A bioinformatics approach to microRNA-sequencing analysis

The rapid expansion of Next Generation Sequencing (NGS) data availability has made exploration of appropriate bioinformatics analysis pipelines a timely issue. Since there are multiple tools and combinations thereof to analyze any dataset, there can be uncertainty in how to best perform an analysis in a robust and reproducible manner. This is especially true for newer omics applications, such as miRNomics, or microRNA-sequencing (miRNA-sequencing). As compared to transcriptomics, there have been far fewer miRNA-sequencing studies performed to date, and those that are reported seldom provide detailed description of the bioinformatics analysis, including aspects such as Unique Molecular Identifiers (UMIs). In this article, we attempt to fill the gap and help researchers understand their miRNA-sequencing data and its analysis. This article will specifically discuss a customizable miRNA bioinformatics pipeline that was developed using miRNA-sequencing datasets generated from human osteoarthritis plasma samples. We describe quality assessment of raw sequencing data files, reference-based alignment, counts generation for miRNA expression levels, and novel miRNA discovery. This report is expected to improve clarity and reproducibility of the bioinformatics portion of miRNA-sequencing analysis, applicable across any sample type, to promote sharing of detailed protocols in the NGS field

Antisense oligonucleotide-based therapies for the treatment of osteoarthritis: Opportunities and roadblocks

Osteoarthritis (OA) is a debilitating disease with no approved disease-modifying therapies. Among the challenges for developing treatment is achieving targeted drug delivery to affected joints. This has contributed to the failure of several drug candidates for the treatment of OA. Over the past 20 years, significant advances have been made in antisense oligonucleotide (ASO) technology for achieving targeted delivery to tissues and cells both in vitro and in vivo. Since ASOs are able to bind specific gene regions and regulate protein translation, they are useful for correcting aberrant endogenous mechanisms associated with certain diseases. ASOs can be delivered locally through intra-articular injection, and can enter cells through natural cellular uptake mechanisms. Despite this, ASOs have yet to be successfully tested in clinical trials for the treatment of OA. Recent chemical modification to ASOs have further improved cellular uptake and reduced toxicity. Among these are locked nucleic acid (LNA)-based ASOs, which have shown promising results in clinical trials for diseases such as hepatitis and dyslipidemia. Recently, LNA-based ASOs have been tested both in vitro and in vivo for their therapeutic potential in OA, and some have shown promising joint-protective effects in preclinical OA animal models. In order to accelerate the testing of ASO therapies in a clinical trial setting for OA, further investigation into delivery mechanisms is required. In this review article, we discuss opportunities for viral-, particle-, biomaterial-, and chemical modification-based therapies, which are currently in preclinical testing. We also address potential roadblocks in the clinical translation of ASO-based therapies for the treatment of OA, such as the limitations associated with OA animal models and the challenges with drug toxicity. Taken together, we review what is known and what would be useful to accelerate translation of ASO-based therapies for the treatment of OA

Shunting of prostanoid biosynthesis in microsomal prostaglandin E synthase‐1 null embryo fibroblasts: regulatory effects on inducible nitric oxide synthase expression and nitrite synthesis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154471/1/fsb2fj066366fje.pd

Racial Differences in Serum Adipokine and Insulin Levels in a Matched Osteoarthritis Sample: A Pilot Study

Background. In an attempt to correlate biomarkers with disease, serum-based biomarkers often are compared between individuals with osteoarthritis (OA) and control subjects. However, variable results have been reported. Some studies have suggested an association between certain adipokines and insulin and OA. We know that there are racial differences in OA prevalence and incidence, and from general population-based studies, those of Asian race consistently demonstrate a unique adipokine/insulin serum concentration profile as compared to Caucasians. Whether similar racial differences exist within OA samples is unknown and may have implications for selecting appropriate controls in comparative studies. Methods. Serum levels of adipokines, leptin, and adiponectin, along with insulin, were determined by ELISA in patients scheduled for total hip or knee replacement surgery for OA. Fifteen Asian patients were matched 1 : 1 on age (±2 years), gender, body mass index (±1.5 kg/m2), and surgical joint with Caucasian patients. Differences in serum concentrations were tested using paired t-tests. Results. Serum leptin and insulin levels were significantly higher in Asians compared to Caucasians (p<0.05). While serum adiponectin levels were lower among Asians, the difference did not reach statistical significance (p=0.12). Conclusion. Findings from this work suggest that when studying serum biomarker concentrations in OA versus controls, race may be an important factor to consider. Our findings warrant confirmation in larger studies

