Biomarkers for tissue engineering of the tendon-bone interface

<div><p>The tendon-bone interface (enthesis) is a highly sophisticated biomaterial junction that allows stress transfer between mechanically dissimilar materials. The enthesis encounters very high mechanical demands and the regenerative capacity is very low resulting in high rupture recurrence rates after surgery. Tissue engineering offers the potential to recover the functional integrity of entheses. However, recent enthesis tissue engineering approaches have been limited by the lack of knowledge about the cells present at this interface. Here we investigated the cellular differentiation of enthesis cells and compared the cellular pattern of enthesis cells to tendon and cartilage cells in a next generation sequencing transcriptome study. We integrated the transcriptome data with proteome data of a previous study to identify biomarkers of enthesis cell differentiation. Transcriptomics detected 34468 transcripts in total in enthesis, tendon, and cartilage. Transcriptome comparisons revealed 3980 differentially regulated candidates for enthesis and tendon, 395 for enthesis and cartilage, and 946 for cartilage and tendon. An asymmetric distribution of enriched genes was observed in enthesis and cartilage transcriptome comparison suggesting that enthesis cells are more chondrocyte-like than tenocyte-like. Integrative analysis of transcriptome and proteome data identified ten enthesis biomarkers and six tendon biomarkers. The observed gene expression characteristics and differentiation markers shed light into the nature of the cells present at the enthesis. The presented markers will foster enthesis tissue engineering approaches by setting a bench-mark for differentiation of seeded cells towards a physiologically relevant phenotype.</p></div

Identified biomarkers of enthesis and tendon.

<p>Biomarkers are presented which were statistically significant enriched in both the proteomics and transcriptomics data sets of enthesis (E) or tendon (T). Orange and blue bars represent enrichment in enthesis and tendon, respectively. Gray boxes correspond to enrichment in proteome and black boxes to enrichment in transcriptome. 13 genes were identified as tendon biomarkers (blue) and 24 genes as enthesis biomarkers (orange). Two genes (COL14A1 and ECM1) were identified that were enriched in the tendon proteome and conversely in the enthesis transcriptome.</p

Comparative transcriptome analysis of enthesis and cartilage.

<p>a, Venn diagram depicts the number of annotated genes that were only present in enthesis (70) or cartilage (100) or present in both tissues (13832). b, Volcano plot of all differentially enriched genes detected in the transcriptomes of enthesis (orange) and cartilage (green). Statistically significant enriched genes were marked red. c, List of genes with highest enrichment of gene expression in enthesis (orange) or cartilage (green). d, Protein-protein interaction network for transcription factors and growth factors enriched in cartilage compared to enthesis. Protein-protein interactions were predicted using the database Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Line thickness indicated strength of data support for protein-protein interaction.</p

Identified biomarkers of enthesis and tendon.

<p>Biomarkers are presented which were statistically significant enriched in both the proteomics and transcriptomics data sets of enthesis (E) or tendon (T). Orange and blue bars represent enrichment in enthesis and tendon, respectively. Gray boxes correspond to enrichment in proteome and black boxes to enrichment in transcriptome. 13 genes were identified as tendon biomarkers (blue) and 24 genes as enthesis biomarkers (orange). Two genes (COL14A1 and ECM1) were identified that were enriched in the tendon proteome and conversely in the enthesis transcriptome.</p

Biomarkers that were at least twofold enriched in both transcriptome and proteome.

<p>The biomarkers as well as the respective functions and log₂ ratios are depicted. Log₂ ratio differences of proteome and transcriptome data sets were calculated for each candidate.</p

Morphological characteristics of enthesis cells.

<p>a, Enthesis cryocut section was stained for cells using SYTO® 13. Cells are depicted cyan, confocal reflection is depicted magenta. Scale bar corresponds to 150 μm. b, Scheme of cell arrangement observed at the enthesis. c, Cells within the interface are round shaped and are often arranged in pairs. Scale bar corresponds 50 μm. d, Cells residing within tendon are longitudinally arranged along the axis of tension in strings between tendon fibers. Scale bar corresponds 50 μm.</p

Top 10 genes with highest and lowest log₂ fold change in enthesis/cartilage transcriptome comparison.

<p>Gene names and functions derive from GeneCards^® and Ensembl.</p

Comparative transcriptome analysis of enthesis and tendon.

<p>a, Venn diagram of differences between enthesis and tendon transcriptomes depicts the number of annotated genes that are only present in tendon (69), enthesis (35) or present in both tissues (13867). b, Volcano plot of all differentially enriched transcripts detected in the transcriptomes of tendon and enthesis. Statistically significant enriched genes were marked red. c, Top 10 genes with highest gene expression difference in tendon (blue) or enthesis (orange).</p

Comparative transcriptome analysis of tendon, enthesis, and cartilage.

<p>a, Experimental design of the transcriptomic study. Tendon (blue), interface region (orange), and cartilage (green) samples were excised, shock-frozen in liquid nitrogen, chemically treated to prevent RNA degradation and cryocut sectioned to enhance RNA extraction. High quality RNA was extracted using a refined protocol including proteinase K and DNase digests. cDNA libraries were produced and sequenced using next generation sequencing on an Illumina platform. Transcriptomes of enthesis, tendon, and cartilage were compared to each other. b, Global differences in the transcriptome of enthesis (E), tendon (T), and cartilage (C). c, Venn diagram of genes detected in the three tissues showing the number of overlapping and differentially expressed genes. Only genes that were annotated and had FPKM > 0 were considered, duplicates were not considered twice.</p

Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies