Response profiles of murine spiral ganglion neurons on multi-electrode arrays.

OBJECTIVE Cochlear implants (CIs) have become the gold standard treatment for deafness. These neuroprosthetic devices feature a linear electrode array, surgically inserted into the cochlea, and function by directly stimulating the auditory neurons located within the spiral ganglion, bypassing lost or not-functioning hair cells. Despite their success, some limitations still remain, including poor frequency resolution and high-energy consumption. In both cases, the anatomical gap between the electrode array and the spiral ganglion neurons (SGNs) is believed to be an important limiting factor. The final goal of the study is to characterize response profiles of SGNs growing in intimate contact with an electrode array, in view of designing novel CI devices and stimulation protocols, featuring a gapless interface with auditory neurons. APPROACH We have characterized SGN responses to extracellular stimulation using multi-electrode arrays (MEAs). This setup allows, in our view, to optimize in vitro many of the limiting interface aspects between CIs and SGNs. MAIN RESULTS Early postnatal mouse SGN explants were analyzed after 6-18 days in culture. Different stimulation protocols were compared with the aim to lower the stimulation threshold and the energy needed to elicit a response. In the best case, a four-fold reduction of the energy was obtained by lengthening the biphasic stimulus from 40 μs to 160 μs. Similarly, quasi monophasic pulses were more effective than biphasic pulses and the insertion of an interphase gap moderately improved efficiency. Finally, the stimulation with an external electrode mounted on a micromanipulator showed that the energy needed to elicit a response could be reduced by a factor of five with decreasing its distance from 40 μm to 0 μm from the auditory neurons. SIGNIFICANCE This study is the first to show electrical activity of SGNs on MEAs. Our findings may help to improve stimulation by and to reduce energy consumption of CIs and thereby contribute to the development of fully implantable devices with better auditory resolution in the future.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow

BACKGROUND AIMS The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

Mode of action of claudin peptidomimetics in the transient opening of cellular tight junction barriers

In epithelial/endothelial barriers, claudins form tight junctions, seal the paracellular cleft, and limit the uptake of solutes and drugs. The peptidomimetic C1C2 from the C-terminal half of claudin-1's first extracellular loop increases drug delivery through epithelial claudin-1 barriers. However, its molecular and structural mode of action remains unknown. In the present study, >100 μM C1C2 caused paracellular opening of various barriers with different claudin compositions, ranging from epithelial to endothelial cells, preferentially modulating claudin-1 and claudin-5. After 6 h incubation, C1C2 reversibly increased the permeability to molecules of different sizes; this was accompanied by redistribution of claudins and occludin from junctions to cytosol. Internalization of C1C2 in epithelial cells depended on claudin-1 expression and clathrin pathway, whereby most C1C2 was retained in recyclosomes >2 h. In freeze-fracture electron microscopy, C1C2 changed claudin-1 tight junction strands to a more parallel arrangement and claudin-5 strands from E-face to P-face association - drastic and novel effects. In conclusion, C1C2 is largely recycled in the presence of a claudin, which explains the delayed onset of barrier and junction loss, the high peptide concentration required and the long-lasting effect. Epithelial/endothelial barriers are specifically modulated via claudin-1/claudin-5, which can be targeted to improve drug delivery.

Methodologies for the Analysis of HCV-Specific CD4(+) T Cells

Virus-specific CD4(+) T cells play a major role in viral infections, such as hepatitis C virus (HCV). Viral clearance is associated with vigorous and multi-specific CD4(+) T-cell responses, while chronic infection has been shown to be associated with weak or absent T-cell responses. Most of these studies have used functional assays to analyze virus-specific CD4(+) T-cell responses; however, these and other detection methods have various limitations. Therefore, the important question of whether virus-specific CD4(+) T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analyzed in detail. A novel assay, in which virus-specific CD4(+) T-cell frequencies can be determined by de novo CD154 (CD40 ligand) expression in response to viral antigens, can help to overcome some of the limitations of functional assays and restrictions of multimer-based methods. This and other current established methods for the detection of HCV-specific CD4(+) T cells will be discussed in this review.

Functional architecture of mammalian horizontal cells

In the rabbit retina, there are two kinds of horizontal cells (HCs). The A-type HC is a large axonless cell which contacts cones exclusively. The B-type HC is an axon bearing cell. While the somatic dendrites of B-type HCs also contact cones, the axon expands into an elaborately branched structure, the axon terminal (AT), which contacts a large number of rods. It is difficult to label the different HCs selectively by immunochemical methods. Therefore, we developed dye injection methods to label each type of HC. Then it was possible, (1) to describe the detailed structure of the AT (2) to identify the glutamate receptors mediating cone input to A and B-type HCs and rod input to ATs and (3) to test the hypothesis that the B-type HCs are coupled via Cx57 gap junctions. ^ To obtain well filled examples of single HCs, it was necessary to block gap junction coupling to stop the spread of Neurobiotin through the network. We used dye coupling in A-type HCs to screen a series of potential gap junction antagonists. One of these compounds, meclofenamic acid (MFA), was potent, water soluble and easily reversible. This compound may be a useful tool to manipulate gap junction coupling. ^ In the presence of MFA, Neurobiotin passed down the axon of B-type HCs to reveal the detailed structure of the AT. We observed that only one AT ending entered each rod spherule invagination. This observation was confirmed by calculation and two dye injections. ^ Glutamate is the neurotransmitter used by both rods and cones. AMPA receptors were colocalized with the dendrites of A and B-type HCs at each cone pedicle. In addition, AMPA receptors were located on the AT ending at each rod spherule. Thus rod and cone input to HCs is mediated by AMPA receptors. ^ A-type and B-type HCs may express different connexins because they have different dye-coupling properties. Recently, we found that connexin50 (Cx50) is expressed by A-type HCs. B-type HCs and B-type ATs are also independently coupled. Cx57 was expressed in the OPL and double label studies showed that Cx 57 was colocalized with the AT matrix but not with the somatic dendrites of B-type HCs. ^ In summary, we have identified a useful gap junction antagonist, MFA. There is one AT ending at each rod spherule, rods inputs to ATs is mediated by AMPA receptors and coupling in the AT matrix is mediated by Cx57. This confirms that HCs with different properties use distinct connexins. The properties of ATs described in this research are consistent. The connections and properties reported here suggest that ATs functions as rod HCs and provide a negative feedback signal to rods. ^

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

TURNOVER RATE OF THE NEURONAL CONNEXIN CX36 IN HELA CELLS

Electrical synapses formed of the gap junction protein Cx36 show a great deal of functional plasticity, much dependent on changes in phosphorylation state of the connexin. However, gap junction turnover may also be important for regulating cell-cell communication, and turnover rates of Cx36 have not been studied. Connexins have relatively fast turnover rates, with short half-lives measured to be 1.5 to 3.5 hours in pulse-chase analyses of connexins (Cx26 and Cx43) in tissue culture cells and whole organs. We utilized HaloTag technology to study the turnover rate of Cx36 in transiently transfected HeLa cells. The HaloTag protein forms irreversible covalent bonds with chloroalkane ligands, allowing pulse-chase experiments to be performed very specifically. The HaloTag open reading frame was inserted into an internal site in the C-terminus of Cx36 designed not to disrupt the regulatory phosphorylation sites and not to block the C-terminal PDZ interaction motif. Functional properties of Cx36-Halo were assessed by Neurobiotin tracer coupling, live cell imaging, and immunostaining. For the pulse-chase study, transiently transfected HeLa cells were pulse labeled with Oregon Green (OG) HaloTag ligand and chase labeled at various times with tetramethylrhodamine (TMR) HaloTag ligand. Cx36-Halo formed large junctional plaques at sites of contact between transfected HeLa cells and was also contained in a large number of intracellular vesicles. The Cx36-Halo transfected HeLa cells supported Neurobiotin tracer coupling that was regulated by activation and inhibition of PKA in the same manner as wild-type Cx36 transfected cells. In the pulse-chase study, junctional protein labeled with the pulse ligand (OG) was gradually replaced by newly synthesized Cx36 labeled with the chase ligand (TMR). The half-life for turnover of protein in junctional plaques was 2.8 hours. Treatment of the pulse-labeled cells with Brefeldin A (BFA) prevented the addition of new connexins to junctional plaques, suggesting that the assembly of Cx36 into gap junctions involves the traditional ER-Golgi-TGN-plasma membrane pathway. In conclusion, Cx36-Halo is functional and has a turnover rate in HeLa cells similar to that of other connexins that have been studied. This turnover rate is likely too slow to contribute substantially to short-term changes in coupling of neurons driven by transmitters such as dopamine, which take minutes to achieve. However, turnover may contribute to longer-term changes in coupling.

Ocean temperature response to idealized Gleissberg and de Vries solar cycles in a comprehensive climate model

The ~90-year Gleissberg and ~200-year de Vries cycles have been identified as two distinctive quasi-periodic components of Holocene solar activity. Evidence exists for the impact of such multi-decadal to centennial-scale variability in total solar irradiance (TSI) on climate, but concerning the ocean, this evidence is mainly restricted to the surface response. Here we use a comprehensive global climate model to study the impact of idealized solar forcing, representing the Gleissberg and de Vries cycles, on global ocean potential temperature at different depth levels, after a recent proxy record indicates a signal of TSI anomalies in the northeastern Atlantic at mid-depth. Potential impacts of TSI anomalies on deeper oceanic levels are climatically relevant due to their possible effect on ocean circulation by altering water mass characteristics. Simulated solar anomalies are shown to penetrate the ocean down to at least deep-water levels. Despite the fact that the two forcing periods differ only by a factor of ~2, the spatial pattern of response is significantly distinctive between the experiments, suggesting different mechanisms for solar signal propagation. These are related to advection by North Atlantic Deep Water flow (200-year forcing), and barotropic adjustment in the South Atlantic in response to a latitudinal shift of the westerly wind belt (90-year forcing).

Alleviation of solar ultraviolet radiation (UVR)-induced photoinhibition in diatom Chaetoceros curvisetus by ocean acidification

The study aimed to unravel the interaction between ocean acidification and solar ultraviolet radiation (UVR) in Chaetoceros curvisetus. Chaetoceros curvisetus cells were acclimated to high CO2 (HC, 1000 ppmv) and low CO2 concentration (control, LC, 380 ppmv) for 14 days. Cell density, specific growth rate and chlorophyll were measured. The acclimated cells were then exposed to PAB (photosynthetically active radiation (PAR) + UV-A + UV-B), PA (PAR + UV-A) or P (PAR) for 60 min. Photochemical efficiency (phi PSII), relative electron transport rate (rETR) and the recovery of ?PSII were determined. HC induced higher cell density and specific growth rate compared with LC. However, no difference was found in chlorophyll between HC and LC. Moreover, phi PSII and rETRs were higher under HC than LC in response to solar UVR. P exposure led to faster recovery of phi PSII, both under HC and LC, than PA and PAB exposure. It appeared that harmful effects of UVR on C. curvisetus could be counteracted by ocean acidification simulated by high CO2 when the effect of climate change is not beyond the tolerance of cells.

A two-dimensional finite element model of front surface current flow in cells under non-uniform, concentrated illumination

A two-dimensional finite element model of current flow in the front surface of a PV cell is presented. In order to validate this model we perform an experimental test. Later, particular attention is paid to the effects of non-uniform illumination in the finger direction which is typical in a linear concentrator system. Fill factor, open circuit voltage and efficiency are shown to decrease with increasing degree of non-uniform illumination. It is shown that these detrimental effects can be mitigated significantly by reoptimization of the number of front surface metallization fingers to suit the degree of non-uniformity. The behavior of current flow in the front surface of a cell operating at open circuit voltage under non-uniform illumination is discussed in detail.

Soiling and other optical losses in solar-tracking PV plants in Navarra

Field data of soiling energy losses on PV plants are scarce. Furthermore, since dirt type and accumulation vary with the location characteristics (climate, surroundings, etc.), the available data on optical losses are, necessarily, site dependent. This paper presents field measurements of dirt energy losses (dust) and irradiance incidence angle losses along 2005 on a solar-tracking PV plant located south of Navarre (Spain). The paper proposes a method to calculate these losses based on the difference between irradiance measured by calibrated cells on several trackers of the PV plant and irradiance calculated from measurements by two pyranometers (one of them incorporating a shadow ring) regularly cleaned. The equivalent optical energy losses of an installation incorporating fixed horizontal modules at the same location have been calculated as well. The effect of dirt on both types of installations will accordingly be compared.

Properties of aluminium nodules foamed with concentrated solar energy

Commercial aluminium foam filled structures and sandwich panels are available for structural applications. As alternative to these materials, small granular foamed pieces are proposed to fill structures as well as sandwich panels. On the present work, foam precursors are obtained by Powder Metallurgy (PM) route, using natural calcium carbonate as foaming agent instead of titanium hydride. Extruded precursor bars were cut into small pieces (around 4.5 mm long and 5mm in diameter). Foaming treatment was carried out on two different ways: electrical preheated furnace and by solar furnace. Foamed nodules presented a low cell size, density e.g. 0.67 g/cm3 to 0.88 g/cm3 and a height/diameter ratio between 0.72 and 0.84 as a function of precursor size. These properties depend on the foaming particle size, foaming cycle and precursor dimensions. Carbonate precursors are easily foamed by concentrated solar energy, due to the lower risk of cell collapse than with hydride precursors, resulting from cell stabilization by oxide skin formation into cells and a low degree of foamed nodules bonding.

On-site measurement of limiting subcell in multijunction solar devices

It is well known that the response of any photovoltaic solar cell is dependent on the spectral characteristics of the incident radiation. This dependency is crucial in the output characteristics of a multijunction (MJ) cell where the spectral composition of the radiation determines the overall photocurrent produced, as either the top or the middle subcell will be limiting its response. The current mismatching between top and middle subcell is translated into energy losses, affecting the yield of the system. For research and commercial purposes it is interesting to measure accurately the incident solar radiation on a MJ cell, in terms of its spectral composition. This measurement will allows us to determine the photocurrent generated in each band of the multijunction device. Nowadays, the only way of measuring the photocurrent generated by each subcell is done with isotype cells or with spectroradiometers but there is no device capable of directly measuring each subcell photocurrent. In this paper it is described a device based on a commercial multijunction solar cell that is capable of measuring the direct irradiance for the top and middle bands thus it offers information of the limiting subcell (top or middle) in outdoors conditions.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals