Cellular Metrology: Scoping for a Value Proposition in Extra- and Intracellular Measurements.

The symptomatic irreproducibility of data in biomedicine and biotechnology prompts the need for higher order measurements of cells in their native and near-native environments. Such measurements may support the adoption of new technologies as well as the development of research programs across different sectors including healthcare and clinic, environmental control and national security. With an increasing demand for reliable cell-based products and services, cellular metrology is poised to help address current and emerging measurement challenges faced by end-users. However, metrological foundations in cell analysis remain sparse and significant advances are necessary to keep pace with the needs of modern medicine and industry. Herein we discuss a role of metrology in cell and cell-related R&D activities to underpin growing international measurement capabilities. Relevant measurands are outlined and the lack of reference methods and materials, particularly those based on functional cell responses in native environments, is highlighted. The status quo and current challenges in cellular measurements are discussed in the light of metrological traceability in cell analysis and applications (e.g., a functional cell count). An emphasis is made on the consistency of measurement results independent of the analytical platform used, high confidence in data quality vs. quantity, scale of measurements and issues of building infrastructure for end-users.

Spatiotemporal distribution of the magnetotactic multicellular prokaryote Candidatus Magnetoglobus multicellularis in a Brazilian hypersaline lagoon and in microcosms

Candidatus Magnetoglobus multicellularis is an unusual morphotype of magnetotactic prokaryotes. These microorganisms are composed of a spherical assemblage of gram-negative prokaryotic cells capable of swimming as a unit aligned along a magnetic field. While they occur in many aquatic habitats around the world, high numbers of Ca. M. multicellularis have been detected in Araruama Lagoon, a large hypersaline lagoon near the city of Rio de Janeiro, in Brazil. Here, we report on the spatiotemporal distribution of one such population in sediments of Araruama Lagoon, including its annual distribution and its abundance compared with the total bacterial community. In microcosm experiments, Ca. M. multicellularis was unable to survive for more than 45 days: the population density gradually decreased coinciding with a shift to the upper layers of the sediment. Nonetheless, Ca. M. multicellularis was detected throughout the year in all sites studied. Changes in the population density seemed to be related to the input of organic matter as well as to salinity. The population density of Ca. M. multicellularis did not correlate with the total bacterial counts; instead, changes in the microbial community structure altered their counts in the environment (AU)

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Spatiotemporal distribution of the magnetotactic multicellular prokaryote Candidatus Magnetoglobus multicellularis in a Brazilian hypersaline lagoon and in microcosms.

Candidatus Magnetoglobus multicellularis is an unusual morphotype of magnetotactic prokaryotes. These microorganisms are composed of a spherical assemblage of gram-negative prokaryotic cells capable of swimming as a unit aligned along a magnetic field. While they occur in many aquatic habitats around the world, high numbers of Ca. M. multicellularis have been detected in Araruama Lagoon, a large hypersaline lagoon near the city of Rio de Janeiro, in Brazil. Here, we report on the spatiotemporal distribution of one such population in sediments of Araruama Lagoon, including its annual distribution and its abundance compared with the total bacterial community. In microcosm experiments, Ca. M. multicellularis was unable to survive for more than 45 days: the population density gradually decreased coinciding with a shift to the upper layers of the sediment. Nonetheless, Ca. M. multicellularis was detected throughout the year in all sites studied. Changes in the population density seemed to be related to the input of organic matter as well as to salinity. The population density of Ca. M. multicellularis did not correlate with the total bacterial counts; instead, changes in the microbial community structure altered their counts in the environment.

Salinity dependence of the distribution of multicellular magnetotactic prokaryotes in a hypersaline lagoon.

