The authors attempted to discover the part the physical setting of the ward plays in patients' recovery by asking patients to take photographs of their ward, its spaces and objects, and then interviewing them about these images in hospital and subsequently in their homes. Photography allowed the patients to identify and capture aspects of the setting that they found salient and provided the photo-elicitation material for the interviews. Based on these data, the authors present (a) a critical discussion of the use of photography as method and its implications for qualitative analysis, (b) an overview of the kinds of image taken with respect to the ward and the course of patients' recovery, and (c) a theoretical analysis, employing Walter Benjamin's use of the concept of mimesis, that understands recovery as a bodily act in response to the shock to the senses that hospitalization and surgery produce.

Two fundamental parameters of the highly dynamic, ultrathin lamellipodia of migrating fibroblasts have been determined-its thickness in living cells (176 +/- 14 nm), by standing-wave fluorescence microscopy, and its F-actin density (1580 +/- 613 microm of F-actin/microm(3)), via image-based photometry. In combination with data from previous studies, we have computed the density of growing actin filament ends at the lamellipodium margin (241 +/- 100/microm) and the maximum force (1.86 +/- 0.83 nN/microm) and pressure (10.5 +/- 4.8 kPa) obtainable via actin assembly. We have used cell deformability measurements (. J. Cell Sci. 44:187-200;. Proc. Natl. Acad. Sci. USA. 79:5327-5331) and an estimate of the force required to stall the polymerization of a single filament (. Proc. Natl. Acad. Sci. USA. 78:5613-5617;. Biophys. J. 65:316-324) to argue that actin assembly alone could drive lamellipodial extension directly.

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGF β 1 is a key upstream transcriptome regulator in AH and induces the use of HNF4 α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4 α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4 α -dependent gene expression. We conclude that targeting TGF β 1 and epigenetic drivers that modulate HNF4 α -dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.

A low-salt extract prepared from human erythrocyte membranes forms a solid gel when purified rabbit muscle G-or F-actin is added to it to give a concentration of -1 mg/ml. This extract contains spectrin, actin, band 4.1, band 4.9, hemoglobin, and several minor components . Pellets obtained by centrifugation of the gelled material at 43,000 g for 10 min contain spectrin, actin, band 4.1, and band 4.9. Although extracts that are diluted severalfold do not gel when actin is added to them, the viscosity of the mixtures increases dramatically over that of G-actin alone, extract alone, or F-actin alone at equivalent concentrations . Heat-denatured extract is completely inactive . Under conditions of physiological ionic strength and pH, formation of this supramolecular structure is inhibited by raising the free calcium ion concentration to micromolar levels . Low-salt extracts prepared by initial extraction at 37°C (and stored at 0°C) gel after actin is added to them only when warmed, whereas extracts prepared by extraction at 0°C are active on ice as well as after warming. Preincubation of the 37'C low-salt extract under conditions that favor conversion of spectrin dimer to tetramer greatly enhances gelation activity at 0°C. Conversely, preincubation of the 0°C low-salt extract under conditions that favor conversion of spectrin tetramer to dimer greatly diminishes gelation activity at 0°C. Spectrin dimers or tetramers are purified from the 37°or 0°C low-salt extract by gel filtration at 4°C over Sepharose 4B . The addition of actin to either purified spectrin dimer (at 32°C) or tetramer (at 0°C or 32°C) results in relatively small increases in viscosity, whereas the addition of actin to a high-molecular-weight complex (HMW complex) containing spectrin, actin, band 4.1, and band 4.9 results in dramatic, calcium-sensitive increases in viscosity. These viscosities are comparable to those obtained with the 37°or 0°C low-salt extracts . The addition of purified band 4.1 to either purified spectrin dimer (at 32°C) or purified spectrin tetramer (at 0°C) plus actin results in large increases in viscosity similar to those observed for the HMW complex and the crude extract, which is in agreement with a recent report by E. Ungewickell, P. M. Bennett, R. Calvert, V. Ohanian, and W. B. Gratzer. 1979 . Nature (Loud.). 280:811-814. We suggest that this spectrin-actin-band 4.1 gel represents a major structural component of the erythrocyte cytoskeleton .J . CELL BIOLOGY