Offen und effektiv? Kommunale Open-Data-Portale für die Stadtentwicklung am Beispiel Berlins

Der Beitrag stellt Ergebnisse einer Studie über Nutzen und Wirkungen offener Datenportale für die Stadtentwicklung in deutschen Städten vor. Anhand der Stadt Berlin werden auf Basis der quantitativen Beschreibung der Entwicklung des Datenangebotes, dessen Erreichbarkeit, Nutzung und Wirkungen herausgearbeitet. Hierzu werden Experteninterviews mit Vertretern unterschiedlicher Akteursgruppen von Verwaltung, Politik und Zivilgesellschaft durchgeführt. Die Ergebnisse zu den Portalen Berlin Open Data und Geoportal Berlin (FIS-Broker) zeigen die gesteigerte Bedeutung offener Daten im Bereich der öffentlichen Verwaltung, insbesondere in der Bürgerbeteiligung. Allerdings spiegeln sie auch die bestehenden Lücken in der Bereitstellung von Daten wider und weisen auf die Einschränkungen hin, die sich aus der fehlenden Berücksichtigung der oft begrenzten Erfahrung eines breiten Nutzerkreises ergeben. Hier gilt es Lösungen zu finden, um Datenportale als Basis für innovative und fundierte Entscheidungen in der Stadtentwicklung weiterzuentwickeln

Connective tissue growth factor promoter activity in normal and wounded skin

In skin, connective tissue growth factor (CTGF/CCN2) is induced during tissue repair. However, what the exact cell types are that express CTGF in normal and wounded skin remain controversial. In this report, we use transgenic knock-in mice in which the Pacific jellyfish Aequorea victoria enhanced green fluorescent protein (E-GFP) gene has been inserted between the endogenous CTGF promoter and gene. Unwounded (day 0) and wounded (days 3 and 7) skin was examined for GFP to detect cells in which the CTGF promoter was active, α-smooth muscle actin (α-SMA) to detect myofibroblasts, and NG2 expression to detect pericytes. In unwounded mice, CTGF expression was absent in epidermis and was present in a few cells in the dermis. Upon wounding, CTGF expression was induced in the dermis. Double immunolabeling revealed that CTGF-expressing cells also expressed α-SMA, indicating the CTGF was expressed in myofibroblasts. A subset (~30%) of myofibroblasts were also NG2 positive, indicating that pericytes significantly contributed to the number of myofibroblasts in the wound. Pericytes also expressed CTGF. Collectively, these results indicate that CTGF expression in skin correlates with myofibroblast induction, and that CTGF-expressing pericytes are significant contributors to myofibroblast activity during cutaneous tissue repair

The non-coding RNA interactome in joint health and disease

Non-coding RNAs have distinct regulatory roles in the pathogenesis of joint diseases including osteoarthritis (OA) and rheumatoid arthritis (RA). As the amount of high-throughput profiling studies and mechanistic investigations of microRNAs, long non-coding RNAs and circular RNAs in joint tissues and biofluids has increased, data have emerged that suggest complex interactions among non-coding RNAs that are often overlooked as critical regulators of gene expression. Identifying these non-coding RNAs and their interactions is useful for understanding both joint health and disease. Non-coding RNAs regulate signalling pathways and biological processes that are important for normal joint development but, when dysregulated, can contribute to disease. The specific expression profiles of non-coding RNAs in various disease states support their roles as promising candidate biomarkers, mediators of pathogenic mechanisms and potential therapeutic targets. This Review synthesizes literature published in the past 2 years on the role of non-coding RNAs in OA and RA with a focus on inflammation, cell death, cell proliferation and extracellular matrix dysregulation. Research to date makes it apparent that \u27non-coding\u27 does not mean \u27non-essential\u27 and that non-coding RNAs are important parts of a complex interactome that underlies OA and RA

