKGROUND There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. DISCUSSION Many terms describe "vitality states" of microorganisms, however, several of them are misleading. Authors define "viable" as "capable to grow". Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial "viability" staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the "viability" kit are dependent on the stains' concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. SUMMARY - The nomenclature regarding "viability" and "vitality" should be used carefully.- The manual of the commercial "viability" kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literature.- Results obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting.- As microbiological parameter the Plating Efficiency should be used for comparison.- Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.

Live−dead comparison of benthic foraminiferal faunas from the Rhône prodelta (Gulf of Lions, NW Mediterranean): Development of a proxy for palaeoenvironmental reconstructions

Dead benthic foraminiferal faunas (> 150 μm) from the Rhône prodelta (Gulf of Lions, NW Mediterranean) were analysed at 41 stations (15–100 m water depth) sampled in June 2005 and September 2006, and compared to the living faunas investigated during previous studies at the same stations. The comparison between dead and living assemblages enhances the understanding of taphonomic processes that may modify the composition of the dead faunas in this area. We observed a loss of individuals from living to dead assemblages of species characterised by a fairly fragile test and therefore more prone to fragmentation or dissolution (e.g., Bolivina alata, Quinqueloculina tenuicollis). Allochthonous dead and/or live specimens may be transported to some parts of the prodelta, particularly the shallowest sites where hydrodynamic processes (i.e., river flood, storm swells, longshore currents) are more intense. These specimens may originate from relict deltaic structures (e.g., Elphidium spp. from the lobe of Bras de Fer) or from surrounding areas (e.g., Ammonia beccarii forma beccarii from the river). Opportunistic species (e.g., Bulimina marginata, Cassidulina carinata) characterised by high reproductive rates have much higher relative abundances in the dead than in the living fauna. Cluster analyses based on dead foraminiferal assemblages divide our study area into four main thanatofacies directly related to distinct local environmental conditions prevailing in the prodelta. Close to the river mouth, Ammonia beccarii forma beccarii and Ammonia tepida are found in sediments subject to a high riverine influence (i.e., bottom currents, high organic and inorganic material input of continental origin). Elphidium species are abundant in the silty-sandy relict deltaic lobe west of the river mouth which is characterised by strong longshore currents that disturb the benthic environment. Nonion fabum, Rectuvigerina phlegeri and Valvulineria bradyana are found along the coast west of the Rhône River mouth, in the area defined as the “river plume” thanatofacies. In the more stable and deeper prodeltaic area, species known to feed on fresh phytodetritus (e.g., Bulimina aculeata/marginata, C. carinata, Hyalinea balthica) dominate the faunas. Since only minor variations in species relative abundances and spatial distributional patterns are observed between the living and the dead faunas, we consider that our thanatofacies have not been influenced by substantial transport of dead tests. This suggests that fossil benthic foraminifera can provide a reliable tool for investigating the development of the palaeo-Rhône prodelta

A SUBSTITUIÇÃO DO TRABALHO VIVO PELO TRABALHO MORTO: UM ESTUDO NOS INTEGRANTES NA REDE DE SERVIÇOS CREDENCIADA DE UMA EMPRESA DO SETOR ELETROELETRÔNICO

O presente estudo confirmou a existência da substituição do trabalho vivo pelo trabalho morto, descrita por Karl Marx e outros importantes estudiosos, pela EC ABC, empresa do setor eletroeletrônico brasileiro (segmento das empresas industriais estabelecidas no Brasil, responsáveis pela produção de equipamentos que atendem a linha marrom que congrega produtos como, Televisores, Monitores de Vídeo, Áudio, reprodutores e/ou gravadores de Disco de Vídeo Versátil - DVD e Disco Compacto - CD), inicialmente no seu Departamento de Serviços Nacional e em seguida, nos Postos de Serviços Credenciados da sua Rede de Serviços Nacional, em função da informatização e mudança do modelo de gestão do conhecimento o que ocasionou a descapitalização intelectual nos Postos de Serviços Credenciados e a perda de qualidade nos serviços oferecidos pelos PSC aos clientes e revendedores. Adotou-se a forma de pesquisa exploratória deste fenômeno, atualmente ainda pouco examinado, em 120 PSC no Brasil.(AU)

