TGF-β1 as a prognostic factor in the process of early osteoarthrosis in the rabbit knee

AbstractObjective To assess changes in knee joint fluid concentrations of transforming growth factor-β1 (TGF-β1) and proteoglycan (PG) fragments during the early course of post-traumatic osteoarthrosis (OA) after meniscectomy in the rabbit knee, and to ascertain whether the concentrations of these substances shortly after operation could be used as prognostic markers for the OA process.Design In 15 rabbits with medial meniscectomy in one knee and a sham operation in the other knee, synovial lavage fluid samples were taken repeatedly, before operation, every third week post-operatively until 12 weeks, thereafter every sixth week, and at death. Five rabbits each were killed at 13, 25 and 40 weeks. Synovial lavage fluid samples from five non-operated rabbits served as controls. At death, two histological scores were formed that characterized the highest (MAX) and the overall (ALL) degree of OA changes in each joint.Results TGF-β1 and PG fragment concentrations in synovial lavage fluid correlated highly (R=0.81, P< 0.001). Both OA scores were higher in meniscectomized than controls (P< 0.05). The synovial lavage fluid concentration of TGF-β1 at 3 weeks, but no other time point, correlated to the histological scores (ALL, R=0.58; MAX, R=0.52;P< 0.001).Conclusion Higher concentrations of TGF-β1 in synovial lavage fluid early after surgery seemed indicative for the later development of more severe OA changes in contrast to lower concentrations. The association between TGF-β1 and the changes found later in the cartilage was underlined by the high correlations between this substance and PG fragment concentrations in synovial lavage fluid at all time points

Simultaneous changes in bone mineral density and articular cartilage in a rabbit meniscectomy model of knee osteoarthrosis

AbstractObjective It was hypothesized that increased bone mineral density of the medial proximal tibia would precede or coincide with the development of more severe cartilage changes after meniscectomy.Methods In a rabbit knee model, mineral density of subchondral bone and changes of articular cartilage were monitored 13 to 40 weeks after medial meniscectomy or a sham operation.Results Both procedures resulted in a decrease of bone mineral density, especially of the medial proximal tibia, which persisted up to 40 weeks (P< 0.02–0.0007). Meniscectomy induced cartilage changes typical for osteoarthrosis (P< 0.009), which progressed over time on the posterior aspect of the medial tibial plateau (P< 0.009), which is physiologically covered by the meniscus, but the procedure also induced iatrogenic changes which were located mainly on the anterior aspect of the concerned compartment, and which did not progress or develop to osteoarthrosis.Conclusions The data suggest that the cartilage changes after meniscectomy in this animal model are caused by the surgical trauma, subsequent limb misuse, and altered load distribution, and initially associated by a decrease not an increase in bone mineral density of the proximal tibia. Moreover, the cartilage changes progressed without a simultaneous increase of the bone mineral density at corresponding sites

Genome-Wide Profiling and Analysis of Arabidopsis siRNAs

Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences

Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants

[EN] In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.We thank Goretti Nguyen for excellent technical assistance. A. C. was supported by a postdoctoral fellowship from the Ministerio de Ciencia e Innovacion (BMC-2008-0188). H.G.-R. was the recipient of a Helen Hay Whitney Postdoctoral fellowship (F-972). 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Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation

SummaryTrans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5′ adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5′ nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps

An automated, high-throughput method for standardizing image color profiles to improve image-based plant phenotyping

High-throughput phenotyping has emerged as a powerful method for studying plant biology. Large image-based datasets are generated and analyzed with automated image analysis pipelines. A major challenge associated with these analyses is variation in image quality that can inadvertently bias results. Images are made up of tuples of data called pixels, which consist of R, G, and B values, arranged in a grid. Many factors, for example image brightness, can influence the quality of the image that is captured. These factors alter the values of the pixels within images and consequently can bias the data and downstream analyses. Here, we provide an automated method to adjust an image-based dataset so that brightness, contrast, and color profile is standardized. The correction method is a collection of linear models that adjusts pixel tuples based on a reference panel of colors. We apply this technique to a set of images taken in a high-throughput imaging facility and successfully detect variance within the image dataset. In this case, variation resulted from temperature-dependent light intensity throughout the experiment. Using this correction method, we were able to standardize images throughout the dataset, and we show that this correction enhanced our ability to accurately quantify morphological measurements within each image. We implement this technique in a high-throughput pipeline available with this paper, and it is also implemented in PlantCV

Expression of Human Beta-Defensins in Children with Chronic Inflammatory Bowel Disease

Background: Human beta-defensins (hBDs) are antimicrobial peptides known to play a major role in intestinal innate host defence. Altered mucosal expression of hBDs has been suggested to be implicated in chronic inflammatory bowel disease pathogenesis. However, little is known about expression of these peptides in children. Methods: Intestinal biopsies were obtained from the duodenum (n = 88), terminal ileum (n = 90) and ascending colon (n = 105) of children with Crohn’s disease (n = 26), ulcerative colitis (n = 11) and healthy controls (n = 16). Quantitative realtime (RT) PCR was performed and absolute mRNA copy numbers analyzed for hBD1-3 as well as inflammatory cytokines IL-8 and TNF-alpha. Results: Significant induction of hBD2 and hBD3 was observed in the inflamed terminal ileum and ascending colon of IBD children. In the ascending colon induction of hBD2 was found to be significantly lower in children with Crohn’s disease compared to ulcerative colitis. A strong correlation was found between inducible defensins hBD2 and 3 and the inflammatory cytokines IL-8 and TNF-alpha, both in the terminal ileum and ascending colon. Conclusion: Our study demonstrates distinct changes in hBD expression throughout the intestinal tract of children with IBD

Biomarker selection for detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT–PCR

Accurate identification of lymph node involvement is critical for successful treatment of patients with colorectal carcinoma (CRC). Real-time quantitative RT–PCR with a specific probe and RNA copy standard for biomarker mRNA has proven very powerful for detection of disseminated tumour cells. Which properties of biomarker mRNAs are important for identification of disseminated CRC cells? Seven biomarker candidates, CEA, CEACAM1-S/L, CEACAM6, CEACAM7-1/2, MUC2, MMP7 and CK20, were compared in a test-set of lymph nodes from 51 CRC patients (Dukes' A–D) and 10 controls. Normal colon epithelial cells, primary tumours, and different immune cells were also analysed. The biomarkers were ranked according to: (1) detection of haematoxylin/eosin positive nodes, (2) detection of Dukes' A and B patients, who developed metastases during a 54 months follow-up period and (3) identification of patients with Dukes' C and D tumours using the highest value of control nodes as cutoff. The following properties appear to be of importance; (a) no expression in immune cells, (b) relatively high and constant expression in tumour tissue irrespective of Dukes' stage and (c) no or weak downregulation in tumours compared to normal tissue. CEA fulfilled these criteria best, followed by CK20 and MUC2

Comparative analysis of miRNAs and their targets across four plant species

BACKGROUND: MicroRNA (miRNA) mediated regulation of gene expression has been recognized as a major posttranscriptional regulatory mechanism also in plants. We performed a comparative analysis of miRNAs and their respective gene targets across four plant species: Arabidopsis thaliana (Ath), Medicago truncatula(Mtr), Brassica napus (Bna), and Chlamydomonas reinhardtii (Cre). RESULTS: miRNAs were obtained from mirBase with 218 miRNAs for Ath, 375 for Mtr, 46 for Bna, and 73 for Cre, annotated for each species respectively. miRNA targets were obtained from available database annotations, bioinformatic predictions using RNAhybrid as well as predicted from an analysis of mRNA degradation products (degradome sequencing) aimed at identifying miRNA cleavage products. On average, and considering both experimental and bioinformatic predictions together, every miRNA was associated with about 46 unique gene transcripts with considerably variation across species. We observed a positive and linear correlation between the number miRNAs and the total number of transcripts across different plant species suggesting that the repertoire of miRNAs correlates with the size of the transcriptome of an organism. Conserved miRNA-target pairs were found to be associated with developmental processes and transcriptional regulation, while species-specific (in particular, Ath) pairs are involved in signal transduction and response to stress processes. Conserved miRNAs have more targets and higher expression values than non-conserved miRNAs. We found evidence for a conservation of not only the sequence of miRNAs, but their expression levels as well. CONCLUSIONS: Our results support the notion of a high birth and death rate of miRNAs and that miRNAs serve many species specific functions, while conserved miRNA are related mainly to developmental processes and transcriptional regulation with conservation operating at both the sequence and expression level

AutoRoot: open-source software employing a novel image analysis approach to support fully-automated plant phenotyping

Background: Computer-based phenotyping of plants has risen in importance in recent years. Whilst much software has been written to aid phenotyping using image analysis, to date the vast majority has been only semi-automatic. However, such interaction is not desirable in high throughput approaches. Here, we present a system designed to analyse plant images in a completely automated manner, allowing genuine high throughput measurement of root traits. To do this we introduce a new set of proxy traits. Results: We test the system on a new, automated image capture system, the Microphenotron, which is able to image many 1000s of roots/h. A simple experiment is presented, treating the plants with differing chemical conditions to produce different phenotypes. The automated imaging setup and the new software tool was used to measure proxy traits in each well. A correlation matrix was calculated across automated and manual measures, as a validation. Some particular proxy measures are very highly correlated with the manual measures (e.g. proxy length to manual length, r2 > 0.9). This suggests that while the automated measures are not directly equivalent to classic manual measures, they can be used to indicate phenotypic differences (hence the term, proxy). In addition, the raw discriminative power of the new proxy traits was examined. Principal component analysis was calculated across all proxy measures over two phenotypically-different groups of plants. Many of the proxy traits can be used to separate the data in the two conditions. Conclusion: The new proxy traits proposed tend to correlate well with equivalent manual measures, where these exist. Additionally, the new measures display strong discriminative power. It is suggested that for particular phenotypic differences, different traits will be relevant, and not all will have meaningful manual equivalent measures. However, approaches such as PCA can be used to interrogate the resulting data to identify differences between datasets. Select images can then be carefully manually inspected if the nature of the precise differences is required. We suggest such flexible measurement approaches are necessary for fully automated, high throughput systems such as the Microphenotron