Ras1^CA-Upregulated <i>bcpi</i> Inhibits Cathepsin Activity to Prevent Tissue Destruction of the <i>Bombyx</i> Posterior Silk Gland

Using the GAL4/UAS transgenic system established in the silkworm, <i>Bombyx mori</i>, we have previously reported that overexpression of the <i>Ras1</i>^CA oncogene specifically in the posterior silk gland (PSG) resulted in improved fibroin synthesis, silk yield, and other phenotypic effects. However, the detailed molecular mechanism remains to be fully elucidated. Using 2D-DIGE-MS/MS analyses, we compared the proteomic profiles of PSGs from the wild type (WT) and <i>Ras1</i>^CA-overexpressed silkworms. Among the 24 <i>Ras1</i>^CA-enhanced proteins, the <i>Bombyx</i> cysteine protease inhibitor (BCPI) was increased 2.4-fold at the protein level and 3.4-fold at the mRNA level. Consistent with the developmental profiles, injection of recombinant BCPI into the WT silkworms at the early wandering stage inhibited cathepsin activity, prevented tissue destruction of the PSG, and delayed pupation. Moreover, injection of small-molecule inhibitors of cathepsin into the WT silkworms prevented PSG destruction and delayed pupation, confirming the role of BCPI in inhibiting cathepsin activity. Furthermore, injection of chemical inhibitors of the Ras downstream effectors into the <i>Ras1</i>^CA-overexpressed and WT silkworms revealed that both Raf-MAPK and PI3K-TORC1 pathways were required for Ras1 to induce <i>bcpi</i> expression. Taken together, we conclude that via the downstream Raf-MAPK and PI3K-TORC1 pathways, <i>Ras1</i>^CA upregulates <i>bcpi</i>, which inhibits cathepsin activity thus preventing PSG destruction in <i>Bombyx</i>

Data_Sheet_1_Exploration of the potential mechanism of Baicalin for hepatic fibrosis based on network pharmacology, gut microbiota, and experimental validation.PDF

Baicalin (BA) is among the most effective and abundant flavonoids extracted from Scutellaria baicalensis that may be utilized to treat diseases associated with hepatic fibrosis (HF). Through network pharmacology, gut microbiota, and experimental validation, this research intends to elucidate the multi-target mechanism of BA on HF. BA targets were screened using databases and literature. As a result, In the anti-HF mechanism, the BA and 191 HF-associated targets interact, with 9 specific targets indicating that the BA’s anti-HF mechanism is closely linked to gut microbiota. Consequently, rat intestinal content samples were obtained and examined using 16S rRNA sequencing. In the BA-treated group, the gut microbiota was positively regulated at the phylum,and genus levels, with Lactobacillus performing significantly. The study concluded that BA has a multi-targeted anti-HF effect and has changed the gut microbial ecosystem.</p

Data_Sheet_2_Exploration of the potential mechanism of Baicalin for hepatic fibrosis based on network pharmacology, gut microbiota, and experimental validation.PDF

Baicalin (BA) is among the most effective and abundant flavonoids extracted from Scutellaria baicalensis that may be utilized to treat diseases associated with hepatic fibrosis (HF). Through network pharmacology, gut microbiota, and experimental validation, this research intends to elucidate the multi-target mechanism of BA on HF. BA targets were screened using databases and literature. As a result, In the anti-HF mechanism, the BA and 191 HF-associated targets interact, with 9 specific targets indicating that the BA’s anti-HF mechanism is closely linked to gut microbiota. Consequently, rat intestinal content samples were obtained and examined using 16S rRNA sequencing. In the BA-treated group, the gut microbiota was positively regulated at the phylum,and genus levels, with Lactobacillus performing significantly. The study concluded that BA has a multi-targeted anti-HF effect and has changed the gut microbial ecosystem.</p

Primers used in this study.

<p>Primers used in this study.</p

<i>H. armigera</i> larvae feeding with transgenic tobacco plants expressing <i>HaEcR</i> dsRNA die with significant molting defects.