A SUBSTITUIÇÃO DO TRABALHO VIVO PELO TRABALHO MORTO: UM ESTUDO NOS INTEGRANTES NA REDE DE SERVIÇOS CREDENCIADA DE UMA EMPRESA DO SETOR ELETROELETRÔNICO

O presente estudo confirmou a existência da substituição do trabalho vivo pelo trabalho morto, descrita por Karl Marx e outros importantes estudiosos, pela EC ABC, empresa do setor eletroeletrônico brasileiro (segmento das empresas industriais estabelecidas no Brasil, responsáveis pela produção de equipamentos que atendem a linha marrom que congrega produtos como, Televisores, Monitores de Vídeo, Áudio, reprodutores e/ou gravadores de Disco de Vídeo Versátil - DVD e Disco Compacto - CD), inicialmente no seu Departamento de Serviços Nacional e em seguida, nos Postos de Serviços Credenciados da sua Rede de Serviços Nacional, em função da informatização e mudança do modelo de gestão do conhecimento o que ocasionou a descapitalização intelectual nos Postos de Serviços Credenciados e a perda de qualidade nos serviços oferecidos pelos PSC aos clientes e revendedores. Adotou-se a forma de pesquisa exploratória deste fenômeno, atualmente ainda pouco examinado, em 120 PSC no Brasil.(AU)

A SUBSTITUIÇÃO DO TRABALHO VIVO PELO TRABALHO MORTO: UM ESTUDO NOS INTEGRANTES NA REDE DE SERVIÇOS CREDENCIADA DE UMA EMPRESA DO SETOR ELETROELETRÔNICO

O presente estudo confirmou a existência da substituição do trabalho vivo pelo trabalho morto, descrita por Karl Marx e outros importantes estudiosos, pela EC ABC, empresa do setor eletroeletrônico brasileiro (segmento das empresas industriais estabelecidas no Brasil, responsáveis pela produção de equipamentos que atendem a linha marrom que congrega produtos como, Televisores, Monitores de Vídeo, Áudio, reprodutores e/ou gravadores de Disco de Vídeo Versátil - DVD e Disco Compacto - CD), inicialmente no seu Departamento de Serviços Nacional e em seguida, nos Postos de Serviços Credenciados da sua Rede de Serviços Nacional, em função da informatização e mudança do modelo de gestão do conhecimento o que ocasionou a descapitalização intelectual nos Postos de Serviços Credenciados e a perda de qualidade nos serviços oferecidos pelos PSC aos clientes e revendedores. Adotou-se a forma de pesquisa exploratória deste fenômeno, atualmente ainda pouco examinado, em 120 PSC no Brasil.(AU)

Inhibitory Effects Of A Cured Antibacterial Bonding System On Viability And Metabolic Activity Of Oral Bacteria.

To evaluate the antimicrobial efficacy of Clearfil SE Protect (CP) and Clearfil SE Bond (CB) after curing and rinsed against five individual oral microorganisms as well as a mixture of bacterial culture prepared from the selected test organisms. Bacterial suspensions were prepared from single species of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Actinomyces viscosus and Lactobacillus lactis, as well as mixed bacterial suspensions from these organisms. Dentin bonding system discs (6 mm×2 mm) were prepared, cured, washed and placed on the bacterial suspension of single species or multispecies bacteria for 15, 30 and 60 min. MTT, Live/Dead bacterial viability (antibacterial effect), and XTT (metabolic activity) assays were used to test the two dentin system's antibacterial effect. All assays were done in triplicates and each experiment repeated at least three times. Data were submitted to ANOVA and Scheffe's f-test (5%). Greater than 40% bacteria killing was seen within 15 min, and the killing progressed with increasing time of incubation with CP discs. However, a longer (60 min) period of incubation was required by CP to achieve similar antimicrobial effect against mixed bacterial suspension. CB had no significant effect on the viability or metabolic activity of the test microorganisms when compared to the control bacterial culture. CP was significantly effective in reducing the viability and metabolic activity of the test organisms. The results demonstrated the antimicrobial efficacy of CP both on single and multispecies bacterial culture. CP may be beneficial in reducing bacterial infections in cavity preparations in clinical dentistry.

