Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM

The β-barrel assembly machinery (BAM) is a ~203 kDa complex of five proteins (BamA-E) which is essential for viability in E. coli. BAM promotes the folding and insertion of β-barrel proteins into the outer membrane via a poorly understood mechanism. Several current models suggest that BAM functions through a ‘lateral gating’ motion of the β-barrel of BamA. Here we present a cryo-EM structure of the BamABCDE complex, at 4.9 Å resolution. The structure is in a laterally open conformation showing that gating is independent of BamB binding. We describe conformational changes throughout the complex, and interactions between BamA, B, D, and E and the detergent micelle that suggest communication between BAM and the lipid bilayer. Finally, using an enhanced reconstitution protocol and functional assays, we show that for the outer membrane protein OmpT, efficient folding in vitro requires lateral gating in BAM

MicroRNA Signature Characterizes Primary Tumors That Metastasize in an Esophageal Adenocarcinoma Rat Model

<div><p>Objective</p><p>To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).</p><p>Background</p><p>The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.</p><p>Methods</p><p>The modified Levrat’s surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.</p><p>Results</p><p>The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.</p><p>Conclusion</p><p><i>In vivo</i> metastasis was confirmed in the modified Levrat’s model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.</p></div

Eleven differentially expressed miRNAs specific to distant organs excluding stomach.

<p>A two-tailed, two-sample equal variance T-test was performed to obtain the p-values.</p><p>Eleven differentially expressed miRNAs specific to distant organs excluding stomach.</p

Cellular interactions between miRNAs and target genes.

<p>Green represents downregulation and red represents upregulation of gene expression. A two-tailed, two-sample equal variance T-test was performed to obtain the p-values.</p

Immunofluorescence staining of rat tissue with MUC5AC.

<p>Panel A to F show immunofluorescence staining for metastatic lung, metastatic liver, metastatic stomach, non-metastatic lung, non-metastatic liver and non-metastatic stomach, respectively for representative cases. Positive MUC5AC cytoplasmic staining was detected in all metastatic samples with the Alexa Fluor 488 secondary antibody, conjugated to a green fluorophore. Metastasis negative liver and lung show the absence of MUC5AC staining. However, metastasis negative stomach stains positive for MUC5AC as gastric mucin M1 antigen is found in mucus cells of gastric epithelium.</p

Study schema outlining the major steps in the experimental design and miRNA analysis.

<p>Study schema outlining the major steps in the experimental design and miRNA analysis.</p

Thirty differentially expressed miRNAs common to all organs (stomach and other distant sites).

<p>A two-tailed, two-sample equal variance T-test was performed to obtain the p-values.</p><p>Thirty differentially expressed miRNAs common to all organs (stomach and other distant sites).</p