HAPTEN-SANDWICH LABELING : I. A GENERAL PROCEDURE FOR SIMULTANEOUS LABELING OF MULTIPLE CELL SURFACE ANTIGENS FOR FLUORESCENCE AND ELECTRON MICROSCOPY

A hapten-sandwich procedure has been developed for specific labeling of cell surface antigens for fluorescence or electron microscopy. Haptens are azo-coupled to immunoglobulins specific for a cell surface antigen; the hapten-modified cell-bound antibodies can then be visualized by adding fluorescent antihapten antibody, or by adding antihapten antibody followed by hapten-modified markers for electron microscopy. Virus or high molecular weight protein markers are lightly cross-linked before conjugation with hapten to prevent their disruption. Such stable hapten-modified markers, and the accessibility of many different purified anti-azophenyl-hapten antibodies, make it feasible to distinguish more than one membrane antigen in a given labeling experiment. When mouse lymphoid cell populations are labeled with separate markers for Ig and for thymus-associated antigens, many cells exhibit the Ig marker exclusively or the thymic marker predominantly, and some cells are completely free of label

From segment to somite: segmentation to epithelialization analyzed within quantitative frameworks

One of the most visually striking patterns in the early developing embryo is somite segmentation. Somites form as repeated, periodic structures in pairs along nearly the entire caudal vertebrate axis. The morphological process involves short- and long-range signals that drive cell rearrangements and cell shaping to create discrete, epithelialized segments. Key to developing novel strategies to prevent somite birth defects that involve axial bone and skeletal muscle development is understanding how the molecular choreography is coordinated across multiple spatial scales and in a repeating temporal manner. Mathematical models have emerged as useful tools to integrate spatiotemporal data and simulate model mechanisms to provide unique insights into somite pattern formation. In this short review, we present two quantitative frameworks that address the morphogenesis from segment to somite and discuss recent data of segmentation and epithelialization

The Incremental Garbage Collection of Processes

Key Words and Phrases: garbage collection, multiprocessing systems, processor scheduling. "lazy evaluation, "eager" evaluation. CR Categories: 3.60, 3.80, 4.13, 4.22, 4.32. This report describes research done at the Artificial Intelligence Laboratory of the Massachusetts Institute of Technology. Support for the laboratory's artificial intelligence research is provided in part by the Advanced Research Projects Agency of the Department of Defense under Office of Naval Research contract N00014-75-C-0522. This paper was presented at the AI*PL Conference at Rochester, N.Y. in August, 1977.This paper investigates some problems associated with an argument evaluation order that we call "future" order, which is different from both call-by-name and call-by-value. In call-by-future, each formal parameter of a function is bound to a separate process (called a "future") dedicated to the evaluation of the corresponding argument. This mechanism allows the fully parallel evaluation of arguments to a function, and has been shown to augment the expressive power of a language. We discuss an approach to a problem that arises in this context: futures which were thought to be relevant when they were created become irrelevant through being ignored in the body of the expression where they were bound. The problem of irrelevant processes also appears in multiprocessing problem-solving systems which start several processors working on the same problem but with different methods, and return with the solution which finishes first. This parallel method strategy has the drawback that the processes which are investigating the losing methods must be identified, stopped, and re-assigned to more useful tasks. The solution we propose is that of garbage collection. We propose that the goal structure of the solution plan be explicitly represented in memory as part of the graph memory (like Lisp's heap) so that a garbage collection algorithm can discover which processes are performing useful work, and which can be recycled for a new task. An incremental algorithm for the unified garbage collection of storage and processes is described.MIT Artificial Intelligence Laboratory Department of Defense Advanced Research Projects Agenc

Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile

<p>Background: Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression.</p> <p>Methods: Vascular transduction capacity was assessed in primary human saphenous vein smooth muscle and endothelial cells using vectors expressing the LacZ reporter gene. The therapeutic capacity of the vectors was compared by measuring smooth muscle cell metabolic activity and migration following infection with vectors that over-express the candidate therapeutic gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3).</p> <p>Results: Compared to Adenovirus serotype 5 (Ad5), the novel vector Ad5T*F35++ demonstrated improved binding and transduction of human vascular cells. Ad5T*F35++ mediated expression of TIMP-3 reduced smooth muscle cell metabolic activity and migration in vitro. We also demonstrated that in human serum samples pre-existing neutralising antibodies to Ad5T*F35++ were less prevalent than Ad5 neutralising antibodies.</p> <p>Conclusions: We have developed a novel vector with improved vascular transduction and improved resistance to human serum neutralisation. This may provide a novel vector platform for human vascular gene transfer.</p&gt

Discovery of a high-z protocluster with tunable filters: the case of 6C0140+326 at z=4.4

We present the first results obtained using a tunable narrowband filter in the search for high-z protoclusters. Using the recently commissioned red tunable filter on the Gran Telescopio Canarias we have searched for Lya emitters in a 75 arcmin^2 field centered on the z=4.413 radio galaxy 6C0140+326. With three different wavelength tunings we find a total of 27 unique candidate Lya emitters. The availability of three different wavelength tunings allows us to make estimates of the redshifts for each of the objects. It also allows us to separate a possible protocluster from structure in the immediate foreground. This division shows that the foreground region contains significantly fewer Lya emitters. Also, the spatial distribution of the objects in the protocluster field deviates from a random distribution at the 2.5 sigma level. The observed redshift distribution of the emitters is different from the expected distribution of a blank field at the ~3 sigma level, with the Lya emitters concentrated near the radio galaxy at z>4.38. The 6C0140+326 field is denser by a factor of 9+/-5 than a blank field, and the number density of Lya emitters close to the radio galaxy is similar to that of the z~4.1 protocluster around TNJ1338-1942. We thus conclude that there is an overdensity of Lya emitters around the radio galaxy 6C0140+326. This is one of few known overdensities at such a high redshift.Comment: 10 pages, 6 figures, accepted for publication in MNRA

