Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format

High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

blique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo-4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure

Entanglement of a Mesoscopic Field with an Atom induced by Photon Graininess in a Cavity

We observe that a mesoscopic field made of several tens of microwave photons exhibits quantum features when interacting with a single Rydberg atom in a high-Q cavity. The field is split into two components whose phases differ by an angle inversely proportional to the square root of the average photon number. The field and the atomic dipole are phase-entangled. These manifestations of photon graininess vanish at the classical limit. This experiment opens the way to studies of large Schrodinger cat states at the quantum-classical boundary

A methodological approach to assess the effect of organic, biodynamic, and conventional production processes on the intrinsic and perceived quality of a typical wine: The case study of chianti docg

The aim of this study was to propose a methodological approach to evaluate the impact of the organic, biodynamic, and conventional production processes on the intrinsic and perceived quality of a typical wine. For this purpose, fourteen commercial Chianti DOCG wines from the 2016 harvest were selected based on the type of production management. A survey was set up to get winemaking information from the estate’s producer of the wines to estimate the carbon dioxide production under the three types of management. The eligibility, identity, and style properties (the intrinsic quality) of the wines were defined. A group of 45 experts evaluated the differences between wines by the Napping test and rated their typicality (perceived quality). The organic and biodynamic management showed a lower level of estimated values of carbon dioxide production. The overall statistical elaboration of the chemical and sensory data highlighted that the registered differences of the intrinsic, perceived quality, and typicality level of the respective wines, did not depend on the type of management. The comparison of the three kinds of wine by SIMCA modeling, put in evidence that the conventional ones showed a greater homogeneity regarding chemical composition, sensory characteristics, and typicality

Stem cell senescence: effects of REAC technology on telomerase-independent and telomerase-dependent pathways

Decline in the gene expression of senescence repressor Bmi1, and telomerase, together with telomere shortening, underlay senescence of stem cells cultured for multiple passages. Here, we investigated whether the impairment of senescence preventing mechanisms can be efficiently counteracted by exposure of human adipose-derived stem cells to radio electric asymmetrically conveyed fields by an innovative technology, named Radio Electric Asymmetric Conveyer (REAC). Due to REAC exposure, the number of stem cells positively stained for senescence associated ß-galactosidase was significantly reduced along multiple culturing passages. After a 90-day culture, REAC-treated cells exhibited significantly higher transcription of Bmi1 and enhanced expression of other stem cell pluripotency genes and related proteins, compared to unexposed cells. Transcription of the catalytic telomerase subunit (TERT) was also increased in REAC-treated cells at all passages. Moreover, while telomere shortening occurred at early passages in both REAC-treated and untreated cells, a significant rescue of telomere length could be observed at late passages only in REAC-exposed cells. Thus, REAC-asymmetrically conveyed radio electric fields acted on a gene and protein expression program of both telomerase-independent and telomerase-dependent patterning to optimize stem cell ability to cope with senescence progression

PTHrP on MCF-7 breast cancer cells: a growth factor or an antimitogenic peptide?

Letter to the Edito

Mechanical Stimulation of Fibroblasts by Extracorporeal Shock Waves: Modulation of Cell Activation and Proliferation Through a Transient Proinflammatory Milieu

Extracorporeal shock waves (ESWTs) are \u201cmechanical\u201d waves, widely used in regenerative medicine, including soft tissue wound repair. Although already being used in the clinical practice, the mechanism of action underlying their biological activities is still not fully understood. In the present paper we tried to elucidate whether a proinflammatory effect may contribute to the regenerative potential of shock waves treatment. For this purpose, we exposed human foreskin fibroblasts (HFF1 cells) to an ESWT treatment (100 pulses using energy flux densities of 0.19 mJ/mm2 at 3 Hz), followed by cell analyses after 5 min, up to 48 h. We then evaluated cell proliferation, reactive oxygen species generation, ATP release, and cytokine production. Cells cultured in the presence of lipopolysaccharide (LPS), to induce inflammation, were used as a positive control, indicating that LPS-mediated induction of a proinflammatory pattern in HFF1 increased their proliferation. Here, we provide evidence that ESWTs affected fibroblast proliferation through the overexpression of selected cytokines involved in the establishment of a proinflammatory program, superimposable to what was observed in LPS-treated cells. The possibility that inflammatory circuits can be modulated by ESWT mechanotransduction may disclose novel hypothesis on their biological underpinning and expand the fields of their biomedical application

Intracrine endorphinergic systems in modulation of myocardial differentiation

A wide variety of peptides not only interact with the cell surface, but govern complex signaling from inside the cell. This has been referred to as an "intracrine" action, and the orchestrating molecules as "intracrines". Here, we review the intracrine action of dynorphin B, a bioactive end-product of the prodynorphin gene, on nuclear opioid receptors and nuclear protein kinase C signaling to stimulate the transcription of a gene program of cardiogenesis. The ability of intracrine dynorphin B to prime the transcription of its own coding gene in isolated nuclei is discussed as a feed-forward loop of gene expression amplification and synchronization. We describe the role of hyaluronan mixed esters of butyric and retinoic acids as synthetic intracrines, controlling prodynorphin gene expression, cardiogenesis, and cardiac repair. We also discuss the increase in prodynorphin gene transcription and intracellular dynorphin B afforded by electromagnetic fields in stem cells, as a mechanism of cardiogenic signaling and enhancement in the yield of stem cell-derived cardiomyocytes. We underline the possibility of using the diffusive features of physical energies to modulate intracrinergic systems without the needs of viral vector-mediated gene transfer technologies, and prompt the exploration of this hypothesis in the near future

One-Dimensional Discrete Stark Hamiltonian and Resonance Scattering by Impurities

A one-dimensional discrete Stark Hamiltonian with a continuous electric field is constructed by extension theory methods. In absence of the impurities the model is proved to be exactly solvable, the spectrum is shown to be simple, continuous, filling the real axis; the eigenfunctions, the resolvent and the spectral measure are constructed explicitly. For this (unperturbed) system the resonance spectrum is shown to be empty. The model considering impurity in a single node is also constructed using the operator extension theory methods. The spectral analysis is performed and the dispersion equation for the resolvent singularities is obtained. The resonance spectrum is shown to contain infinite discrete set of resonances. One-to-one correspondence of the constructed Hamiltonian to some Lee-Friedrichs model is established.Comment: 20 pages, Latex, no figure

International migration of unaccompanied minors: trends, health risks, and legal protection

The global population of unaccompanied minors—children and adolescents younger than 18 years who migrate without their legal guardians—is increasing. However, as data are not systematically collected in any region, if collected at all, little is known about this diverse group of young people. Compared with adult migrants, unaccompanied minors are at greater risk of harm to their health and integrity because they do not have the protection provided by a family, which can affect their short-term and long-term health. This Review summarises evidence regarding the international migration and health of unaccompanied minors. Unaccompanied minors are entitled to protection that should follow their best interests as a primary consideration; however, detention, sometimes under the guise of protection, is a widespread practice. If these minors are provided with appropriate forms of protection, including health and psychosocial care, they can thrive and have good long-term outcomes. Instead, hostile immigration practices persist, which are not in the best interests of the child