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At least 55 records · Page 3

Distribution of expansins in graviresponding maize roots

To test if expansins, wall loosening proteins that disrupt binding between microfibrils and cell wall matrix, participate in the differential elongation of graviresponding roots, Zea mays L. cv. Merit roots were gravistimulated and used for immunolocalization with anti-expansin. Western blots showed cross-reaction with two proteins of maize, one of the same mass as cucumber expansin (29 kDa), the second slightly larger (32 kDa). Maize roots contained mainly the larger protein, but both were found in coleoptiles. The expansin distribution in cucumber roots and hypocotyls was similar to the distribution in maize. Roots showed stronger expansin signals on the expanding convex side than the concave flank as early as 30 min after gravistimulation. Treatment with brefeldin A, a vesicle transport inhibitor, or the auxin transport inhibitor, naphthylphthalamic acid, showed delayed graviresponse and the appearance of differential staining. Our results indicate that expansins may be transported and secreted to cell walls via vesicles and function in wall expansion.

Non-NASA Center↗

Growing B Lymphocytes in a Three-Dimensional Culture System

A three-dimensional (3D) culture system for growing long-lived B lymphocytes has been invented. The capabilities afforded by the system can be expected to expand the range of options for immunological research and related activities, including testing of immunogenicity of vaccine candidates in vitro, generation of human monoclonal antibodies, and immunotherapy. Mature lymphocytes, which are the effectors of adaptive immune responses in vertebrates, are extremely susceptible to apoptotic death, and depend on continuous reception of survival-inducing stimulation (in the forms of cytokines, cell-to-cell contacts, and antigen receptor signaling) from the microenvironment. For this reason, efforts to develop systems for long-term culture of functional, non-transformed and non-activated mature lymphocytes have been unsuccessful until now. The bone-marrow microenvironment supports the growth and differentiation of many hematopoietic lineages, in addition to B-lymphocytes. Primary bone-marrow cell cultures designed to promote the development of specific cell types in vitro are highly desirable experimental systems, amenable to manipulation under controlled conditions. However, the dynamic and complex network of stromal cells and insoluble matrix proteins is disrupted in prior plate- and flask-based culture systems, wherein the microenvironments have a predominantly two-dimensional (2D) character. In 2D bone-marrow cultures, normal B-lymphoid cells become progressively skewed toward precursor B-cell populations that do not retain a normal immunophenotype, and such mature B-lymphocytes as those harvested from the spleen or lymph nodes do not survive beyond several days ex vivo in the absence of mitogenic stimulation. The present 3D culture system is a bioreactor that contains highly porous artificial scaffolding that supports the long-term culture of bone marrow, spleen, and lymph-node samples. In this system, unlike in 2D culture systems, B-cell subpopulations developing within 3D cultures that have been modified to foster lymphopoiesis retain an immunophenotype that closely recapitulates cells in fresh bone marrow harvests. The 3D culture system has been found to be capable of supporting long-lived (8 weeks) populations of B and T lymphocytes from peripheral lymphoid organs, in the absence of activation signals, to an extent not achievable by conventional culture techniques. Interestingly, it has been found that 3D-culture B cells display a phenotype that has characteristics of both B1a and B2 cells. These promising preliminary observations suggest that the 3D culture system could be used with success in the study of peripheral-B-lymphocyte biology and in the development of biotechnological techniques and processes.

Wu, J. H. David↗

Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

Non-NASA Center↗

Hydrodynamically induced fluid transfer and non-convective double-diffusion in microgravity sliding solvent diffusion cells

Microgravity can provide a diffusion-dominated environment for double-diffusion and diffusion-reaction experiments otherwise disrupted by buoyant convection or sedimentation. In sliding solvent diffusion cells, a diffusion interface between two liquid columns is achieved by aligning two offset sliding wells. Fluid in contact with the sliding lid of the cavities is subjected to an applied shear stress. The momentum change by the start/stop action of the well creates an additional hydrodynamical force. In microgravity, these viscous and inertial forces are sufficiently large to deform the diffusion interface and induce hydrodynamic transfer between the wells. A series of KC-135 parabolic flight experiments were conducted to characterize these effects and establish baseline data for microgravity diffusion experiments. Flow visualizations show the diffusion interface to be deformed in a sinusoidal fashion following well alignment. After the wells were separated again in a second sliding movement, the total induced liquid transfer was determined and normalized by the well aspect ratio. The normalized transfer decreased linearly with Reynolds number from 3.3 to 4.0% (w/v) for Re = 0.4 (Stokes flow) to a minimum of 1.0% for Re = 23 to 30. Reynolds numbers that provide minimum induced transfers are characterized by an interface that is highly deformed and unsuitable for diffusion measurements. Flat diffusion interfaces acceptable for diffusion measurements are obtained with Reynolds numbers on the order of 7 to 10. Microgravity experiments aboard a sounding rocket flight verified counterdiffusion of different solutes to be diffusion dominated. Ground control experiments showed enhanced mixing by double-diffusive convection. Careful selection of experimental parameters improves initial conditions and minimizes induced transfer rates.

Pollmann, Konrad W.↗

Rapid flow-induced responses in endothelial cells

Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

Non-NASA Center↗

Isoperiodic neuronal activity in suprachiasmatic nucleus of the rat

A subpopulation of neurons in the suprachiasmatic nucleus (SCN) is shown here to exhibit isoperiodic bursting activity. The period of discharge in these cells may be lengthened or the periodicity may be transiently disrupted by photic stimulation. It is suggested that many, if not all, of these cells are vasoactive intestinal polypeptide (VIP) neurons. It is shown that the ultradian periodicity of these cells, estimates of the VIP neuron population size in the SCN, effects of partial lesions on tau (period), and estimates of the phase stability of SCN-driven circadian rhythms are consistent with a strongly coupled, multioscillator model of circadian rhythmicity, in which the oscillator population constitutes a restricted subset of the SCN neuronal population.

NASA Discipline Regulatory Physiology↗

Three-dimensional co-culture process

The present invention relates to a 3-dimensional co-culture process, more particularly to methods or co-culturing at least two types of cells in a culture environment, either in space or in unit gravity, with minimum shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region to form 3-dimensional tissue-like structures. Several examples of multicellular 3-dimensional experiences are included. The protocol and procedure are also set forth. The process allows simultaneous culture of multiple cell types and supporting substrates in a manner which does not disrupt the 3-dimensional spatial orientation of these components. The co-cultured cells cause a mutual induction effect which mimics the natural hormonal signals and cell interactions found in the intact organism. This causes the tissues to differentiate and form higher 3-dimensional structures such as glands, junctional complexes polypoid geometries, and microvilli which represent the corresponding in-vitro structures to a greater degree than when the cell types are cultured individually or by conventional processes. This process was clearly demonstrated for the case of two epithelial derived colon cancer lines, each co-cultured with normal human fibroblasts and with microcarrier bead substrates. The results clearly demonstrate increased 3-dimensional tissue-like structure and biochemical evidence of an increased differentiation state. With the present invention a variety of cells may be co-cultured to produce tissue which has 3-dimensionality and has some of the characteristics of in-vitro tissue. The process provides enhanced 3-dimensional tissue which create a multicellular organoid differentiation model.

Wolf, David A.↗

Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells

The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985. c 2001. Elsevier Science Ltd. All rights reserved.

NASA Discipline Cell Biology↗

Transduction-like gene transfer in the methanogen Methanococcus voltae

Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

Methanococcus/genetics/growth & development/metabo↗

Columella cells revisited: novel structures, novel properties, and a novel gravisensing model

A hundred years of research has not produced a clear understanding of the mechanism that transduces the energy associated with the sedimentation of starch-filled amyloplast statoliths in root cap columella cells into a growth response. Most models postulate that the statoliths interact with microfilaments (MF) to transmit signals to the plasma membrane (or ER), or that sedimentation onto these organelles produces the signals. However, no direct evidence for statolith-MF links has been reported, and no asymmetric structures of columella cells have been identified that might explain how a root turned by 90 degrees knows which side is up. To address these and other questions, we have (1) quantitatively examined the effects of microgravity on the size, number, and spatial distribution of statoliths; (2) re-evaluated the ultrastructure of columella cells in high-pressure frozen/freeze-substituted roots; and (3) followed the sedimentation dynamics of statolith movements in reoriented root tips. The findings have led to the formulation of a new model for the gravity-sensing apparatus of roots, which envisages the cytoplasm pervaded by an actin-based cytoskeletal network. This network is denser in the ER-devoid central region of the cell than in the ER-rich cell cortex and is coupled to receptors in the plasma membrane. Statolith sedimentation is postulated to disrupt the network and its links to receptors in some regions of the cell cortex, while allowing them to reform in other regions and thereby produce a directional signal.

NASA Discipline Cell Biology↗

Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation

Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.