Candidatus Magnetoglobus multicellularis is an unusual magnetotactic multicellular microorganism composed of a highly organized assemblage of gram-negative bacterial cells. In this work, the salinity dependence of Ca. M. multicellularis and its abundance in the hypersaline Araruama Lagoon, Brazil were studied. Viability experiments showed that Ca. M. multicellularis died in salinities upper than 55 per thousand and lower than 40 per thousand. Low salinities were also observed to modify the cellular assemblage. In microcosms prepared with different salinities, the microorganism grew better at intermediate salinities whereas in high or low salinities, the size of the population did not increase over time. The concentrations of Ca. M. multicellularis in the lagoon were related to salinity; sites with lower and higher salinities than the lagoon average contained less Ca. M. multicellularis. These results demonstrate the influence of salinity on the survival and distribution of Ca. M. multicellularis in the environment. In sediments, the abundance of Ca. M. multicellularis ranged from 0 to 103 microorganisms/ml, which represented 0.001% of the counts of total bacteria. The ability of Ca. M. multicellularis to accumulate iron and sulfur in high numbers of magnetosomes (up to 905 per microorganism) suggests that its impact on the sequestration of these elements (0.1% for biogenic bacterial iron) is not proportional to its abundance in the lagoon.

Salinity dependence of the distribution of multicellular magnetotactic prokaryotes in a hypersaline lagoon

Candidatus Magnetoglobus multicellularis is an unusual magnetotactic multicellular microorganism composed of a highly organized assemblage of gram-negative bacterial cells. In this work, the salinity dependence of Ca. M. multicellularis and its abundance in the hypersaline Araruama Lagoon, Brazil were studied. Viability experiments showed that Ca. M. multicellularis died in salinities >55 per-mille and <40 per-mille. Low salinities were also observed to modify the cellular assemblage. In microcosms prepared with different salinities, the microorganism grew better at intermediate salinities whereas in high or low salinities, the size of the population did not increase over time. The concentrations of Ca. M. multicellularis in the lagoon were related to salinity; sites with lower and higher salinities than the lagoon average contained less Ca. M. multicellularis. These results demonstrate the influence of salinity on the survival and distribution of Ca. M. multicellularis in the environment. In sediments, the abundance of Ca. M. multicellularis ranged from 0 to 103 microorganisms/ml, which represented 0.001% of the counts of total bacteria. The ability of Ca. M. multicellularis to accumulate iron and sulfur in high numbers of magnetosomes (up to 905 per microorganism) suggests that its impact on the sequestration of these elements (0.1% for biogenic bacterial iron) is not proportional to its abundance in the lagoon (AU)

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Grazing protozoa and magnetosome dissolution in magnetotactic bacteria.

Magnetotactic bacteria show an ability to navigate along magnetic field lines because of magnetic particles called magnetosomes. All magnetotactic bacteria are unicellular except for the multicellular prokaryote (recently named 'Candidatus Magnetoglobus multicellularis'), which is formed by an orderly assemblage of 17-40 prokaryotic cells that swim as a unit. A ciliate was used in grazing experiments with the M. multicellularis to study the fate of the magnetosomes after ingestion by the protozoa. Ciliates ingested M. multicellularis, which were located in acid vacuoles as demonstrated by confocal laser scanning microscopy. Transmission electron microscopy and X-ray microanalysis of thin-sectioned ciliates showed the presence of M. multicellularis and magnetosomes inside vacuoles in different degrees of degradation. The magnetosomes are dissolved within the acidic vacuoles of the ciliate. Depending on the rate of M. multicellularis consumption by the ciliates the iron from the magnetosomes may be recycled to the environment in a more soluble form.

Cell viability in magnetotactic multicellular prokaryotes / Viabilidad celular en procariotas multicelulares magnetotácticos

A magnetotactic multicellular prokaryote (MMP) is an assembly of bacterial cells organized side by side in a hollow sphere in which each cell faces both the external environment and an internal acellular compartment in the center of the multicellular organism. MMPs swim as a unit propelled by the coordinated beating of the many flagella on the external surface of each cell. At every stage of its life cycle, MMPs are multicellular. Initially, a spherical MMP grows by enlarging the size of each of its cells, which then divide. Later, the cells separate into two identical spheres. Swimming individual cells of MMPs have never been observed. Here we have used fluorescent dyes and electron microscopy to study the viability of individual MMP cells. When separated from the MMP, the cells cease to move and they no longer respond to magnetic fields. Viability tests indicated that, although several cells could separate from a MMP before completely losing their motility and viability, all of the separated cells were dead. Our data show that the high level of cellular organization in MMPs is essential for their motility, magnetotactic behavior, and viability (AU)

