Assembly and movement of a plant virus carrying a green fluorescent protein overcoat.

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.

Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera.

A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the rat glucocorticoid receptor (GR). When GFP-GR is expressed in living mouse cells, it is competent for normal transactivation of the GR-responsive mouse mammary tumor virus promoter. The unliganded GFP-GR resides in the cytoplasm and translocates to the nucleus in a hormone-dependent manner with ligand specificity similar to that of the native GR receptor. Due to the resistance of the mutant GFP to photobleaching, the translocation process can be studied by time-lapse video microscopy. Confocal laser scanning microscopy showed nuclear accumulation in a discrete series of foci, excluding nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although concentration into nuclear foci is not observed. This reproducible pattern of transactivation-competent GR reveals a previously undescribed intranuclear architecture of GR target sites.

Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells.

Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13- to 14-day-old EBs (diameter 510-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.

Circuit-specific alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys.

Age-associated memory impairment occurs frequently in primates. Based on the established importance of both the perforant path and N-methyl-D-aspartate (NMDA) receptors in memory formation, we investigated the glutamate receptor distribution and immunofluorescence intensity within the dentate gyrus of juvenile, adult, and aged macaque monkeys with the combined use of subunit-specific antibodies and quantitative confocal laser scanning microscopy. Here we demonstrate that aged monkeys, compared to adult monkeys, exhibit a 30.6% decrease in the ratio of NMDA receptor subunit 1 (NMDAR1) immunofluorescence intensity within the distal dendrites of the dentate gyrus granule cells, which receive the perforant path input from the entorhinal cortex, relative to the proximal dendrites, which receive an intrinsic excitatory input from the dentate hilus. The intradendritic alteration in NMDAR1 immunofluorescence occurs without a similar alteration of non-NMDA receptor subunits. Further analyses using synaptophysin as a reflection of total synaptic density and microtubule-associated protein 2 as a dendritic structural marker demonstrated no significant difference in staining intensity or area across the molecular layer in aged animals compared to the younger animals. These findings suggest that, in aged monkeys, a circuit-specific alteration in the intradendritic concentration of NMDAR1 occurs without concomitant gross structural changes in dendritic morphology or a significant change in the total synaptic density across the molecular layer. This alteration in the NMDA receptor-mediated input to the hippocampus from the entorhinal cortex may represent a molecular/cellular substrate for age-associated memory impairments.

Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons.

Temporal and spatial changes in the intracellular Ca2+ concentration ([Ca2+]i) were examined in dendrites and somata of rat cerebellar Purkinje neurons by combining whole-cell patch-clamp recording and fast confocal laser-scanning microscopy. In cells loaded via the patch pipette with the high-affinity Ca2+ indicator Calcium Green-1 (Kd approximately 220 nM), a single synaptic climbing fiber response, a so-called complex spike, resulted in a transient elevation of [Ca2+]i that showed distinct differences among various subcellular compartments. With conventional imaging, the Ca2+ signals were prominent in the dendrites and almost absent in the soma. Confocal recordings from the somatic region, however, revealed steep transient increases in [Ca2+]i that were confined to a submembrane shell of 2- to 3-microns thickness. In the central parts of the soma [Ca2+]i increases were much slower and had smaller amplitudes. The kinetics and amplitudes of the changes in [Ca2+]i were analyzed in more detail by using the fast, low-affinity Ca2+ indicator Calcium Green-5N (Kd approximately 17 microM). We found that brief depolarizing pulses produced [Ca2+]i increases in a narrow somatic submembrane shell that resembled those seen in the dendrites. These results provide direct experimental evidence that the surface-to-volume ratio is a critical determinant of the spatiotemporal pattern of Ca2+ signals evoked by synaptic activity in neurons.

Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells.

A hair cell, the sensory receptor of the internal ear, transduces mechanical stimuli into electrical responses. Transduction results from displacement of the hair bundle, a cluster of rod-shaped stereocilia extending from the cell's apical surface. Biophysical experiments indicate that, by producing shear between abutting stereocilia, a bundle displacement directly opens cation-selective transduction channels. Specific models of gating depend on the location of these channels, which has been controversial: although some physiological and immunocytochemical experiments have situated the transduction channels at the hair bundle's top, monitoring of fluorescence signals from the Ca2+ indicator fura-2 has instead suggested that Ca2+ traverses channels at the bundle's base. To examine the site of Ca2+ entry through transduction channels, we used laser-scanning confocal microscopy, with a spatial resolution of < 1 micron and a temporal resolution of < 2 ms, to observe hair cells filled with the indicator fluo-3. An unstimulated hair cell showed a "tip blush" of enhanced fluorescence at the hair bundle's top, which we attribute to Ca2+ permeation through transduction channels open at rest. Upon mechanical stimulation, individual stereocilia displayed increased fluorescence that originated near their tips, then spread toward their bases. Our results confirm that mechanoelectrical transduction occurs near stereociliary tips.

Two types of calcium response limited to single spines in cerebellar Purkinje cells.

Of fundamental importance in understanding neuronal function is the unambiguous determination of the smallest unit of neuronal integration. It was recently suggested that a whole dendritic branchlet, including tens of spines, acts as the fundamental unit in terms of dendritic calcium dynamics in Purkinje cells. By contrast, we demonstrate that the smallest such unit is the single spine. The results show, by two-photon excited fluorescence laser scanning microscopy, that individual spines are capable of independent calcium activation. Moreover, two distinct spine populations were distinguished by their opposite response to membrane hyperpolarization. Indeed, in a subpopulation of spines calcium entry can also occur through a pathway other than voltage-gated channels. These findings challenge the assumption of a unique parallel fiber activation mode and prompt a reevaluation of the level of functional complexity ascribed to single neurons.

