Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.

Rapid and quantitative detection of the microbial spoilage of meat by Fourier Transform Infrared Spectroscopy and machine learning

Ellis, D. I., Broadhurst, D., Kell, D. B., Rowland, J. J., Goodacre, R. (2002). Rapid and quantitative detection of the microbial spoilage of meat by Fourier Transform Infrared Spectroscopy and machine learning. ? Applied and Environmental Microbiology, 68, (6), 2822-2828 Sponsorship: BBSRC

PHOTOREFRACTIVE CRYSTAL-BASED ACOUSTO-OPTIC IMAGING IN THE NEAR-INFRARED AND ITS APPLICATIONS

Acousto-optic (AO) sensing and imaging (AOI) is a dual-wave modality that combines ultrasound with diffusive light to measure and/or image the optical properties of optically diffusive media, including biological tissues such as breast and brain. The light passing through a focused ultrasound beam undergoes a phase modulation at the ultrasound frequency that is detected using an adaptive interferometer scheme employing a GaAs photorefractive crystal (PRC). The PRC-based AO system operating at 1064 nm is described, along with the underlying theory, validating experiments, characterization, and optimization of this sensing and imaging apparatus. The spatial resolution of AO sensing, which is determined by spatial dimensions of the ultrasound beam or pulse, can be sub-millimeter for megahertz-frequency sound waves.A modified approach for quantifying the optical properties of diffuse media with AO sensing employs the ratio of AO signals generated at two different ultrasound focal pressures. The resulting “pressure contrast signal” (PCS), once calibrated for a particular set of pressure pulses, yields a direct measure of the spatially averaged optical transport attenuation coefficient within the interaction volume between light and sound. This is a significant improvement over current AO sensing methods since it produces a quantitative measure of the optical properties of optically diffuse media without a priori knowledge of the background illumination. It can also be used to generate images based on spatial variations in both optical scattering and absorption. Finally, the AO sensing system is modified to monitor the irreversible optical changes associated with the tissue heating from high intensity focused ultrasound (HIFU) therapy, providing a powerful method for noninvasively sensing the onset and growth of thermal lesions in soft tissues. A single HIFU transducer is used to simultaneously generate tissue damage and pump the AO interaction. Experimental results performed in excised chicken breast demonstrate that AO sensing can identify the onset and growth of lesion formation in real time and, when used as feedback to guide exposure parameters, results in more predictable lesion formation.

EVALUATION OF HARMONIC MOTION ELASTOGRAPHY AND ACOUSTO-­OPTIC IMAGING FOR MONITORING LESION FORMATION BY HIGH INTENSITY FOCUSED ULTRASOUND

Malignant or benign tumors may be ablated with high‐intensity focused ultrasound (HIFU). This technique, known as focused ultrasound surgery (FUS), has been actively investigated for decades, but slow to be implemented and difficult to control due to lack of real‐time feedback during ablation. Two methods of imaging and monitoring HIFU lesions during formation were implemented simultaneously, in order to investigate the efficacy of each and to increase confidence in the detection of the lesion. The first, Acousto‐Optic Imaging (AOI) detects the increasing optical absorption and scattering in the lesion. The intensity of a diffuse optical field in illuminated tissue is mapped at the spatial resolution of an ultrasound focal spot, using the acousto‐optic effect. The second, Harmonic Motion Imaging (HMI), detects the changing stiffness in the lesion. The HIFU beam is modulated to force oscillatory motion in the tissue, and the amplitude of this motion, measured by ultrasound pulse‐echo techniques, is influenced by the stiffness. Experiments were performed on store‐bought chicken breast and freshly slaughtered bovine liver. The AOI results correlated with the onset and relative size of forming lesions much better than prior knowledge of the HIFU power and duration. For HMI, a significant artifact was discovered due to acoustic nonlinearity. The artifact was mitigated by adjusting the phase of the HIFU and imaging pulses. A more detailed model of the HMI process than previously published was made using finite element analysis. The model showed that the amplitude of harmonic motion was primarily affected by increases in acoustic attenuation and stiffness as the lesion formed and the interaction of these effects was complex and often counteracted each other. Further biological variability in tissue properties meant that changes in motion were masked by sample‐to‐sample variation. The HMI experiments predicted lesion formation in only about a quarter of the lesions made. In simultaneous AOI/HMI experiments it appeared that AOI was a more robust method for lesion detection.