Circulating microRNAs differentiate fast-progressing from slow-progressing and non-progressing knee osteoarthritis in the Osteoarthritis Initiative cohort

INTRODUCTION: The objective of this study is to identify circulating microRNAs that distinguish fast-progressing radiographic knee osteoarthritis (OA) in the Osteoarthritis Initiative cohort by applying microRNA-sequencing. METHODS: Participants with Kellgren-Lawrence (KL) grade 0/1 at baseline were included (N = 106). Fast-progressors were defined by an increase to KL 3/4 by 4-year follow-up (N = 20), whereas slow-progressors showed an increase to KL 2/3/4 only at 8-year follow-up (N = 35). Non-progressors remained at KL 0/1 by 8-year follow-up (N = 51). MicroRNA-sequencing was performed on plasma collected at baseline and 4-year follow-up from the same participants. Negative binomial models were fitted to identify differentially expressed (DE) microRNAs. Penalized logistic regression (PLR) analyses were performed to select combinations of DE microRNAs that distinguished fast-progressors. Area under the receiver operating characteristic curves (AUC) were constructed to evaluate predictive ability. RESULTS: DE analyses revealed 48 microRNAs at baseline and 2 microRNAs at 4-year follow-up [false discovery rate (FDR) \u3c 0.05] comparing fast-progressors with both slow-progressors and non-progressors. Among these were hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, and hsa-miR-320e, which were predicted to target gene families, including members of the 14-3-3 gene family, involved in signal transduction. PLR models included miR-320 members as top predictors of fast-progressors and yielded AUC ranging from 82.6 to 91.9, representing good accuracy. CONCLUSION: The miR-320 family is associated with fast-progressing radiographic knee OA and merits further investigation as potential biomarkers and mechanistic drivers of knee OA

Association of cartilage-specific deletion of peroxisome proliferator-activated receptor γ with abnormal endochondral ossification and impaired cartilage growth and development in a murine model

Objective Long bones develop through the strictly regulated process of endochondral ossification within the growth plate, resulting in the replacement of cartilage by bone. Defects in this process can result in skeletal abnormalities and a predisposition to degenerative joint diseases such as osteoarthritis (OA). Studies suggest that activation of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is an important therapeutic target in OA. To devise PPARγ-related therapies in OA, it is critical to identify the role of this transcription factor in cartilage biology. Therefore, this study sought to determine the in vivo role of PPARγ in endochondral ossification and cartilage development, using cartilage-specific PPARγ-knockout (KO) mice. Methods Cartilage-specific PPARγ-KO mice were generated using the Cre/loxP system. Histomorphometric and immunohistochemical analyses were performed to assess the patterns of ossification, proliferation, differentiation, and hypertrophy of chondrocytes, skeletal organization, bone density, and calcium deposition in the KO mice. Results PPARγ-KO mice exhibited reductions in body length, body weight, length of the long bones, skeletal growth, cellularity, bone density, calcium deposition, and trabecular bone thickness, abnormal organization of the growth plate, loss of columnar organization, shorter hypertrophic zones, and delayed primary and secondary ossification. Immunohistochemical analyses for Sox9, 5-bromo-2\u27-deoxyuridine, p57, type X collagen, and platelet endothelial cell adhesion molecule 1 revealed reductions in the differentiation, proliferation, and hypertrophy of chondrocytes and in vascularization of the growth plate in mutant mice. Isolated chondrocytes and cartilage explants from mutant mice showed aberrant expression of Sox9 and extracellular matrix markers, including aggrecan, type II collagen, and matrix metalloproteinase 13. In addition, chondrocytes from mutant mice exhibited enhanced phosphorylation of p38 and decreased expression of Indian hedgehog. Conclusion The presence of PPARγ is required for normal endochondral ossification and cartilage development in vivo. Copyright © 2012 by the American College of Rheumatology