<p>Fifty <i>H. armigera</i> larvae were fed with a detached mature leaf maintained in an 80 mm sterile plastic flask. Three similar leaves from the same plant were repeated in a feeding bioassay. The other conditions are the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038572#pone-0038572-g003" target="_blank">Figure 3</a>. (A) Leaves from transgenic tobacco plants expressing <i>HaEcR</i> dsRNA were ingested significantly less by <i>H. armigera</i> larvae after 5 days of incubation in comparison with those expressing <i>GFP</i> dsRNA. (B) The growth of <i>H. armigera</i> larvae feeding with transgenic tobacco leaves expressing <i>HaEcR</i> dsRNA was significantly delayed and their body sizes reduced. (C) Feeding with transgenic tobacco leaves expressing <i>HaEcR</i> dsRNA (No. 1–4) caused significantly higher lethality than in the control.</p

<i>HaEcR</i> expression in <i>H. armigera</i> is suppressed by feeding with leaves of transgenic tobacco plants expressing <i>HaEcR</i> dsRNA.

<p><i>HaEcR</i> expression in <i>H. armigera</i> is suppressed by feeding with leaves of transgenic tobacco plants expressing <i>HaEcR</i> dsRNA.</p

Ingestion of bacterial-expressed <i>HaEcR</i> dsRNA results in molting defects and lethality in <i>H. armigera</i> larvae.

<p>(A) The L4440<i>-HaEcR</i> construct producing <i>HaEcR</i> dsRNA in <i>E. coli</i> HT115. (B) Expression of <b><i>HaEcR</i></b> dsRNA was confirmed by electrophoresis on 1% agarose gel. (C) Ingestion of bacterial-expressed <i>HaEcR</i> dsRNA caused up to 60% larval lethality in <i>H. armigera</i>. (D) Some <i>H. armigera</i> larvae died as larval-pupal intermediates.</p

DataSheet_1_Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan.pdf

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.</p

MET Is Required for the Maximal Action of 20-Hydroxyecdysone during <em>Bombyx</em> Metamorphosis

<div><p>Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone (20E; the molting hormone) action. Here we report that two MET proteins found in the silkworm, <em>Bombyx mori</em>, participate in 20E signal transduction. <em>Met</em> is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of <em>EcR</em>-<em>USP</em> (the ecdysone receptor genes), RNAi knockdown of <em>Met</em> at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling (including autophagy, apoptosis and destruction of larval tissues and generation of adult structures) and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during <em>Bombyx</em> metamorphosis.</p> </div

Physical interaction between MET and EcR-USP.

<p>(A) The CytoTrap yeast two-hybrid analyses revealed direct associations among MET1, MET2, EcR and USP. Strong associations between bait and prey proteins led to more yeast colonies. (B) When the <i>HA-EcR</i>, <i>FLAG-USP</i>, and <i>V5-Met1</i> constructs were co-transfected into human HEK 293 cells, 20E treatment for 6 hr at a final concentration of 1 µM had little or no stimulating effects on the physical interactions between MET and EcR-USP. In the immunoprecipation experiments, the bottom Western blot is input. IP, immunoprecipitate; Blot, Western blot. (C) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into the human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated on a 5% native PAGE gel followed by EMSA. Addition of the HA or FLAG antibody resulted in a shift of EcRE. In (C) and (D), the shift was indicated by a black arrow in comparison with a gray arrow. (D) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated 5% native PAGE followed by EMSA. When the V5 or cMyc antibody was added, binding of EcR-USP-EcRE was shifted by MET1 and MET2 in EMSA showing that MET, EcR-USP and EcRE form a protein-DNA complex. (E) <i>Met1</i> RNAi and transfection were simultaneously conducted in <i>Bombyx</i> DZNU-Bm-12 cells for 48 hr, followed by 20E treatment for 6 hr at a final concentration of 1 µM, and measurements of EcRE-driven luciferase activity were done. MET is required for 20E function to induce gene expression via the ecdysone receptor and EcRE. The bars labeled with different lowercase letters are significantly different (P<0.05, ANOVA).</p