Discontinuity Set Extractor

Disponible en Github: https://github.com/adririquelme/DSE

Imaging liver-stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High-speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver-stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.

Using Terrestrial LiDAR to Monitor Erosion within the Gold Basin Landslide Complex, Verlot, WA

In 2014 the United States Forest Service closed the Gold Basin Campground of western Washington in an effort to protect the public from unstable hillslopes directly adjacent to the campground. The Gold Basin Landslide Complex (GBLC) is actively eroding via block fall, dry ravel, and debris flows, which contribute sediment into the South Fork of the Stillaguamish River. This sediment diminishes the salmonid population within the South Fork of the Stillaguamish River by reducing habitable spawning grounds, which is a big concern to the Stillaguamish Tribe of Indians. In this investigation, I quantified patterns of degradation and total volume of sediment erosion from the middle lobe of the GBLC over the period of July 2015 through January 2016 using terrestrial (ground-based) LiDAR (TLS). I characterized site specific stratigraphy and geomorphic processes, and laid the groundwork for future, long-term monitoring of this site. Results of this investigation determined that ~ 4,800m3 of sediment was eroded from the middle lobe of the GBLC during the 6 month study period (July 2015 – January 2016). This erosion likely occurred from debris flows, raveling of poorly sorted sand and gravel deposits and block failures of high plasticity silts and clays, and/or other mass wasting mechanisms. The generalized stratigraphic sequence in the GBLC consists of alternating massive beds of sand and gravel with silts and clays. The low permeability of these silts and clays provide a perfect venue for groundwater to percolate, as I observed during field investigations, which likely contributes to the active instability of the hillslopes. Continued monitoring and mapping of this complex will lead to viable information that could help both the United States Forest Service and the Stillaguamish Tribe.

Observations of Joints Present in the Lawton Clay Member of the Vashon Stade, Exposed at a Coastal Bluff in Discovery Park, Seattle, Washington

The southwest-facing coastal bluff present at Discovery Park, Seattle, Washington, displays distinctive joints throughout the exposed Lawton Clay Member. Exhibiting a characteristic local stratigraphy of permeable advance outwash over the impermeable proglacial lacustrine clay, this bluff is located in an area of Seattle at high risk from landslides. This project addressed the relationship between the joints observed at this coastal bluff and the coherency of the bluff as a whole, through remote sensing and field measurements. Aerial drone photography taken of the bluff was processed through a photogrammetry software to produce a 3-dimensional Structure from Motion model, allowing for a digital manipulation and broad examination of the bluff not possible by foot. Stereonet plots produced from these measurements provided insight into patterns of varying joint strike along a horizontal transect of the observed bluff face. Taken together, these two visualizations provided a better picture of the possible chicken-and-egg interaction of the joints and bluff topography; they demonstrated the likelihood that the joint formation at the bluff was most likely to be primarily influenced by the local topography of the bluff over other sources of possible tensional stress in the immediate area.

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-Lanaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm. (C) 2005 Wiley Periodicals, Inc.

Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development

Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. in the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (K-m 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. in expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression.

ESTUDO DAS DIMENSÕES TRANSVERSAIS DOS ARCOS DENTAIS MANDIBULARES EM INDIVÍDUOS COM DIFERENTES PADRÕES FACIAIS

O presente estudo avaliou as dimensões transversais dos arcos dentais mandibulares em indivíduos com diferentes padrões faciais. A amostra foi constituída por telerradiografias em norma lateral direita e modelos em gesso de 33 indivíduos, leucodermas, em ambos os sexos, com idade entre 13 e 25 anos, na fase de dentição permanente. O Padrão Facial foi obtido pela análise facial subjetiva em fotografias frontal e de perfil de 1500 documentações ortodonticas, foi utilizada análise cefalométrica por meio do ângulo ANB para confirmar o padrão esquelético, o qual deveria coincidir com a classificação de maloclusão de Angle. A amostra foi dividida em três grupos: Grupo I Padrão I, Classe I de Angle e ANB 2,0 o ±0,5o; Grupo II Padrão II, Classe II divisão 1 de Angle e ANB ≥ 4,0, e Grupo III Padrão III, Classe III de Angle e ANB ≥ - 4,5o. As dimensões transversais do arco foram mensuradas após a digitalização dos modelos em gesso pelo Scanner Dental Wings (3D), a partir dos quais foram estabelecidas as distâncias transversais intercanino, inter 1º PM, inter 2º PM, inter 1º M (cúspide mesial e distal), inter 2º M (cúspide mesial e distal), com o auxílio do software Geomagic Studio® 12. As médias e desvio padrão das dimensões transversais foram obtidas, e, para comparação entre os três grupos foi utilizado a Análise de Variância e teste de Tukey. Em todos os testes estatísticos foi adotado nível de significância de 5% (p<0,05). Houve diferença estatística em duas dimensões transversais das 14 avaliadas no arco maxilar na região mesial do segundo molar (p=0,024) e no mandibular na região distal do primeiro molar (p=0,047). Os arcos dentais mandibulares foram semelhantes nos três grupos estudados.