Recent improvements in target discovery and high throughput screening (HTS) have increased the pressure at key points along the drug discovery pipeline. High-content screening (HCS) was developed to ease bottlenecks that have formed at target validation and lead optimization points in the pipeline. HCS defines the role of targets in cell functions by combining fluorescence-based reagents with the ArrayScan™ System to automatically extract temporal and spatial information about target activities within cells. The ArrayScan System is a tabletop instrument that includes optics for subcellular resolution of fluorescence signals from many cells in a field within a well of a microtiter plate. One demonstrated application is a high-content screen designed to measure the drug-induced transport of a green fluorescent protein-human glucocorticoid receptor chimeric protein from the cytoplasm to the nucleus of human tumor cells. A high-content screen is also described for the multiparametric measurement of apoptosis. This single screen provides measurements of nuclear size and shape changes, nuclear DNA content, mitochondrial potential, and actin-cytoskeletal rearrangements during drug-induced programmed cell death. The next generation HCS system is a miniaturized screening platform, the CellChip™ System, that will increase the throughput of HCS, while integrating HCS with HTS on the same platform.

Abstract. The formation of protrusions at the leading edge of the cell is an essential step in fibroblast locomotion. Using fluorescent analogue cytochemistry, ratio imaging, multiple parameter analysis, and fluorescence photobleaching recovery, the distribution of actin and myosin was examined in the same protrusions at the leading edge of live, locomoting cells during wound-healing in vitro. We have previously defined two temporal stages of the formation of protrusions: (a) initial protrusion and (b) established protrusion (Fisher et al., 1988). Actin was slightly concentrated in initial protrusions, while myosin was either totally absent or present at extremely low levels at the base of the initial protrusions. In contrast, established protrusions contained diffuse actin and actin microspikes, as well as myosin in both diffuse and structured forms. Actin and myosin were also localized along concave transverse fibers near the base of initial and established protrusions. The dynamics of myosin penetration into a relatively stable, established protrusion was demonstrated by recording sequential images over time. Myosin was shown to be absent from an initial protrusion, but diffuse and punctate myosin was detected in the same protrusion within 1-2 min. Fluorescence photobleaching recovery indicated that myosin was 100% immobile in the region behind the leading edge containing transverse fibers, in comparison to the 21% immobile fraction detected in the perinuclear region. Possible explanations of the delayed penetration of myosin into established protrusions and the implications on the mechanism of protrusion are discussed.

A fundamental problem in cell motility is to define the state of assembly and interaction of the major cytoskeletal and contractile proteins both in vitro and in vivo . One example is the role played by actin assembly and disassembly during motile events . To date, actin assembly has usually been assayed indirectly both in vitro and in vivo. Experiments using viscosity, light scattering, and electron microscopy yield results that could be interpreted in several ways (2,10,12,13,20,44; see Taylor and Condeelis [32] and Uyemura and Spudich [37] for reviews) . Furthermore, more direct measurements of assembly using sedimentation assays (3,25) can only be applied in vitro. Therefore, techniques must be developed that would yield direct evidence for the state of actin assembly and interaction with specific molecules even in living cells.Fluorescence spectroscopy has already generated some information in vitro on the assembly of actin (4, 6, 40), the structure of actin (14,15,19,21,31), and the interaction of actin with associated proteins (6,23,24).I Recently, the technique of molecular cytochemistry (33,40; see Taylor and Wang [39] for a review) has opened the possibility of making spectroscopic measurements of fluorescently labeled actin in living cells. Actin labeled with 5-iodoacetamidofluorescein has been studied initially, because the site of labeling is known, the extinction coefficient is high, and it is excited by a relatively long wavelength of light (495 nm), which minimizes autofluorescence and cellular damage (40) .Dr. P. Detmers kindly provided D. L. Taylor with a copy of her manuscript (6) before publication. ABSTRACTFluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide) . Donor-labeled actin and acceptor-labeled actin were coassembled . The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is -35 A, which means that this site is located near the outer surface of the filament . The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.Fluorescence energy transfer (7, 28) is a powerful technique for studying the structure and dynamics of molecular...

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