Servo-controlled intravital microscope system

A microscope system is described for viewing an area of a living body tissue that is rapidly moving, by maintaining the same area in the field-of-view and in focus. A focus sensing portion of the system includes two video cameras at which the viewed image is projected, one camera being slightly in front of the image plane and the other slightly behind it. A focus sensing circuit for each camera differentiates certain high frequency components of the video signal and then detects them and passes them through a low pass filter, to provide dc focus signal whose magnitudes represent the degree of focus. An error signal equal to the difference between the focus signals, drives a servo that moves the microscope objective so that an in-focus view is delivered to an image viewing/recording camera

Automatically-focusing microscope system for live tissue observation

System includes focus-sensing arrangement which controls servo to keep microscope constantly focused on target. Microscope objective is moved along optical axis. System includes two video cameras that are used as transducers for sensing focus. Incoming visual image is split by beam splitter so that one-half of information is fed to each camera

QUANTITATION OF HUMAN RED BLOOD CELL FIXATION BY GLUTARALDEHYDE

The uptake of glutaraldehyde by human red blood cells has been measured as a function of time by a freezing point osmometer. The rate of attachment of glutaraldehyde to the cell proteins is high over the first hour, declining to zero over a period of a few days. The number of glutaraldehyde molecules cross-linking with each hemoglobin molecule is of the order of 200, in reasonable agreement with the calculated number of attachment sites. The cell membrane is immediately highly permeable to glutaraldehyde. Selective permeability to ions is lost during fixation. Ionic equilibrium is obtained only after a few hours. An optimum fixation technique for shape preservation is suggested

Alternative structures for water rights markets: Overview and hypothetical case study

The design of systems of marketable permits for water consumption from natural watercourses is examined through a qualitative overview and a quantitative quasi-empirical, quasi-hypothetical case study. For the work reported upon here, the most important considerations are those associated with: 1) uncertainty of future streamflows and economic conditions, 2) locational issues, and 3) efficient and effective functioning of the markets. Particular attention is given to the problem of implementing marketable rights systems in regions presently following the riparian doctrine. In these regions the most important design decisions include: the basis of definition of permits, the means for initially distributing them, the type of market mechanism used for their transfer after they are issued, and the restrictions placed on their use and transfer. These design decisions are examined here with respect to program objectives including: economic efficiency, equity, ease of administration and implementation, and maintenance of instream flows. Alternative approaches to the design problems are discussed and trade-offs implied by the decisions are identified. The efficiency of two marketable water rights systems in a lentic (lake-like) structure is assessed quantitatively for a case study based on hypothetical irrigation water use. Water rights markets are simulated on the bases of no foresight and perfect foresight on the parts of users, and the economic outcomes of these markets are evaluated from both ex ante and ex post perspectives. The market outcomes are compared to the optimal (efficient) scheme and to two alternative non-market policies. Distributional aspects of markets are examined on the basis of individual financial gain to the users. Simulation results show that higher efficiency is obtained for the two market systems than for the non-market policies and that the market systems recoup about 95% of the economic value of the optimal distribution. The results suggest that most of the 5% efficiency loss should be attributed to the restrictions imposed by the definition of the rights, rather than the users' inability to predict future events.U.S. Department of the InteriorU.S. Geological SurveyOpe

Thai lexical tone perception in native speakers of Thai, English and Mandarin Chinese: An event-related potentials training study

<p>Abstract</p> <p>Background</p> <p>Tone languages such as Thai and Mandarin Chinese use differences in fundamental frequency (F₀, pitch) to distinguish lexical meaning. Previous behavioral studies have shown that native speakers of a non-tone language have difficulty discriminating among tone contrasts and are sensitive to different F₀dimensions than speakers of a tone language. The aim of the present ERP study was to investigate the effect of language background and training on the non-attentive processing of lexical tones. EEG was recorded from 12 adult native speakers of Mandarin Chinese, 12 native speakers of American English, and 11 Thai speakers while they were watching a movie and were presented with multiple tokens of low-falling, mid-level and high-rising Thai lexical tones. High-rising or low-falling tokens were presented as deviants among mid-level standard tokens, and vice versa. EEG data and data from a behavioral discrimination task were collected before and after a two-day perceptual categorization training task.</p> <p>Results</p> <p>Behavioral discrimination improved after training in both the Chinese and the English groups. Low-falling tone deviants versus standards elicited a mismatch negativity (MMN) in all language groups. Before, but not after training, the English speakers showed a larger MMN compared to the Chinese, even though English speakers performed worst in the behavioral tasks. The MMN was followed by a late negativity, which became smaller with improved discrimination. The High-rising deviants versus standards elicited a late negativity, which was left-lateralized only in the English and Chinese groups.</p> <p>Conclusion</p> <p>Results showed that native speakers of English, Chinese and Thai recruited largely similar mechanisms when non-attentively processing Thai lexical tones. However, native Thai speakers differed from the Chinese and English speakers with respect to the processing of late F₀contour differences (high-rising versus mid-level tones). In addition, native speakers of a non-tone language (English) were initially more sensitive to F₀onset differences (low-falling versus mid-level contrast), which was suppressed as a result of training. This result converges with results from previous behavioral studies and supports the view that attentive as well as non-attentive processing of F₀contrasts is affected by language background, but is malleable even in adult learners.</p

Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue

Multiple functional neurosteroid binding sites on GABAA receptors

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action