Synchronized Real-time Measurement of Sec-mediated Protein Translocation.

The Sec translocon, consisting of a heterotrimeric transmembrane channel (SecYEG) and an associated ATPase (SecA), catalyzes the export of unfolded proteins from the cytosol in bacteria. Kinetically resolving protein translocation at high resolution yields mechanistic insight into the process. Translocation is typically followed by measuring the protection of proteins transported into lipid vesicles, which only allows visualization of translocation after it has already been completed and limits time resolution. Here, we describe the implementation of an assay for measuring translocation in real-time. By priming the reconstituted translocon with suitably engineered substrate proteins, the kinetics of the actual translocation process can be resolved at high resolution. To analyze translocation kinetics, we developed a detailed kinetic model of the process that includes on-pathway and off-pathway processes. Together, this experimental protocol and model permit detailed mechanistic analyses of Sec-dependent protein translocation. Graphic abstract: Synchronized real-time measurements, combined with a detailed kinetic model, enable a mechanistic analysis of protein transport.

The SecA motor generates mechanical force during protein translocation.

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 Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting 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 single-molecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.

Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy.

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.

MR T(1)ρ quantification of cartilage focal lesions in acutely injured knees: correlation with arthroscopic evaluation.

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.

CdS nanoclusters doped with divalent atoms.

ZnS and CdS small nanoclusters have been predicted to trap alkali metals and halogen atoms. However would this kind of nanocompounds be able to encapsulate dianions and dications? This would be very interesting from an experimental point of view, since it would allow the isolation of such divalent ions. Moreover, the resulting endohedral complexes would serve as building blocks for new cluster-assembled materials, with enhanced stability arising from the electrostatic interaction between the incarcerated ions. In this work we have studied the structure and stability of (X@(CdS)i)(±2) with X = Be, Mg, Ca, O, S, Se and i = 9, 12, 15, 16 on the basis of Density Functional Theory and Quantum Molecular Dynamics simulations. Most of the nanoclusters are found to trap both chalcogen and alkaline earth atoms. Furthermore, the chalcogen doped clusters are calculated to be both thermodynamically and thermally stable. However, only a few of alkaline earth metal doped structures are predicted to be thermally stable. Therefore, the charge of the dopant atom appears to be crucial in the endohedral doping. Additionally, the absorption spectra of the title compounds have been simulated by means of Time Dependent Density Functional Theory (TDDFT) calculations. The calculated optical features show a blueshift with respect to the bulk CdS wurtzite. Furthermore, doping modifies notably the optical spectra of nanoclusters, as the absorption spectra shift to lower energies upon encapsulation.