A Neural Model of Surface Perception: Lightness, Anchoring, and Filling-in

This article develops a neural model of how the visual system processes natural images under variable illumination conditions to generate surface lightness percepts. Previous models have clarified how the brain can compute the relative contrast of images from variably illuminate scenes. How the brain determines an absolute lightness scale that "anchors" percepts of surface lightness to us the full dynamic range of neurons remains an unsolved problem. Lightness anchoring properties include articulation, insulation, configuration, and are effects. The model quantatively simulates these and other lightness data such as discounting the illuminant, the double brilliant illusion, lightness constancy and contrast, Mondrian contrast constancy, and the Craik-O'Brien-Cornsweet illusion. The model also clarifies the functional significance for lightness perception of anatomical and neurophysiological data, including gain control at retinal photoreceptors, and spatioal contrast adaptation at the negative feedback circuit between the inner segment of photoreceptors and interacting horizontal cells. The model retina can hereby adjust its sensitivity to input intensities ranging from dim moonlight to dazzling sunlight. A later model cortical processing stages, boundary representations gate the filling-in of surface lightness via long-range horizontal connections. Variants of this filling-in mechanism run 100-1000 times faster than diffusion mechanisms of previous biological filling-in models, and shows how filling-in can occur at realistic speeds. A new anchoring mechanism called the Blurred-Highest-Luminance-As-White (BHLAW) rule helps simulate how surface lightness becomes sensitive to the spatial scale of objects in a scene. The model is also able to process natural images under variable lighting conditions.

A Neuromorphic Model for Achromatic and Chromatic Surface Representation of Natural Images

This study develops a neuromorphic model of human lightness perception that is inspired by how the mammalian visual system is designed for this function. It is known that biological visual representations can adapt to a billion-fold change in luminance. How such a system determines absolute lightness under varying illumination conditions to generate a consistent interpretation of surface lightness remains an unsolved problem. Such a process, called "anchoring" of lightness, has properties including articulation, insulation, configuration, and area effects. The model quantitatively simulates such psychophysical lightness data, as well as other data such as discounting the illuminant, the double brilliant illusion, and lightness constancy and contrast effects. The model retina embodies gain control at retinal photoreceptors, and spatial contrast adaptation at the negative feedback circuit between mechanisms that model the inner segment of photoreceptors and interacting horizontal cells. The model can thereby adjust its sensitivity to input intensities ranging from dim moonlight to dazzling sunlight. A new anchoring mechanism, called the Blurred-Highest-Luminance-As-White (BHLAW) rule, helps simulate how surface lightness becomes sensitive to the spatial scale of objects in a scene. The model is also able to process natural color images under variable lighting conditions, and is compared with the popular RETINEX model.

Fixational Instability and Natural Image Statistics: Implications for Early Visual Representations

Under natural viewing conditions small movements of the eye, head, and body prevent the maintenance of a steady direction of gaze. It is known that stimuli tend to fade when they a restabilized on the retina for several seconds. However; it is unclear whether the physiological motion of the retinal image serves a visual purpose during the brief periods of natural visual fixation. This study examines the impact of fixational instability on the statistics of the visua1 input to the retina and on the structure of neural activity in the early visual system. We show that fixational instability introduces a component in the retinal input signals that in the presence of natural images, lacks spatial correlations. This component strongly influences neural activity in a model of the LGN. It decorrelates cell responses even if the contrast sensitivity functions of simulated cells arc not perfectly tuned to counterbalance the power-law spectrum of natural images. A decorrelation of neural activity at the early stages of the visual system has been proposed to be beneficial for discarding statistical redundancies in the input signals. The results of this study suggest that fixational instability might contribute to establishing efficient representations of natural stimuli.

Temporal Dynamics of Decision-Making during Motion Perception in the Visual Cortex

How does the brain make decisions? Speed and accuracy of perceptual decisions covary with certainty in the input, and correlate with the rate of evidence accumulation in parietal and frontal cortical "decision neurons." A biophysically realistic model of interactions within and between Retina/LGN and cortical areas V1, MT, MST, and LIP, gated by basal ganglia, simulates dynamic properties of decision-making in response to ambiguous visual motion stimuli used by Newsome, Shadlen, and colleagues in their neurophysiological experiments. The model clarifies how brain circuits that solve the aperture problem interact with a recurrent competitive network with self-normalizing choice properties to carry out probablistic decisions in real time. Some scientists claim that perception and decision-making can be described using Bayesian inference or related general statistical ideas, that estimate the optimal interpretation of the stimulus given priors and likelihoods. However, such concepts do not propose the neocortical mechanisms that enable perception, and make decisions. The present model explains behavioral and neurophysiological decision-making data without an appeal to Bayesian concepts and, unlike other existing models of these data, generates perceptual representations and choice dynamics in response to the experimental visual stimuli. Quantitative model simulations include the time course of LIP neuronal dynamics, as well as behavioral accuracy and reaction time properties, during both correct and error trials at different levels of input ambiguity in both fixed duration and reaction time tasks. Model MT/MST interactions compute the global direction of random dot motion stimuli, while model LIP computes the stochastic perceptual decision that leads to a saccadic eye movement.

Simulations of X and Y Retinal Ganglion Cell Behavior with a Nonlinear Push-pull Model of Spatiotemporal Retinal Processing

This article describes a nonlinear model of neural processing in the vertebrate retina, comprising model photoreceptors, model push-pull bipolar cells, and model ganglion cells. Previous analyses and simulations have shown that with a choice of parameters that mimics beta cells, the model exhibits X-like linear spatial summation (null response to contrast-reversed gratings) in spite of photoreceptor nonlinearities; on the other hand, a choice of parameters that mimics alpha cells leads to Y-like frequency doubling. This article extends the previous work by showing that the model can replicate qualitatively many of the original findings on X and Y cells with a fixed choice of parameters. The results generally support the hypothesis that X and Y cells can be seen as functional variants of a single neural circuit. The model also suggests that both depolarizing and hyperpolarizing bipolar cells converge onto both ON and OFF ganglion cell types. The push-pull connectivity enables ganglion cells to remain sensitive to deviations about the mean output level of nonlinear photoreceptors. These and other properties of the push-pull model are discussed in the general context of retinal processing of spatiotemporal luminance patterns.

Miniature Eye Movements Enhance Fine Spatial Details

Our eyes are constantly in motion. Even during visual fixation, small eye movements continually jitter the location of gaze. It is known that visual percepts tend to fade when retinal image motion is eliminated in the laboratory. However, it has long been debated whether, during natural viewing, fixational eye movements have functions in addition to preventing the visual scene from fading. In this study, we analysed the influence in humans of fixational eye movements on the discrimination of gratings masked by noise that has a power spectrum similar to that of natural images. Using a new method of retinal image stabilization18, we selectively eliminated the motion of the retinal image that normally occurs during the intersaccadic intervals of visual fixation. Here we show that fixational eye movements improve discrimination of high spatial frequency stimuli, but not of low spatial frequency stimuli. This improvement originates from the temporal modulations introduced by fixational eye movements in the visual input to the retina, which emphasize the high spatial frequency harmonics of the stimulus. In a natural visual world dominated by low spatial frequencies, fixational eye movements appear to constitute an effective sampling strategy by which the visual system enhances the processing of spatial detail.

Neuroprotection resulting from insufficiency of RANBP2 is associated with the modulation of protein and lipid homeostasis of functionally diverse but linked pathways in response to oxidative stress.

Oxidative stress is a deleterious stressor associated with a plethora of disease and aging manifestations, including neurodegenerative disorders, yet very few factors and mechanisms promoting the neuroprotection of photoreceptor and other neurons against oxidative stress are known. Insufficiency of RAN-binding protein-2 (RANBP2), a large, mosaic protein with pleiotropic functions, suppresses apoptosis of photoreceptor neurons upon aging and light-elicited oxidative stress, and promotes age-dependent tumorigenesis by mechanisms that are not well understood. Here we show that, by downregulating selective partners of RANBP2, such as RAN GTPase, UBC9 and ErbB-2 (HER2; Neu), and blunting the upregulation of a set of orphan nuclear receptors and the light-dependent accumulation of ubiquitylated substrates, light-elicited oxidative stress and Ranbp2 haploinsufficiency have a selective effect on protein homeostasis in the retina. Among the nuclear orphan receptors affected by insufficiency of RANBP2, we identified an isoform of COUP-TFI (Nr2f1) as the only receptor stably co-associating in vivo with RANBP2 and distinct isoforms of UBC9. Strikingly, most changes in proteostasis caused by insufficiency of RANBP2 in the retina are not observed in the supporting tissue, the retinal pigment epithelium (RPE). Instead, insufficiency of RANBP2 in the RPE prominently suppresses the light-dependent accumulation of lipophilic deposits, and it has divergent effects on the accumulation of free cholesterol and free fatty acids despite the genotype-independent increase of light-elicited oxidative stress in this tissue. Thus, the data indicate that insufficiency of RANBP2 results in the cell-type-dependent downregulation of protein and lipid homeostasis, acting on functionally interconnected pathways in response to oxidative stress. These results provide a rationale for the neuroprotection from light damage of photosensory neurons by RANBP2 insufficiency and for the identification of novel therapeutic targets and approaches promoting neuroprotection.

Intraoperative spectral domain optical coherence tomography for vitreoretinal surgery.

We demonstrate in vivo human retinal imaging using an intraoperative microscope-mounted optical coherence tomography system (MMOCT). Our optomechanical design adapts an Oculus Binocular Indirect Ophthalmo Microscope (BIOM3), suspended from a Leica ophthalmic surgical microscope, with spectral domain optical coherence tomography (SD-OCT) scanning and relay optics. The MMOCT enables wide-field noncontact real-time cross-sectional imaging of retinal structure, allowing for SD-OCT augmented intrasurgical microscopy for intraocular visualization. We experimentally quantify the axial and lateral resolution of the MMOCT and demonstrate fundus imaging at a 20Hz frame rate.

Automatic segmentation of seven retinal layers in SDOCT images congruent with expert manual segmentation.

Segmentation of anatomical and pathological structures in ophthalmic images is crucial for the diagnosis and study of ocular diseases. However, manual segmentation is often a time-consuming and subjective process. This paper presents an automatic approach for segmenting retinal layers in Spectral Domain Optical Coherence Tomography images using graph theory and dynamic programming. Results show that this method accurately segments eight retinal layer boundaries in normal adult eyes more closely to an expert grader as compared to a second expert grader.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein).

The beta-adrenergic receptor kinase is an enzyme, possibly analogous to rhodopsin kinase, that multiply phosphorylates the beta-adrenergic receptor only when it is occupied by stimulatory agonists. Since this kinase may play an important role in mediating the process of homologous, or agonist-specific, desensitization, we investigated the functional consequences of receptor phosphorylation by the kinase and possible analogies with the mechanism of action of rhodopsin kinase. Pure hamster lung beta 2-adrenergic receptor, reconstituted in phospholipid vesicles, was assessed for its ability to mediate agonist-promoted stimulation of the GTPase activity of coreconstituted stimulatory guanine nucleotide-binding regulatory protein. When the receptor was phosphorylated by partially (approximately 350-fold) purified preparations of beta-adrenergic receptor kinase, as much as 80% inactivation of its functional activity was observed. However, the use of more highly purified enzyme preparations led to a dramatic decrease in the ability of phosphorylation to inactivate the receptor such that pure enzyme preparations (approximately 20,000-fold purified) caused only minimal (approximately 1off/- 7%) inactivation. Addition of pure retinal arrestin (48-kDa protein or S antigen), which is involved in enhancing the inactivating effect of rhodopsin phosphorylation by rhodopsin kinase, led to partial restoration of the functional effect of beta-adrenergic receptor kinase-promoted phosphorylation (41 +/- 3% inactivation). These results suggest the possibility that a protein analogous to retinal arrestin may exist in other tissues and function in concert with beta-adrenergic receptor kinase to regulate the activity of adenylate cyclase-coupled receptors.