Immuno flow cytometry in marine phytoplankton research

The developments in the combination of flow cytometry and immunology as a tool to identify, count and examine marine phytoplankton cells are reviewed. The concepts of immunology and flow cytometry are described. A distinction is made between quantitative and qualitative immunofluorescence. Quantitative immunofluorescence, the identification and enumeration of phytoplankton cells, is the research area that has advanced rapidly in the past decade, and is reviewed extensively. Key steps of quantitative immunofluorescence, fixation and immunolabel intensity, are discussed in more detail. Qualitative immunofluorescence is a new, hardly explored but highly interesting development in which qualitative -physiological- variables related to e.g. nutrient limitation or primary production are measured in individual cells instead of phytoplankton populations as a whole. Several combinations of immunological probes, both for species identification and for physiological measurements, are proposed. A special case of qualitative immunofluorescence is the measurement of phytoplankton toxins in single cells from natural populations. It is anticipated that the future use of semiconductor nanocrystals or quantum dots as fluorophores will greatly enhance signal detection in flow cytometry, and hence in both quantitative and qualitative immunofluorescence applications

THE EFFECT OF LABELING INTENSITY, ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY, ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real-time confocal laser scanning microscopy using an anti-rabbit IgG/FITC-conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti-rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells

Novel Crystalline SiO2 Nanoparticles via Annelids Bioprocessing of Agro-Industrial Wastes

The synthesis of nanoparticles silica oxide from rice husk, sugar cane bagasse and coffee husk, by employing vermicompost with annelids (Eisenia foetida) is reported. The product (humus) is calcinated and extracted to recover the crystalline nanoparticles. X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS) show that the biotransformation allows creating specific crystalline phases, since equivalent particles synthesized without biotransformation are bigger and with different crystalline structure

Efficient screening for ‘genetic pollution’ in an anthropogenic crested newt hybrid zone

Genetic admixture between endangered native and non-native invasive species poses a complex conservation problem. Decision makers often need to quickly screen large numbers of individuals and distinguish natives from morphologically similar invading species and their genetically admixed offspring. We describe a protocol using the fast and economical Kompetitive Allele Specific PCR (KASP) technology for genotyping on a large scale. We apply this protocol to a case study of hybridization between a native and an invasive crested newt species. Using previously published data, we designed a panel of ten nuclear and one mitochondrial diagnostic SNP markers. We observed only minor differences between KASP and next-generation sequencing data previously produced with the Ion Torrent platform. We briefly discuss practical considerations for tackling the insidious conservation problem of genetic admixture between native and invasive species. The KASP genotyping protocol facilitates policy decision making for the crested newt case and is generally applicable to invasive hybridization with endangered taxa

Energy cost of ambulation in trans-tibial amputees using a dynamic-response foot with hydraulic versus rigid 'ankle': insights from body centre of mass dynamics.

BACKGROUND:Previous research has shown that use of a dynamic-response prosthetic foot (DRF) that incorporates a small passive hydraulic ankle device (hyA-F), provides certain biomechanical benefits over using a DRF that has no ankle mechanism (rigA-F). This study investigated whether use of a hyA-F in unilateral trans-tibial amputees (UTA) additionally provides metabolic energy expenditure savings and increases the symmetry in walking kinematics, compared to rigA-F. METHODS:Nine active UTA completed treadmill walking trials at zero gradient (at 0.8, 1.0, 1.2, 1.4, and 1.6 of customary walking speed) and for customary walking speed only, at two angles of decline (5° and 10°). The metabolic cost of locomotion was determined using respirometry. To gain insights into the source of any metabolic savings, 3D motion capture was used to determine segment kinematics, allowing body centre of mass dynamics (BCoM), differences in inter-limb symmetry and potential for energy recovery through pendulum-like motion to be quantified for each foot type. RESULTS:During both level and decline walking, use of a hyA-F compared to rigA-F significantly reduced the total mechanical work and increased the interchange between the mechanical energies of the BCoM (recovery index), leading to a significant reduction in the metabolic energy cost of locomotion, and hence an associated increase in locomotor efficiency (p < 0.001). It also increased inter-limb symmetry (medio-lateral and progression axes, particularly when walking on a 10° decline), highlighting the improvements in gait were related to a lessening of the kinematic compensations evident when using the rigA-F. CONCLUSIONS:Findings suggest that use of a DRF that incorporates a small passive hydraulic ankle device will deliver improvements in metabolic energy expenditure and kinematics and thus should provide clinically meaningful benefits to UTAs' everyday locomotion, particularly for those who are able to walk at a range of speeds and over different terrains

Immunocytochemical evidence for acidic thylakoids in intact diatom cells

Using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyI propylamine (DAMP) in in situ probing experiments, ultrastructural evidence for the acidic nature of the thylakoids in whole cells has been obtained in three unicellular pennate diatom species, with special emphasis on Navicula salinarum. Positive internal controls were provided by compartments known to be acidic (microbodies, lysosomal active vacuoles, and silica deposition vesicle). Negative internal controls were circumneutral compartments (nucleus and mitochondria). The predictable labelling of these compartments demonstrated the translocation of unprotonated DAMP over membranes and into acidic environments, where it became protonated. In cells that were treated with ionophores to disrupt pH gradients after incubation with DAMP, DAMP was no longer confined specifically to thylakoids and appeared to be more randomly distributed within the chloroplasts, indicating that the pH gradient of the thylakoids was affected. After densitometry, the transthylakoid DeltapH was estimated to vary between 1.0 and 2.0, coinciding with a moderate lumen acidity. The results obtained provide what we believe is the first clear evidence of the acidic nature of the thylakoid lumen in living cells at an ultrastructural level