Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc

For next generation tissue-engineered constructs and regenerative medicine to succeed clinically, the basic biology and extracellular matrix composition of tissues that these repair techniques seek to restore have to be fully determined. Using the latest reagents coupled with tried and tested methodologies, we continue to uncover previously undetected structural proteins in mature intervertebral disc. In this study we show that the embryonic type IIA procollagen isoform (containing a cysteine-rich amino propeptide) was biochemically detectable in the annulus fibrosus of both calf and mature steer caudal intervertebral discs, but not in the nucleus pulposus where the type IIB isoform was predominantly localized. Specifically, the triple-helical type IIA procollagen isoform immunolocalized in the outer margins of the inner annulus fibrosus. Triple helical processed type II collagen exclusively localized within the inter-lamellae regions and with type IIA procollagen in the intra-lamellae regions. Mass spectrometry of the α1(II) collagen chains from the region where type IIA procollagen localized showed high 3-hydroxylation of Proline-944, a post-translational modification that is correlated with thin collagen fibrils as in the nucleus pulposus. The findings implicate small diameter fibrils of type IIA procollagen in select regions of the annulus fibrosus where it likely contributes to the organization of collagen bundles and structural properties within the type I-type II collagen transition zone

Deep sequencing of the vaginal microbiota of women with HIV

Background:Women living with HIV and co-infected with bacterial vaginosis (BV) are at higher risk for transmitting HIV to a partner or newborn. It is poorly understood which bacterial communities constitute BV or the normal vaginal microbiota among this population and how the microbiota associated with BV responds to antibiotic treatment. Methods and Findings: The vaginal microbiota of 132 HIV positive Tanzanian women, including 39 who received metronidazole treatment for BV, were profiled using Illumina to sequence the V6 region of the 16S rRNA gene. Of note, Gardnerella vaginalis and Lactobacillus iners were detected in each sample constituting core members of the vaginal microbiota. Eight major clusters were detected with relatively uniform microbiota compositions. Two clusters dominated by L. iners or L. crispatus were strongly associated with a normal microbiota. The L. crispatus dominated microbiota were associated with low pH, but when L. crispatus was not present, a large fraction of L. iners was required to predict a low pH. Four clusters were strongly associated with BV, and were dominated by Prevotella bivia, Lachnospiraceae, or a mixture of different species. Metronidazole treatment reduced the microbial diversity and perturbed the BV-associated microbiota, but rarely resulted in the establishment of a lactobacilli-dominated microbiota. Conclusions: Illumina based microbial profiling enabled high though-put analyses of microbial samples at a high phylogenetic resolution. The vaginal microbiota among women living with HIV in Sub-Saharan Africa constitutes several profiles associated with a normal microbiota or BV. Recurrence of BV frequently constitutes a different BV-associated profile than before antibiotic treatment

Observations of Past Lunar Landing Sites by the D-CIXS X-Ray Spectrometer on SMART-1

D-CIXS initial observations show a first unambiguous remote sensing of calcium in the lunar regolith. Data obtained are broadly consistent with current understanding of mare and highland composition. Ground truth is provided by the returned Apollo and Luna sample sets

ESET histone methyltransferase is essential to hypertrophic differentiation of growth plate chondrocytes and formation of epiphyseal plates

AbstractThe ESET (also called SETDB1) protein contains an N-terminal tudor domain that mediates protein–protein interactions and a C-terminal SET domain that catalyzes methylation of histone H3 at lysine 9. We report here that ESET protein is transiently upregulated in prehypertrophic chondrocytes in newborn mice. To investigate the in vivo effects of ESET on chondrocyte differentiation, we generated conditional knockout mice to specifically eliminate the catalytic SET domain of ESET protein only in mesenchymal cells. Such deletion of the ESET gene caused acceleration of chondrocyte hypertrophy in both embryos and young animals, depleting chondrocytes that are otherwise available to form epiphyseal plates for endochondral bone growth. ESET-deficient mice are thus characterized by defective long bone growth and trabecular bone formation. To understand the underlying mechanism for ESET regulation of chondrocytes, we carried out co-expression experiments and found that ESET associates with histone deacetylase 4 to bind and inhibit the activity of Runx2, a hypertrophy-promoting transcription factor. Repression of Runx2-mediated gene transactivation by ESET is dependent on its H3–K9 methyltransferase activity as well as its associated histone deacetylase activity. In addition, knockout of ESET is associated with repression of Indian hedgehog gene in pre- and early hypertrophic chondrocytes. Together, these results provide clear evidence that ESET controls hypertrophic differentiation of growth plate chondrocytes and endochondral ossification during embryogenesis and postnatal development

Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads al- lowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an ob- servation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bac- terial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.Comment: 28 pages, 13 figure

Environmental cold exposure increases blood flow and affects pain sensitivity in the knee joints of CFA-induced arthritic mice in a TRPA1-dependent manner

BACKGROUND: The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor. METHODS: Mono-arthritis was induced by unilateral intra-articular injection of complete Freund’s adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of (99m)Technetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK(1)) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP(8–37)). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples. RESULTS: Cold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints. CONCLUSIONS: We provide evidence that environmental cold exposure enhances pain and increases blood flow in a mono-arthritis model. These changes are dependent on TRPA1. Thus, TRPA1 may act locally within the joint to influence blood flow via sensory nerves, in addition to its established nociceptive actions

A resolved, au-scale gas disk around the B[e] star HD 50138

articleHD 50138 is a B[e] star surrounded by a large amount of circumstellar gas and dust. Its spectrum shows characteristics which may indicate either a pre- or a post-main-sequence system. Mapping the kinematics of the gas in the inner few au of the system contributes to a better understanding of its physical nature. We present the first high spatial and spectral resolution interferometric observations of the Brγ line of HD 50138, obtained with VLTI/AMBER. The line emission originates in a region more compact (up to 3 au) than the continuum-emitting region. Blue- and red-shifted emission originates from the two different hemispheres of an elongated structure perpendicular to the polarization angle. The velocity of the emitting medium decreases radially. An overall offset along the NW direction between the line- and continuum-emitting regions is observed. We compare the data with a geometric model of a thin Keplerian disk and a spherical halo on top of a Gaussian continuum. Most of the data are well reproduced by this model, except for the variability, the global offset and the visibility at the systemic velocity. The evolutionary state of the system is discussed; most diagnostics are ambiguous and may point either to a post-main-sequence or a pre-main-sequence nature.National Science FoundationGeorgia State UniversityW.M. Keck FoundationDavid and Lucile Packard FoundationFizeau ProgramCNRS-PICS progra

The wide-field infrared survey explorer properties of complete samples of radio-loud active galactic nucleus

We present an analysis of four complete samples of radio-loud active galactic nucleus (AGN; 3CRR, 2Jy, 6CE and 7CE) using near- and mid-IR data taken by theWide-field Infrared Survey Explorer (WISE). The combined sample consists of 79 quasars and 273 radio galaxies, and covers a redshift range 0.003<z<3.395. The dichotomy in the mid-IR properties of low- and high-excitation radio galaxies (LERGs and HERGs) is analysed for the first time using large complete samples. Our results demonstrate that a division in the accretion modes of LERGs and HERGs clearly stands out in the mid-IR-radio plane (L22μm = 5 × 1043 erg s-1). This means that WISE data can be effectively used to diagnose accretion modes in radio-loud AGN. The mid-IR properties of all objects were analysed to test the unification between quasars and radio galaxies, consistent with earlier work, and we argue that smooth torus models best reproduce the observation. Quasars are found to have higher mid-IR luminosities than radio galaxies. We also studied all the sources in the near-IR to gain insights into evolution of AGN host galaxies. A relation found between the near-IR luminosity and redshift, well known in the near-IR, is apparent in the two near-IR WISE bands, supporting the idea that radio sources are hosted by massive elliptical galaxies that formed their stars at high redshifts and evolved passively thereafter. Evaluation of the positions of the sample objects in WISE colour-colour diagrams shows that widely used WISE colour cuts are not completely reliable in selecting AGN.Peer reviewe

CRTAP Is Required for Prolyl 3- Hydroxylation and Mutations Cause Recessive Osteogenesis Imperfecta

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