Human chondrocytes in tridimensional culture.

peer reviewedCartilage was taken from the macroscopically normal part of human femoral heads immediately after orthopedic surgical operations for total prothesis consecutive to hip arthrosis. After clostridial collagenase digestion and repeated washings, chondrocytes (10(6) cells) were cultivated in a gyrotory shaker (100 rpm). Under these conditions, cells were kept in suspension and after 3 to 5 d formed a flaky aggregate which, on Day 10, became dense. These chondrocytes were morphologically differentiated: they had a round shape, were situated inside cavities, and were surrounded by a new matrix. Histochemical methods showed the presence of collagen and polysaccharides in cell cytoplasm and in intercellular matrix, and the immunofluorescence method using specific antisera (anticartilage proteoglycans and anti-type II collagen) showed that these two constituents were in intercellular matrix. The measurement of the amounts of proteoglycans (PG) released into culture medium and those present in chondrocyte aggregate (by a specific PG radioimmunoassay) showed a maximum production on Days 3 to 5 of culture, then the production decreased and stabilized (from Day 10 to the end of culture). The observed difference between the amounts of PG in aggregates after 20 d and those after 2 h of culture demonstrated that PG neosynthesis did occur during cultivation. This conclusion was supported by other results obtained by [14C]glucosamine incorporation in chondrocyte aggregates. Moreover, the aggregate fresh weight related to cell number (appreciated by DNA assay) increased significantly with culture duration. Three-dimensional chondrocyte culture represents an interesting model: chondrocytes were differentiated morphologically as well as biosynthetically and synthesized a new cartilage matrix

The guider and wavefront curvature sensor subsystem for the Large Synoptic Survey Telescope

The Large Synoptic Survey Telescope instrument include four guiding and wavefront sensing subsystems called corner raft subsystems, in addition to the main science array of 189 4K x 4K CCDs. These four subsystems are placed at the four corners of the instrumented field of view. Each wavefront/guiding subsystem comprises a pair of 4K x 4K guide sensors, capable of producing 9 frames/second, and a pair of offset 2K x 4K wavefront curvature sensors from which the images are read out at the cadence of the main camera system, providing 15 sec integrations. These four guider/wavefront corner rafts are mechanically and electrically isolated from the science sensor rafts and can be installed or removed independently from any other focal plane subsystem. We present the implementation of this LSST subsystem detailing both hardware and software development and status.Astronom

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases

A Novel Unstable Duplication Upstream of HAS2 Predisposes to a Breed-Defining Skin Phenotype and a Periodic Fever Syndrome in Chinese Shar-Pei Dogs

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (praw = 2.3×10−6, pgenome = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p<0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation

Chondroitin sulfates and their binding molecules in the central nervous system

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in the central nervous system (CNS) matrix. Its sulfation and epimerization patterns give rise to different forms of CS, which enables it to interact specifically and with a significant affinity with various signalling molecules in the matrix including growth factors, receptors and guidance molecules. These interactions control numerous biological and pathological processes, during development and in adulthood. In this review, we describe the specific interactions of different families of proteins involved in various physiological and cognitive mechanisms with CSs in CNS matrix. A better understanding of these interactions could promote a development of inhibitors to treat neurodegenerative diseases

The On-orbit Calibrations for the Fermi Large Area Telescope

The Large Area Telescope (LAT) on--board the Fermi Gamma ray Space Telescope began its on--orbit operations on June 23, 2008. Calibrations, defined in a generic sense, correspond to synchronization of trigger signals, optimization of delays for latching data, determination of detector thresholds, gains and responses, evaluation of the perimeter of the South Atlantic Anomaly (SAA), measurements of live time, of absolute time, and internal and spacecraft boresight alignments. Here we describe on orbit calibration results obtained using known astrophysical sources, galactic cosmic rays, and charge injection into the front-end electronics of each detector. Instrument response functions will be described in a separate publication. This paper demonstrates the stability of calibrations and describes minor changes observed since launch. These results have been used to calibrate the LAT datasets to be publicly released in August 2009.Comment: 60 pages, 34 figures, submitted to Astroparticle Physic

Defective processing of keratan sulfate in Macular corneal dystrophy

macular corneal dystrophy is a human genetic disorder characterized by corneal opacities that arise, in part, from a failure to synthesize mature keratan sulfate proteoglycans. The macromolecules in macular corneas and in keratoconus corneas, an abnormality not involving proteoglycans, were biosynthetically labeled with [3H]mannose and [14C]glucosamine in organ culture, and the keratan sulfate proteoglycans were immunoprecipitated with antibodies against the protein core of monkey keratan sulfate proteoglycan. The chondroitin sulfate proteoglycans, which did not react with the antibody, were oversulfated in corneas from patients with macular corneal dystrophy. Characterization of the immunoprecipitates showed that macular corneas did not make keratan sulfate proteoglycan but did synthesize an immunoreactive glycoprotein in nearly equal amounts as keratan sulfate proteoglycan was synthesized by the keratoconus cornea. The oligosaccharides on the immunoprecipitated macular glycoprotein appeared to be normal. However, the macromolecules contained an unsulfated glycoconjugate that was nearly as large as the normal keratan sulfate chains isolated from the keratoconus keratan sulfate-proteoglycan and contained the same relative proportions of labeled glucosamine, mannose, and fucose. This glycoconjugate was resistant to digestion with keratanase. These observations indicate that macular corneal dystropy is caused by an error in the synthesis of keratan sulfate, possibly involving the specific sulfotransferases involved in sulfation of the lactosaminoglycan backbone of the chains