Non-NASA Center↗

Three-Dimensional Human Bronchial-Tracheal Epithelial Tissue-Like Assemblies (TLAs) as Hosts for Severe Acute Respiratory Syndrome (SARS)-CoV Infection

A three-dimensional (3-D) tissue-like assembly (TLA) of human bronchial-tracheal mesenchymal (HBTC) cells with an overlay of human bronchial epithelial (BEAS-2B) cells was constructed using a NASA Bioreactor to survey the infectivity of SARS-CoV. This TLA was inoculated with a low passage number Urbani strain of SARS-CoV. At selected intervals over a 10-day period, media and cell aliquots of the 3-D TLA were harvested for viral titer assay and for light and electron microscopy examination. All viral titer assays were negative in both BEAS-2B two-dimensional monolayer and TLA. Light microscopy immunohistochemistry demonstrated antigen-antibody reactivity with anti-SARS-CoV polyclonal antibody to spike and nuclear proteins on cell membranes and cytoplasm. Coronavirus Group 2 cross-reactivity was demonstrated by positive reaction to anti-FIPV 1 and anti-FIPV 1 and 2 antibodies. TLA examination by transmission electron microscopy indicated increasing cytoplasmic vacuolation with numerous electron-dense bodies measuring 45 to 270 nm from days 4 through 10. There was no evidence of membrane blebbing, membrane duplication, or fragmentation of organelles in the TLAs. However, progressive disruption of endoplasmic reticulum was observed throughout the cells. Antibody response to SARS-CoV specific spike and nucleocapsid glycoproteins, cross-reactivity with FIPV antibodies, and the cytoplasmic pathology suggests this HBTE TLA model is permissive to SARS-CoV infection.

Suderman, M. T.↗

Experiment K-6-09. Morphological and biochemical investigation of microgravity-induced nerve and muscle breakdown. Part 1: Investigation of nerve and muscle breakdown during spaceflight; Part 2: Biochemical analysis of EDL and PLT muscles

The present findings on rat hindlimb muscles suggest that skeletal muscle weakness induced by prolonged spaceflight can result from a combination of muscle fiber atrophy, muscle fiber segmental necrosis, degeneration of motor nerve terminals and destruction of microcirculatory vessels. Damage was confined to the red adductor longus (AL) and soleus muscles. The midbelly region of the AL muscle had more segmental necrosis and edema than the ends. Macrophages and neutrophils were the major mononucleated cells infiltrating and phagocytosing the cellular debris. Toluidine blue-positive mast cells were significantly decreased in Flight AL muscles compared to controls; this indicated that degranulation of mast cells contributed to tissue edema. Increased ubiquitination of disrupted myofibrils may have promoted myofilament degradation. Overall, mitochondria content and SDH activity were normal, except for a decrease in the subsarcolemmal region. The myofibrillar ATPase activity shifted toward the fast type in the Flight AL muscles. Some of the pathological changes may have occurred or been exacerbated during the 2 day postflight period of readaptation to terrestrial gravity. While simple atrophy should be reversible by exercise, restoration of pathological changes depends upon complex processes of regeneration by stem cells. Initial signs of muscle and nerve fiber regeneration were detected. Even though regeneration proceeds on Earth, the space environment may inhibit repair and cause progressive irreversible deterioration during long term missions. Muscles obtained from Flight rats sacrificed immediately (within a few hours) after landing are needed to distinguish inflight changes from postflight readaptation.

Riley, D. A.↗

Skeletal muscle fiber, nerve, and blood vessel breakdown in space-flown rats

Histochemical and ultrastructural analyses were performed postflight on hind limb skeletal muscles of rats orbited for 12.5 days aboard the unmanned Cosmos 1887 biosatellite and returned to Earth 2 days before sacrifice. The antigravity adductor longus (AL), soleus, and plantaris muscles atrophied more than the non-weight-bearing extensor digitorum longus, and slow muscle fibers were more atrophic than fast fibers. Muscle fiber segmental necrosis occurred selectively in the AL and soleus muscles; primarily, macrophages and neutrophils infiltrated and phagocytosed cellular debris. Granule-rich mast cells were diminished in flight AL muscles compared with controls, indicating the mast cell secretion contributed to interstitial tissue edema. Increased ubiquitination of disrupted myofibrils implicated ubiquitin in myofilament degradation. Mitochondrial content and succinic dehydrogenase activity were normal, except for subsarcolemmal decreases. Myofibrillar ATPase activity of flight AL muscle fibers shifted toward the fast type. Absence of capillaries and extravasation of red blood cells indicated failed microcirculation. Muscle fiber regeneration from activated satellite cells was detected. About 17% of the flight AL end plates exhibited total or partial denervation. Thus, skeletal muscle weakness associated with spaceflight can result from muscle fiber atrophy and segmental necrosis, partial motor denervation, and disruption of the microcirculation.

