Search

Microalgae are one of the most suitable subjects for testing the potentiality of light microscopy and image analysis, because of the size of single cells, their endogenous chromaticity, and their metabolic and physiological characteristics. Microscope observations and image analysis can use microalgal cells from lab cultures or collected from water bodies as model to investigate metabolic processes, behavior/reaction of cells under chemical or photic stimuli, and dynamics of population in the natural environment in response to changing conditions. In this paper we will describe the original microscope we set up together with the image processing techniques we improved to deal with these topics. Our system detects and recognizes in-focus cells, extracts their features, measures cell concentration in multi-algal samples, reconstructs swimming cell tracks, monitors metabolic processes, and measure absorption and fluorescent spectra of subcellular compartments. It can be used as digital microscopy station for algal cell biology and behavioral studies, and field analysis applications.

A novel procedure for deriving the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its image is presented. Any digital image acquired by a microscope can be used; typical applications are the analysis of cellular/subcellular metabolic processes under physiological conditions and in response to environmental stressors (e.g. heavy metals), and the measurement of chromophore composition, distribution and concentration in cells. In this paper, we challenged the procedure with images of algae, acquired by means of a CCD camera mounted onto a microscope. The many colours algae display result from the combinations of chromophores whose spectroscopic information is limited to organic solvents extracts that suffers from displacements, amplifications, and contraction/dilatation respect to spectra recorded inside the cell. Hence, preliminary processing is necessary, which consists of in vivo measurement of the absorption spectra of photosynthetic compartments of algal cells and determination of spectra of the single chromophores inside the cell. The final step of the procedure consists in the reconstruction of the absorption spectrum of the cell spot from the colour values of the corresponding pixel(s) in its digital image by minimization of a system of transcendental equations based on the absorption spectra of the chromophores under physiological conditions.

The proceedings contain 190 papers. The topics discussed include: hybrid modeling for testing intelligent software for lunar-mars closed life support; image analysis and vision systems for processing plants; from fuzzy set theory to computational intelligence - special European experiences; computer simulation and information management systems for material processing; simulation of springback with the draw/bend test; logical rule extraction from data by maximum neural networks; a systematic and reliable approach to pattern classification; dynamic associative memory using chaotic neural networks; trends in intelligent process control in the primary aluminum industry; and the heredity and control of microstructures of liquid metals during rapid cooling processes.

Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 ?-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy.

The ability to sense light can be considered the most fundamental and presumably the most ancient property of visual systems. This ability is the basis of phototaxis, one of the most striking behavioral responses of motile photosynthetic microorganisms (i.e. microalgae) to light stimuli, which allows them to move toward or away directional light. In order to fully exploit the information content of light (intensity, direction, distribution) microorganisms need proper perceiving devices, termed photoreceptors, which must act as sensors, to perceive wavelength and direction of light, as transducers, to convert the light signal into chemical and/or electrical information, but also as amplifiers and eventually as transmitters. This review describes the universal structural, behavioral and physiological features necessary for the proper functioning of these devices in algae, and how these features have been investigated by means of different analytical techniques such as for example microspectroscopy, digital fluorescence microscopy, two photons FLIM. The insight of the photoreceptive response mechanism is explained using the unicellular alga Euglena gracilis, in which the different structural, behavioral and physiological features combine to achieve a concerted, efficient response to light stimuli

Se evaluó la estabilidad del ADN en espermatozoides de alpaca mediante citometría de flujo en muestras congeladas con antioxidantes análogos de superóxido dismutasa (Tempo y Tempol). Doce muestras de semen de alpaca fueron congeladas utilizando un dilutor en base a leche descremada, fructosa, yema de huevo y etilenglicol. Cada muestra fue dividida en tres porciones: grupo control, grupo Tempo (1 mM) y grupo Tempol (1 mM). Los antioxidantes fueron agregados al dilutor durante la curva de enfriamiento (10 °C). Las muestras fueron descongeladas y fijadas en una solución de formaldehido al 2% y permeabilizadas utilizando una solución de Triton X-100 al 0.8%. La integridad del ADN espermático se evaluó con la técnica de TUNEL y una sonda fluorescente para determinar la viabilidad celular (Ioduro de propidio [PI]). Los espermatozoides marcados con la sonda del TUNEL y PI fueron considerados células permeabilizadas con el ADN fragmentado. Las muestras fueron analizadas mediante citometría de flujo utilizando un láser de 488 nm, contando un mínimo de 10 000 células por muestra y utilizando microscopía de fluorescencia. La frecuencia de espermatozoides con ADN fragmentado en el grupo control (38.8 ± 16.2%) fue significativamente mayor (p

Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglenagracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A(498,) non fluorescent and B(462), fluorescent. Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption consists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A(498) becomes B(462)), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B(462) form acts as donor for the non-fluorescent A(498) form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell.

Se evaluó el efecto del tratamiento superovulatorio en las dos fases del ciclo ovárico sobre la respuesta folicular y la calidad embrionaria en 45 llamas hembras adultas. Se incluyeron en el estudio aquellos animales que a la ecografía presentaron un folículo preovulatorio >7 mm. Los animales se distribuyeron en tres grupos: T0 (no estimulado), T1 (tratamiento superovulatorio en fase no luteal) y T2 (tratamiento superovulatorio en fase luteal). Los animales de T1 y T2 recibieron 1 ml de LH (día 0) para sincronizar la onda folicular y 1000 UI de eCG (día 3) como tratamiento superovulatorio. Se utilizaron esponjas vaginales impregnadas con progesterona entre el día 3 y 7 para simular la fase luteal en el T2. La inducción de la ovulación se hizo mediante monta natural y aplicación de 1 ml de GnRH (día 8). La colección y evaluación de embriones se realizó 7 días post cópula (día 15) en T1 y T2. En el grupo T0 se realizó monta natural y aplicación de GnRH y 7 días después se realizó la colección de embriones. El número de folículos preovulatorios fue mayor en T1 (11.07 ± 7.53) y T2 (6.13 ± 7.11) con respecto a T0 (1.07 ± 0.26) (p7 mm follicle, observed by ultrasonography, were selected and allocated into 3 groups: T0 (non-stimulated), T1 (superovulatory treatment during the non luteal phase), and T2 (superovulatory treatment during the luteal phase). Animals in groups T1 and T2 received 1 ml of LH (day 0) for synchronization of the follicular wave and 1000 IU of eCG (day 3) as superovulatory treatment. Vaginal sponges impregnated with progesterone were used on days 3 to 7 in T2 to simulate the luteal phase. The induction of the ovulation (day 8) was done through natural mating and the application of GnRH (1 ml). Embryo recovery was done 7 days after natural mating (day 15) on T1 and T2. Similarly, embryo recovery was done 7 days after natural mating and application of GnRH in T0. The number of preovulatory follicles was larger in T1 (11.07 ± 7.53) and T2 (6.13 ± 7.11) than in T0 (1.07 ± 0.26) (p

A relatively small number of freshwater dinoflagellates are involved in symbiotic association with cryptophytes. The chloroplasts of the cryptophytes are retained by the dinoflagellate and give it the characteristic phycobilin pigmentation, either phycoerythrin or phycocyanin. The pigment characterization of the retained chloroplasts can give precise and accurate information about the type of cryptophyte preyed upon by the dinoflagellate. For this purpose, we performed microspectrophotometric evaluation of the pigments of Gymnodinium acidotum Nygaard and three different cryptophytes present in samples collected from a tributary of the river Arno, in Tuscany (Italy). The comparison of the different spectroscopic data allowed us to discriminate effectively among the cryptophytes preyed upon by the dinoflagellate.

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.

In microorganisms and plants, chromium (Cr) is not essential for any metabolic process, and can ultimately prove highly deleterious. Due to its widespread industrial use, chromium has become a serious pollutant in diverse environmental settings. The presence of Cr leads to the selection of specific algal populations able to tolerate high levels of Cr compounds. The varying Cr-resistance mechanisms displayed by microorganisms include biosorption, diminished accumulation, precipitation, reduction of Cr(6+) to Cr(3+), and chromate efflux. In this paper we describe the effects of Cr(6+) (the most toxic species) on the photosynthetic and photoreceptive apparatus of two fresh water microalgae, Eudorina unicocca and Chlorella kessleri. We measured the effect of this heavy metal by means of in vivo absorption microspectroscopy of both the thylakoid compartments and the eyespot. The decomposition of the overall absorption spectra in pigment constituents indicates that Cr(6+) effects are very different in the two algae. In E. unicocca the metal induced a complete pheophinitization of the chlorophylls and a modification of the carotenoids present in the eyespot after only 120 h of exposition at a concentration equal or greater than 40 microM, which is the limit for total Cr discharge established by US EPA regulations. In C. kessleri, chromium concentrations a hundred times higher than this limit had no effect on the photosynthetic machinery. The different tolerance level of the two algae is suggested to be due to the different properties of the mucilaginous envelope and cell wall covering, respectively, the colonies of Eudorina and the single cells of Chlorella, which binds chromium cations to a different extent.