Gene expression changes in the human diaphragm after cardiothoracic surgery

ObjectiveWe examined the effects of cardiothoracic surgery, including cardiopulmonary bypass and controlled mechanical ventilation, on messenger RNA gene expression in human diaphragm. We hypothesized that genes responsible for stress response, redox regulation, protein turnover, energy metabolism, and contractile function would be altered by cardiothoracic surgery.MethodsPaired diaphragm biopsy samples were obtained from 5 male patients (67 ± 11 years) during cardiothoracic surgery, the first as soon as the diaphragm was exposed and the second as late in surgery as possible (4.9 ± 1.8 hours between samples). We profiled messenger RNA from 5 specimen pairs with microarray analysis (Hu U133 plus 2.0; Affymetrix UK Ltd, High Wycombe, UK). Quantitative reverse transcriptase polymerase chain reaction was performed with a select set of genes exhibiting differential expression for validation.ResultsMicroarray analysis identified 779 differentially expressed (early vs late samples) unique gene products (P < .005). Postoperatively, genes related to stress response and redox regulation were upregulated. Additionally, we found significantly upregulated expression of cathepsin C (2.7-fold), cathepsin L1 (2.0-fold), various ubiquitin-conjugating enzymes (E2, approximately 1.8-fold), proinflammatory cytokine interleukin 6 (15.6-fold), and muscle-specific ubiquitin ligase (MuRF-1, 2.6-fold). Comparison of fold change values obtained by quantitative reverse transcriptase polymerase chain reaction and microarray yielded significant correlation (r = 0.95, P < .0001).ConclusionsCardiothoracic surgery results in rapid changes in human diaphragm gene expression in the operating room, including genes related to stress response, inflammation, redox regulation, and proteolysis. These results may provide insight into diaphragm muscle biology after prolonged cardiothoracic procedures

A pivotal role for starch in the reconfiguration of 14C-partitioning and allocation in Arabidopsis thaliana under short-term abiotic stress.

Plant carbon status is optimized for normal growth but is affected by abiotic stress. Here, we used 14C-labeling to provide the first holistic picture of carbon use changes during short-term osmotic, salinity, and cold stress in Arabidopsis thaliana. This could inform on the early mechanisms plants use to survive adverse environment, which is important for efficient agricultural production. We found that carbon allocation from source to sinks, and partitioning into major metabolite pools in the source leaf, sink leaves and roots showed both conserved and divergent responses to the stresses examined. Carbohydrates changed under all abiotic stresses applied; plants re-partitioned 14C to maintain sugar levels under stress, primarily by reducing 14C into the storage compounds in the source leaf, and decreasing 14C into the pools used for growth processes in the roots. Salinity and cold increased 14C-flux into protein, but as the stress progressed, protein degradation increased to produce amino acids, presumably for osmoprotection. Our work also emphasized that stress regulated the carbon channeled into starch, and its metabolic turnover. These stress-induced changes in starch metabolism and sugar export in the source were partly accompanied by transcriptional alteration in the T6P/SnRK1 regulatory pathway that are normally activated by carbon starvation

Somitogenesis Clock-Wave Initiation Requires Differential Decay and Multiple Binding Sites for Clock Protein

Somitogenesis is a process common to all vertebrate embryos in which repeated blocks of cells arise from the presomitic mesoderm (PSM) to lay a foundational pattern for trunk and tail development. Somites form in the wake of passing waves of periodic gene expression that originate in the tailbud and sweep posteriorly across the PSM. Previous work has suggested that the waves result from a spatiotemporally graded control protein that affects the oscillation rate of clock-gene expression. With a minimally constructed mathematical model, we study the contribution of two control mechanisms to the initial formation of this gene-expression wave. We test four biologically motivated model scenarios with either one or two clock protein transcription binding sites, and with or without differential decay rates for clock protein monomers and dimers. We examine the sensitivity of wave formation with respect to multiple model parameters and robustness to heterogeneity in cell population. We find that only a model with both multiple binding sites and differential decay rates is able to reproduce experimentally observed waveforms. Our results show that the experimentally observed characteristics of somitogenesis wave initiation constrain the underlying genetic control mechanisms

CMB Telescopes and Optical Systems

The cosmic microwave background radiation (CMB) is now firmly established as a fundamental and essential probe of the geometry, constituents, and birth of the Universe. The CMB is a potent observable because it can be measured with precision and accuracy. Just as importantly, theoretical models of the Universe can predict the characteristics of the CMB to high accuracy, and those predictions can be directly compared to observations. There are multiple aspects associated with making a precise measurement. In this review, we focus on optical components for the instrumentation used to measure the CMB polarization and temperature anisotropy. We begin with an overview of general considerations for CMB observations and discuss common concepts used in the community. We next consider a variety of alternatives available for a designer of a CMB telescope. Our discussion is guided by the ground and balloon-based instruments that have been implemented over the years. In the same vein, we compare the arc-minute resolution Atacama Cosmology Telescope (ACT) and the South Pole Telescope (SPT). CMB interferometers are presented briefly. We conclude with a comparison of the four CMB satellites, Relikt, COBE, WMAP, and Planck, to demonstrate a remarkable evolution in design, sensitivity, resolution, and complexity over the past thirty years.Comment: To appear in: Planets, Stars and Stellar Systems (PSSS), Volume 1: Telescopes and Instrumentatio