Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM.

Objective Quantitative T1ρ MRI has been suggested as a promising tool to detect changes in cartilage composition that are characteristic of cartilage damage and degeneration. The objective of this study was to evaluate the capability of MR T1ρ to detect cartilage lesions as evaluated by arthroscopy in acutely ACL-injured knees and to compare with the Whole-Organ Magnetic Resonance Imaging Score (WORMS) using clinical standard MRI. Method Ten healthy controls (mean age 35) with no ACL injury or history of osteoarthritis (OA) and 10 patients with acute ACL injuries (mean age 39) were scanned at 3 Tesla (3T). ACL patients underwent ACL reconstruction, where focal lesions were graded according to an Outerbridge grading system during arthroscopic evaluation. Normalized MR T1ρ values (T1ρ z-scores normalized to control values in matched regions) in full thickness, and superficial and deep layers of cartilage were compared between defined sub-compartments with and without focal lesions. Intraclass (ICC) correlation and the root mean square coefficient of variation (RMS-CV) were performed to evaluate the inter-observer reproducibility of T1ρ quantification. Sub-compartments of cartilage were also evaluated using WORMS scoring and compared to their Outerbridge score respectively. Results The inter-observer ICC and the RMS-CV of the sub-compartment T1ρ quantification were 0.961 and 3.9%, respectively. The average T1ρ z-scores were significantly increased in sub-compartments with focal lesions compared to those without focal lesions and to the control cohort (p < 0.05). Conclusion Our results indicate that T1ρ provided a better diagnostic capability than clinical standard MRI grading in detecting focal cartilage abnormalities after acute injuries. Quantitative MRI may have great potential in detecting cartilage abnormalities and degeneration non-invasively, which are occult with standard morphological MRI.

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Here, we kinetically dissect Secdependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the ratelimiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and singlemolecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Monitoring translocation of a folded substrate protein with tunable stability at high time resolution allowed us to kinetically dissect Secdependent translocation. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we have also defined the response of the protein to mechanical force. Relating the kinetic and force measurements revealed that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements has thus allowed us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.