miR-92b-5p negatively regulates IKK through targeting its ORF region in the innate immune responses of amphioxus (Branchiostoma belcheri).

Cephalochordate (Amphioxus), situated at a key phylogenetic position in the phylum Chordata, serves as a model organism for studying the origin and evolution of the vertebrate innate immune. In this study, five members of precursor miR-92 family (miR-92a-1, miR-92a-2, miR-92b, miR-92c and miR-92d) are identified in Branchiostoma belcheri, and their evolutionary conservation and potential molecular functions in innate immunity are analyzed. Among them, miR-92b-5p was validated in HEK293 cells to target the coding region but not classic 3'UTR of IKK (inhibitor of nuclear factor kappa-B kinase) mRNA, one integral component of MAPK and TLR4 immune signaling. Furthermore, the spatiotemporal expression patterns of miR-92b-5p and IKK were examined in different tissues or different time points (2 h, 4 h, 8 h, 12 h, 24 h and 48 h) post LPS stimulation at RNA and protein level in vivo. The seemingly inverse expression pattern between miR-92b-5p and IKK supports the involvement of miR-92b-5p in Branchiostoma belcheri innate immune response. In conclusion, our work not only illustrates the evolutionary pattern of Branchiostoma belcheri miR-92 family across chordates, but also reveals that miR-92b-5p could target IKK expression to regulate innate immune response.

miR-2013 negatively regulates phylogenetically conserved PIP5K involved in TLR4 mediated immune responses of amphioxus (Branchiostoma belcheri Tsingtaunese).

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a catalytic kinase that performs multiple functions in organisms. Recent studies have shown that PIP5Kα in mammals can directly participate in the TLR-mediated innate immune regulation by controlling the production of PIP2. However, the PIP5K homologous gene has not been identified in Cephalochordata to date. In this study, we firstly identify and characterize a new PIP5K family member (designed as AmphiPIP5K) from Cephalochordata amphioxus (Branchiostoma belcheri tsingtaunese), particularly AmphiPIP5K is orthologous with vertebrate PIP5Kα and paralogous with PIP5Kß and PIP5Kγ. Secondly, we find that the AmphiPIP5K is involved in amphioxus innate immune response to LPS stimulation. Thirdly, our results demonstrate that miR-2013 can inhibit AmphiPIP5K expression by binding to the CDS and 3' UTR regions of AmphiPIP5K. Collectively, our work not only demonstrates the evolutionary pattern of amphioxus PIP5K, but also reveals that miR-2013 negatively regulate PIP5K expression to involve in amphioxus innate immune response.

A protease-insensitive feruloyl esterase from China Holstein cow rumen metagenomic library: expression, characterization, and utilization in ferulic acid release from wheat straw.

A metagenomic library of China Holstein cow rumen microbes was constructed and screened for novel gene cluster. A novel feruloyl esterase (FAE) gene was identified with a length of 789 bp and encoded a protein displaying 56% identity to known esterase sequences. The gene was functionally expressed in Escherichia coli BL21 (DE3), and the total molecular weight of the recombined protein was 32.4 kDa. The purified enzyme showed a broad specificity against the four methyl esters of hydroxycinnamic acids and high activity (259.5 U/mg) to methyl ferulate at optimum conditions (pH 8.0, 40 °C). High thermal and pH stability were also observed. Moreover, the enzyme showed broad resistance to proteases. FAE-SH1 can enhance the release of ferulic acid from wheat straw with cellulase, ß-1,4-endoxylanase, ß-1,3-glucanase, and pectase. These features suggest FAE-SH1 as a good candidate to enhance biomass degradation and improve the health effects of food and forage.