Agronomic-productive Characteristics of Two Genotype of Stevia Rebaudiana in Central Italy

Stevia rebaudiana Bertoni produces a variety of high-potency low calorie sweeteners in its' leaf tissue. The aim of this work was to evaluate the productive potential of two different Stevia rebaudiana genotypes and the characteristics of the production of the plant obtained through different cultivation methods, in central Italy. For several years (1992-2000) agronomic trials on Stevia rebaudiana cultivated in the littoral area near Pisa (Italy) carried out. In 1992 two different genotypes of Stevia (B1 and B2) transplanted in silt-loam soil; in 1996 other plants from B2 genotype, produced by vegetative process (micropropagation), were transplanted in the same field. In the period from 1992-1999 the production of leaves from both genotypes increase as of the third year and the ratio leaf-aerial plant, after the peak in the third year, tends to diminish. Except the first year (1997) there were not recorded statistically significant differences between the two propagation methods employed in relation to leaf production. The quantity of leaves produced from a single harvest was less than resulting from two cuttings and the micropropagation plants produced a larger amount of leaves than those from cutting. The leaf-stem ratio was to become an interesting morphological and production characteristic parameter of the plant. Stevia rebaudiana appears particularly suited for the cultivation environment of central Italy. A particular positive aspect that must be considered in these regions is that Stevia can be grown successfully as poliannual species because crop survival over the winter is high. The results obtained show that this species is economically profitable until the 5th or 6th year of cultivation. At our latitudes is also necessary to establish a program of genetic improvement in order to develop earlier varieties that can guaranty an optimum qualitative and quantitative seed production

AFM macro-probes to investigate whole 3D cardiac spheroids

In its many applications, the Atomic Force Microscope (AFM) is a promising tool in cardiac mechanobiology because it can unravel the viscoelastic and mechano-dynamic properties of individual cardiomyocytes. However, the biophysical investigation of more accurate 3D models is hampered by commercial probes, which typically operate at the cell sub-compartmental resolution. We have previously shown how flat macro-probes can overcome these limitations by extending the AFM mechanical measurements to multicellular aggregates. Such macro-probes are fabricated by standard micromachining and carry a flat polymeric wedge to offset the AFM mounting tilt. Therefore, the AFM is upgraded to a micro-parallel plate rheometer with unmatched force range and sensitivity. In this article, we show how these macro-probes can be applied to reveal the global rheology of primary cardiomyocytes spheroids, by performing stress-relaxation tests. More importantly, we demonstrate that these macro-probes can be used as passive sensors capable of monitoring the spheroid beating force and beating pattern, and to perform a “micro-CPR” on the spheroid itself

From Awareness to Action: Energy Literacy and Household Energy Use

peer reviewe

Atomic force spectroscopy-based essay to evaluate oocyte postovulatory aging

Postovulatory aging is a process occurring in the mature (MII) oocyte leading the unfertilized ones to apoptosis. The optimal time window of fertility for different mammalian species after oocytes maturation depends on its timeliness: the higher the time elapsed from the accomplishment of the MII stage, the lower are the chances of fertilization and of development of a viable embryo. In the in vitro fertilization, the selection of competent oocytes for intracytoplasmic sperm injection (ICSI) is mostly made by the visual inspection of the MII oocyte morphology, which does not allow to determine the oocyte postovulatory age. On the other hand, more specific tests usually involve some kind of staining, thus compromising the viability of the oocyte for reproductive purposes. Hence, the need of a noninvasive analysis of oocyte aging to improve the success rate of in vitro fertilization procedures. Here, we exploit atomic force microscopy to examine the evolution of the mechanical properties of mouse oocytes during in vitro postovulatory aging. Three hours before the occurrence of any visual morphological feature related to degradation, we observe a sudden change of the mechanical parameters: the elastic modulus doubles its initial value, while the viscosity decreases significantly. These mechanical variations are temporally correlated with the release of the cortical granules, investigated by fluorescence microscopy. Interestingly, the oocyte mechanics correlates as well with the yield of embryo formation, evaluated up to the blastocyst formation stage. These results demonstrate that minimally invasive mechanical measurements are very sensitive to the aging of the oocyte and can be used as a label-free method to detect the age of the postovulatory oocytes

Effects of Temperature, Light and Pre-Chilling on Seed Germination of Stevia rebaudiana (Bertoni) Bertoni Accessions

Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub of the Asteraceae family native to Paraguay and Brazil where it has been used for several years as a sweetener. It is a short-day species, with a critical light requirement for flowering of roughly 13 hours. In plants whose biological cycle is strongly photoperiod-dependent, latitude is one of the major factors influencing reproduction. Late flowering may adversely affect seed production if this occurs during a season that is unfavorable to pollination. At Italian latitudes, this species often gives scanty seed production, with low germination rate and poor germination energy of seeds produced. In 2001 four accessions have been grown for seed production in a field plot experiment in Central Italy. The various accessions were found to exhibit noticeably different photoperiod requirements, which affected flowering time (from late August to the end of September) and consequently also the conditions of achene filling and ripening. Late flowering and seed ripening occurred during autumn season, unfavourable to complete seed formation, leading to an increase in the empty seed percentage recorded for each accession. Detailed germination trials were therefore undertaken using seeds collected from plants of the different accessions in order to assess the quality of the seeds produced. Various germination methods have been tested in a controlled environment adopting four different temperatures (20° C, 25° C constant temperature and 15/25° C, 20/30° C (16/8h) alternating temperature) in light or darkness with or without pre-chilling. Germination rates varied over an extensive range (germination percentage from 9 to 83%), mainly due to the divergent specific characteristics of the material examined and the different treatments studied. At all temperatures tested, the most earlier accession, showed the higher germination percentages (54-83%) while the latest accession was among those with the lowest germination values (9-44%). Overall, at the constant temperatures assayed, pre-chilling gave a higher germination percentage as compared to no pre-chilling, while at alternating temperatures higher germination rates were obtained in the absence of pre-chilling. The great variability observed between accessions for photoperiodic requirement as well as in seed germinative characteristics was probably due to the fact that this species was not subjected to any genetic improvement program

The application of scanning near field optical imaging to the study of human sperm morphology

BackgroundThe morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures.ResultsIn this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols.ConclusionsSuch findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together

Solid state molecular rectifier based on self organized metalloproteins

Recently, great attention has been paid to the possibility of implementing hybrid electronic devices exploiting the self-assembling properties of single molecules. Impressive progress has been done in this field by using organic molecules and macromolecules. However, the use of biomolecules is of great interest because of their larger size (few nanometers) and of their intrinsic functional properties. Here, we show that electron-transfer proteins, such as the blue copper protein azurin (Az), can be used to fabricate biomolecular electronic devices exploiting their intrinsic redox properties, self assembly capability and surface charge distribution. The device implementation follows a bottom-up approach in which the self assembled protein layer interconnects nanoscale electrodes fabricated by electron beam lithography, and leads to efficient rectifying behavior at room temperature.Comment: 13 pages including two figures. Accepted for publication in Advanced Material

Mapping of FcεRI immunoglobulin E receptor in activated mast cells by scanning near-field optical microscopy

Introduction. The cell membrane has a dynamic role that enables the reorganization of receptors and signal molecules in response to signaling processes. To visualize these phenomena we can nowadays benefit from techniques that allow subdiffraction optical resolution.(1) Among these, scanning near-field optical microscopy (SNOM) exploits the evanescent field exiting at the probe fibre apex. The lateral resolution depends essentially on the subwavelength aperture size of the optical fibre (typically better than 100 nm). This makes SNOM particularly suited for nanoscale study on intact biological membrane. Recently, we demonstrated that SNOM combined with immunolabelling and diaminobenzidine (DAB) staining is a valuable non-invasive approach for investigating nanostructures components within intact oligodendrocites.(2) Here we extend this approach to the study of reorganization of FcεRI immunoglobulin E (IgE) receptor in intact mast cells upon antigen-induced degranulation by IgE cross-linking. Materials and Methods. Rat basophilic leukemia RBL-2H3 cells were grown on coverslips, incubated overnight with anti-2,4-Dinitrophenol (DNP) IgE and degranulated by adding DNP. After 30’ cells were fixed and immunolabelled with anti-FcεRI monoclonal antibody. DAB staining was performed with VECTASTAIN® UNIVERSAL Elite ABC kit. TriA-SNOM microscope (A.P.E. Research, Trieste) was used for near-field measurements. Results and discussions. Resting and activated cells with and without DAB staining immunolabelling for the internal portion of FcεRI are analyzed by SNOM. Topography of activated cells shows membrane ridges over surface and in some cases a considerable cell flattening, in general accordance with the morphology observed as result of the degranulation process. The optical transmission images of DAB stained activated mast cell display numerous very dark circular areas, not observed in the unlabelled activated cells. Such features are likely due to a strong light adsorption for the presence of localized DAB reaction. These areas (lateral size about 300 nm) appear to be in agreement with IgE receptor FcεRI patches observed on cytoplasmatic side of membrane sheets of activated mast cells.(3) In conclusion these results demonstrate that SNOM combined with immunolabelling and DAB staining holds great potential for investigating the organization of proteins into micro- or nanodomains in cell membrane. 1. Bioch. Biophys. Acta 2010, 1798: 77. 2. Neuroimage 2010, 49: 517. 3. Biophys. J. 2006, 90: 2404