Primers used for RT-PCR, qPCR and 3′RACE.

<p>Primers used for RT-PCR, qPCR and 3′RACE.</p

Lentiviral-Mediated RNAi Knockdown of Cbfa1 Gene Inhibits Endochondral Ossification of Antler Stem Cells in Micromass Culture

<div><p>Articular cartilage (AC) lacks ability to repair defects due to its avascular nature as healing process relies on cells being brought in by blood vessels. Multiple approaches have been taken to facilitate cartilage repair in clinics, to date there is no effective treatment available that can restores the AC lesion to a normally functioning level over extended periods. In this regard, antler cartilage is unique in being richly vascularised and hence can effectively repair and regenerate. Interestingly, antler stem cells, from which the vascularised cartilage is derived, can form avascular cartilage when taken away from their original niche, suggesting that the vascular or avascular state of antler cartilage is controlled by extrinsic factors. Understanding the mechanisms underlying this phenotype switch may help us to devise a way to trigger the effective intrinsic repair of AC. However, adoption of antler cartilage model for AC repair requires the demonstration that the cartilage specific signalling pathways also prevail in antler chondrogenesis. To achieve this, in the present study we silenced expression of Cbfa1, a key factor regulatingendochondral ossification, using RNAi, and showed that expression of the downstream genes type I collagen and osteocalcin were suppressed which, in turn, inhibited endochondral ossification process taking place in the antler stem cell-formed nodules. Therefore, we provided further evidence at molecular level that antler could be developed as novel model for the study of AC repair. The eventual identification of the extrinsic factors dictating the phenotype switch between the vascular and avascular state of antler cartilage will open up a new avenue for the cure of osteoarthritis.</p> </div

Effects of Cbfa 1 silencing on chondrogenic and osteogenic differentiation in antler stem cell-derived nodules.

<p>A and B: Histology (200X). A, From the uninfected nodule, remodelling process (asterisk), chondroclast-like cells (arrow) and typical chondrocytes residing in the lacunae (inset) could be observed in the central region of the tissue. B, From the S6-infected nodule, no sign of EO in the S6-infected nodule could be detected. C and D: Alcian blue staining (400X). C, From the uninfected nodule. B, From the S6-infected nodule. Note that the section from the uninfected nodule was heavily stained, whereas from the S6-infected nodule was barely stained. E and F: Alizarin red staining (400X). E, From the uninfected nodule. F, From the S6-infected nodule. Note that two foci on the tissue section from the uninfected nodule were heavily stained (arrows), whereas the tissue section from the S6-infected nodule was not stained.</p

Interfering sequences of siRNA targeting for Cbfa1.

<p>Interfering sequences of siRNA targeting for Cbfa1.</p

Morphology and GFP expression of antler stem cell-derived nodules.

<p>A and B: Nodule morphology. A, Nodule from the S6-infected group. B, Nodule from the control (scrambled sequence) group. Note that there was essentially no difference in shape and size between theS6-infected and the control groups. C and D: GFP expression (50x). C, Nodule from the S6-infected group. D, Nodule from the control group. Note that numerous fluorescent dots could be observed from the S6-infected nodule, but the control nodule only showed faint auto-fluorescence.</p

Effects of Cbfa 1 silencing on the expression of type I collagen and osteocalcin genes.

<p>A: qPCRof type I collagen. Note that all of the siRNA Cbfa1-target sequences(S1–S6) suppressed expression of downstream gene type I collagen compared to the scrambled sequence (C), particularly the S6 sequence that had most dramatic effects and knocked down expression of type I collagen gene up to 86.8%. B–D: Immunohistochemistry of osteocalcin (400X). B, Section from the uninfected nodule. C, Section from the S6-infected nodule. D, Section from the S6-infected nodule but in the absence of the primary antibody. Note that uninfected nodule had the strongest staining, and the staining of the S6-infected nodule was significantly weaker, but still stronger than the S6-infected nodule in the absence of the primary antibody.</p

Relationships between microbial populations and fermentation products in the rumen of sika deer fed tannin rich plants.

<p>(A) Correlation between microbial populations and fermentation products. Strong correlations are indicated by large circles, whereas weak correlations are indicated by small circles. The colors of the scale bar denote the nature of the correlation with 1 indicating perfect positive correlation (dark blue) and -1 indicating the negative correlation (dark red). (B) Co-occurrence network analysis among microbial populations and fermentation products. Bright blue circle nodes represent microbial populations at genus level, and rounded rectangle nodes represent fermentation products. Each co-occurring pair among microbial populations at genus level and fermentation products has an absolute Spearman rank correlation above 0.70 [Gold line: positive correlation (R >0.70); Gray line: negative correlation (R <-0.70)] with an false discovery rate-corrected significance level under 0.05. Different groups of microorganisms and fermentation products were indicated by various colors: Bacteria (blue), Methanogens (green), Protozoa (purple), Fungi (orange), Fermentation products (red). Par = Paraprevotellaceae, <i>Mbr</i> = <i>Methanobrevibacter</i>, <i>Msp</i> = <i>Methanosphaera</i>, <i>Ep</i> = <i>Epidinium</i>, <i>Ent</i> = <i>Entodinium</i>, <i>Eud</i> = <i>Eudiplodinium</i>.</p

The composition of rumen fungi in the two groups.

<p>OLH = High group, OLL = Low group.</p

The composition of rumen methanogens in the two groups.

<p><i>Mbr</i> = <i>Methanobrevibacter</i>, <i>Msp</i> = <i>Methanosphaera</i>, <i>Mth</i> = <i>Methanomethylophilus</i>, OTUs = operational taxonomic units, OLH = High group, OLL = Low group.</p

The bacterial community composition in the two groups at phylum level (A) and genus level (B).

<p>OLH = High group, OLL = Low group. The relative abundance of Firmicutes*, <i>Pseudobutyrivibrio</i>*, and unidentified bacteria and <i>Prevotella</i> belonging to the family Paraprevotellaceae* was significantly different between the two groups, with * means the significance at <i>P</i><0.05.</p