NASA Experiment Number COS 1887-6↗

Cell prestress. II. Contribution of microtubules

The tensegrity model hypothesizes that cytoskeleton-based microtubules (MTs) carry compression as they balance a portion of cell contractile stress. To test this hypothesis, we used traction force microscopy to measure traction at the interface of adhering human airway smooth muscle cells and a flexible polyacrylamide gel substrate. The prediction is that if MTs balance a portion of contractile stress, then, upon their disruption, the portion of stress balanced by MTs would shift to the substrate, thereby causing an increase in traction. Measurements were done first in maximally activated cells (10 microM histamine) and then again after MTs had been disrupted (1 microM colchicine). We found that after disruption of MTs, traction increased on average by approximately 13%. Because in activated cells colchicine induced neither an increase in intracellular Ca(2+) nor an increase in myosin light chain phosphorylation as shown previously, we concluded that the observed increase in traction was a result of load shift from MTs to the substrate. In addition, energy stored in the flexible substrate was calculated as work done by traction on the deformation of the substrate. This result was then utilized in an energetic analysis. We assumed that cytoskeleton-based MTs are slender elastic rods supported laterally by intermediate filaments and that MTs buckle as the cell contracts. Using the post-buckling equilibrium theory of Euler struts, we found that energy stored during buckling of MTs was quantitatively consistent with the measured increase in substrate energy after disruption of MTs. This is further evidence supporting the idea that MTs are intracellular compression-bearing elements.

Non-NASA Center↗

Retinal changes in rats flown on Cosmos 936 - A cosmic ray experiment

Ten rats, five centrifuged during flight to simulate gravity and five stationary in flight and experiencing hypogravity, orbited the Earth. No differences were noted between flight-stationary and flight-centrifuged animals, but changes were seen between these two groups and ground controls. Morphological alterations were observed comparable to those in the experiment flown on Cosmos 782 and to the retinal cells exposed to high-energy particles at Berkeley. Affected cells in the outer nuclear layer showed swelling, clearing of cytoplasm, and disruption of the membranes. Tissue channels were again found, similar to those seen on 782. After space flight, preliminary data indicated an increase in cell size in montages of the nuclear layer of both groups of flight animals. This experiment shows that weightlessness and environmental conditions other than cosmic radiation do not contribute to the observed damage of retinal cells.

Philpott, D. E.↗

High aspect reactor vessel and method of use

An improved bio-reactor vessel and system useful for carrying out mammalian cell growth in suspension in a culture media are presented. The main goal of the invention is to grow and maintain cells under a homogeneous distribution under acceptable biochemical environment of gas partial pressures and nutrient levels without introducing direct agitation mechanisms or associated disruptive mechanical forces. The culture chamber rotates to maintain an even distribution of cells in suspension and minimizes the length of a gas diffusion path. The culture chamber design is presented and discussed.

Wolf, David A.↗

Effect of hexylene glycol-altered microtubule distributions on cytokinesis and polar lobe formation in fertilized eggs of Ilyanassa obsoleta

Some effects of gravity on early morphogenesis are correlated with microtubule locations within cells. During first cleavage in Ilyanassa obsoleta embryos, a transitory polar lobe constriction forms and then relaxes, allowing the polar lobe to merge with one daughter cell. If the polar lobe is equally divided or removed, morphogenesis is severely disrupted. To examine microtuble locations during early Ilyanassa development, eggs were fixed and stained for polymerized alpha-tubulin during first cleavage. The mitotic apparatus assembles at the animal pole. The cleavage furrow forms between the asters, constricting to a stabilized intercellular bridge encircling midbody-bound microtubules, whereas the polar lobe constriction forms below and parallel to the spindle, constricting to a transitory intercellular bridge encircling no detectable microtubules. At metaphase an alpha-tubulin epitope is distributed throughout the spindle, whereas a beta-tubulin epitope is present predominantly in the asters. Incubation in hexylene glycol, a drug that increases microtubule polymerization, during mitosis causes the polar lobe constriction to tighten around polymerized alpha-tubulin and remain stably constricted. If hexylene glycol is removed, alpha-tubulin staining disappears from the polar lobe constriction, which relaxes, whereas microtubules remain in the cleavage furrow, which remains constricted. These observations suggest that asymmetric distribution of microtubules affects early Ilyanassa cleavage patterns, and that continued presence of microtubules extending through an intercellular bridge is important for stabilization of the bridge constriction prior to completion of cytokinesis. These data provide the basis for further analysis of the role of microtubules in possible microgravity disruptions of Ilyanassa development.

NASA Discipline Cell Biology↗