Modulation of the secretome of hBMSCs by tailoring the macromolecular gradient in hydrogels to generate tissue-to-tissue interfaces

Tissue-to-tissue interfaces are commonly present in all tissues exhibiting structural, biological and chemical gradients serving a wide range of physiological functions. These interfaces are responsible for mediation of load transfer between two adjacent tissues. They are also important structures in sustaining the cellular communications to retain tissueâ s functional integration and homeostasis. [1] All cells have the capacity to sense and respond to physical and chemical stimulus and when cultured in three-dimensional (3D) environments they tend to perform their function better than in two-dimensional (2D) environments. Spatial and temporal 3D gradient hydrogels better resemble the natural environment of cells in mimicking their extracellular matrix. [2] In this study we hypothesize that differential functional properties can be engineered by modulation of macromolecule gradients in a cell seeded threedimensional hydrogel system. Specifically, differential paracrine secretory profiles can be engineered using human Bone Marrow Stem Cells (hBMSCâ s). Hence, the specific objectives of this study are to: assemble the macromolecular gradient hydrogels to evaluate the suitablity for hBMSCâ s encapsulation by cellular viability and biofunctionality by assessing the paracrine secretion of hBMSCâ s over time. The gradient hydrogels solutions were prepared by blend of macromolecules in one solution such as hyaluronic (HA) acid and collagen (Col) at different ratios. The gradient hydrogels were fabricated into cylindrical silicon moulds with higher ratio solutions assembled at the bottom of the mould and adding the two solutions consecutively on top of each other. The labelling of the macromolecules was performed to confirm the gradient through fluorescence microscopy. Additionally, AFM was conducted to assess the gradient hydrogels stiffness. Gradient hydrogels characterization was performed by HA and Col degradation assay, degree of crosslinking and stability. hBMSCâ s at P3 were encapsulated into each batch solution at 106 cells/ml solution and gradient hydrogels were produced as previously described. The hBMSCâ s were observed under confocal microscopy to assess viability by Live/Dead® staining. Cellular behaviour concerning proliferation and matrix deposition was also performed. Secretory cytokine measurement for pro-inflammatory and angiogenesis factors was carried out using ELISA. At genomic level, qPCR was carried out. The 3D gradient hydrogels platform made of different macromolecules showed to be a suitable environment for hBMSCâ s. The hBMSCâ s gradient hydrogels supported high cell survival and exhibited biofunctionality. Besides, the 3D gradient hydrogels demonstrated differentially secretion of pro-inflammatory and angiogenic factors by the encapsulated hBMSCâ s. References: 1. Mikos, AG. et al., Engineering complex tissues. Tissue Engineering 12,3307, 2006 2. Phillips, JE. et al., Proc Natl Acad Sci USA, 26:12170-5, 2008

Polymicrobial interactions in infections: the case Pseudomonas aeruginosa and Candida albicans in ventilator-associated pneumonia

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clinica)

Quantitative fidelity of recent freshwater mollusk assemblages from the Touro Passo River, Rio Grande do Sul, Brazil

This study represents one of the first contributions to the knowledge on the quantitative fidelity of the recent freshwater molluscan assemblages in subtropical rivers. Thanatocoenoses and biocoenoses were studied in straight and meandering to braided sectors, in the middle course of the Touro Passo River, a fourth-order tributary of the Uruguay River, located in the westernmost part of the State of Rio Grande do Sul. Samplings were carried out through quadrats of 5 m², five in each sector. A total area of 50 m² was sampled. Samplings were also made in a lentic environment (abandoned meander), with intermittent communication with the Touro Passo River, aiming to record out-of-habitat shell transportation from the lentic communities to the main river channel. The results show that, despite the frequent oscillation of the water level, the biocoenosis of the Touro Passo River shows high ecological fidelity and undergoes little influence from the lentic vicinal environments. The taxonomic composition and some features of the structure of communities, especially the dominant species, also reflect some ecological differences between the two main sectors sampled, such as the complexity of habitats in the meandering-sector. Regarding the quantitative fidelity, 60% of the species found alive were also found dead and 47.3% of the species found dead were also found alive, at river-scale. However, 72% of the dead individuals belong to species also found alive. This value might be related with the good rank order correlation obtained for live/dead assemblages. Consequently, the dominant species of the thanatocoenoses could be used to infer the ecological attributes of the biocoenoses. The values of all the indexes analyzed were very variable in small-scale samplings (quadrat), but were more similar to others registered in previous studies, when they were analyzed in a station and river scale.

A flow cytometry based oligotrophic pollutant exposure test to detect bacterial growth inhibition and cell injury.

Toxicity of chemical pollutants in aquatic environments is often addressed by assays that inquire reproductive inhibition of test microorganisms, such as algae or bacteria. Those tests, however, assess growth of populations as a whole via macroscopic methods such as culture turbidity or colony-forming units. Here we use flow cytometry to interrogate the fate of individual cells in low-density populations of the bacterium Pseudomonas fluorescens SV3 exposed or not under oligotrophic conditions to a number of common pollutants, some of which derive from oil contamination. Cells were stained at regular time intervals during the exposure assay with fluorescent dyes that detect membrane injury (i.e., live-dead assay). Reduction of population growth rates was observed upon toxicant insult and depended on the type of toxicant. Modeling and cell staining indicate that population growth rate decrease is a combined effect of an increased number of injured cells that may or may not multiply, and live cells dividing at normal growth rates. The oligotrophic assay concept presented here could be a useful complement for existing biomarker assays in compliance with new regulations on chemical effect studies or, more specifically, for judging recovery after exposure to fluctuating toxicant conditions.

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

Identification and Characterization of Natural Anti-Breast Cancer Compound: Costunolide

Breast cancer is the most common cancer among women, 23% (1.3 million) of the total of new cases and the second leading cause of cancer death in women exceeded only by lung cancer. Natural medicines have been proven to be a central source of narrative agents with a pharmaceutical potential. Costunolide is sesquiterpene lactones consisting of diverse plant chemicals that exhibit anti cancer action through cytotoxic effects on various cancer cells. The objectives of present study were to explore the effects of natural compounds on the proliferation of MCF-7 cells and to determine the role of ROS in natural compounds-induced apoptosis in breast cancer cells with a therapeutic potential. Results showed that costunolide screened, possess potent anticancer properties against breast cancer MCF-7 cells, Costunolide was observed as strong anti-proliferative agent with IC50 = 50µM. The anti-proliferative effect of costunolide on MCF cells was confirmed by live/dead assay using fluorescent probes calcein AV/PI. The results demonstrated that treatment of cells with costunolide decreased the viability of MCF-7 cells in a dose-dependent manner. To determine the costunolide-induced apoptosis, flow cytometric analysis was carried out. The results showed that costunolide induced apoptosis in a dose-dependent manner in breast cancer MCF-7cells. ROS are well known mediators of intracellular signaling of cascades. The excessive generation of ROS can induce oxidative stress, loss of cell functioning, and apoptosis. In the present study, we assumed that costunolide might arouse ROS level, which could be involved in induction of apoptosis. Therefore, the intracellular ROS level was measured using the ROS-detecting fluorescence dye 2, 7-dichlorofluorescein diacetate (DCF-DA). Interestingly these effects were significantly abrogated when the cells were pretreated with N-acetyl- cysteine (NAC), a specific ROS inhibitor. Costunolide induces apoptosis through extrinsic pathway in MCF-7 breast cancer cells, In order to examine whether costunolide suppresses cell growth inducing apoptotic cell death, we analyzed DNA contents and apoptosis-related proteins expression level by flow cytometry and western blot, respectively in MCF-7 breast cancer cells we investigated whether costunolide activates extrinsic apoptotic pathway. We examined the expression levels of death receptor signaling-related proteins, caspase-3, and PARP. The results showed that procaspase-3 was cleaved to yield 17 and 20kDa fragments and activation of PARP in treated cells with 25 and 50μM of costunolide. Costunolide induce apoptosis through intrinsic mitochondria pathway in MCF-7 breast cancer Cells. We examined the expression levels of mitochondrial apoptotic pathway related proteins such as anti-apoptotic protein, B-cell lymphoma protein-2 (Bcl2), and pro-apoptotic protein Bax. Costunolide involved in the down regulation of Bcl-2 and up regulation of Bax. These results suggest that costunolide may have beneficial effects for the reduction of breast cancer growth, and new therapeutic strategy for the treatment of human cancers.