Un procariota multicelular magnetotáctico (MMP en inglés) es un conjunto de células bacterianas dispuestas una al lado de otra en una esfera hueca en la que cada célula se enfrenta tanto hacia el ambiente externo como hacia un compartimento acelular interno en el centro del organismo multicelular. Los MMPs pueden nadar como una unidad propulsadas por el batir coordinado de los numerosos flagelos de la superficie exterior de cada célula. Todas las fases del ciclo vital de los MMPs son multicelulares. Inicialmente, un MMP esférico crece debido al aumento del tamaño de cada célula, y entonces las células se dividen. Después, las células se separan, formando dos esferas idénticas. Nunca se han observado células individuales de MMPs nadando. Describimos el estudio de la viabilidad de las células individuales de MMP realizado con tinciones fluorescentes y el microscopio electrónico. Cuando se separan del MMP, las células dejan de moverse y ya no responden a campos magnéticos. Las pruebas de viabilidad indican que todas las células separadas están muertas. Varias células se pueden separar de un MMP antes de que ésta pierda completamente su movilidad y viabilidad. Nuestros datos indican que el alto nivel de organización celular de los MMPs es esencial para su movilidad, comportamiento magnetotáctico y viabilidad (AU)

Antileishmanial activity of MDL 28170, a potent calpain inhibitor.

Several calpain inhibitors are under development and some are useful agents against important human pathogens. We therefore investigated the effect of MDL 28170, a potent calpain inhibitor, on the growth of Leishmania amazonensis. After 48 h of treatment, the inhibitor exhibited a dose-dependent antileishmanial activity, with a 50% lethal dose (LD(50)) of 23.3 microM. The inhibitor promoted cellular alterations, such as the parasites becoming short and round. A calpain-like protein migrating at 80 kDa was identified by Western blotting. In addition, the calpain-like molecules were identified on the cell surface of the flagellate. These results add new in vitro insights into the exploitation of calpain inhibitors in treating parasitic infections and add this family of peptidases to the list of potential targets for development of more potent and specific inhibitors against trypanosomatids.

Cell viability in magnetotactic multicellular prokaryotes.

A magnetotactic multicellular prokaryote (MMP) is an assembly of bacterial cells organized side by side in a hollow sphere in which each cell faces both the external environment and an internal acellular compartment in the center of the multicellular organism. MMPs swim as a unit propelled by the coordinated beating of the many flagella on the external surface of each cell. At every stage of its life cycle, MMPs are multicellular. Initially, a spherical MMP grows by enlarging the size of each of its cells, which then divide. Later, the cells separate into two identical spheres. Swimming individual cells of MMPs have never been observed. Here we have used fluorescent dyes and electron microscopy to study the viability of individual MMP cells. When separated from the MMP, the cells cease to move and they no longer respond to magnetic fields. Viability tests indicated that, although several cells could separate from a MMP before completely losing their motility and viability, all of the separated cells were dead. Our data show that the high level of cellular organization in MMPs is essential for their motility, magnetotactic behavior, and viability.

Multicellular life cycle of magnetotactic prokaryotes.

Most multicellular organisms, prokaryotes as well as animals, plants, and algae have a unicellular stage in their life cycle. Here, we describe an uncultured prokaryotic magnetotactic multicellular organism that reproduces by binary fission. It is multicellular in all the stages of its life cycle, and during most of the life cycle the cells organize into a hollow sphere formed by a functionally coordinated and polarized single-cell layer that grows by increasing the cell size. Subsequently, all the cells divide synchronously; the organism becomes elliptical, and separates into two equal spheres with a torsional movement in the equatorial plane. Unicellular bacteria similar to the cells that compose these organisms have not been found. Molecular biology analysis showed that all the organisms studied belong to a single genetic population phylogenetically related to many-celled magnetotactic prokaryotes in the delta sub-group of the proteobacteria. This appears to be the first report of a multicellular prokaryotic organism that proliferates by dividing into two equal multicellular organisms each similar to the parent one.