Detecting molecular fingerprints in single molecule force spectroscopy using pattern recognition

Single molecule force spectroscopy has given experimental access to the mechanical properties of protein molecules. Typically, less than 1% of the experimental recordings reflect true single molecule events due to abundant surface and multiple-molecule interactions. A key issue in single molecule force spectroscopy is thus to identify the characteristic mechanical ''fingerprint'' of a specific protein in noisy data sets. Here, we present an objective pattern recognition algorithm that is able to identify fingerprints in such noisy data sets

Bioorthogonal protein-DNA conjugation methods for force spectroscopy.

Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible

Dissection of Kinesin's Processivity

The protein family of kinesins contains processive motor proteins that move stepwise along microtubules. This mechanism requires the precise coupling of the catalytic steps in the two heads, and their precise mechanical coordination. Here we show that these functionalities can be uncoupled in chimera of processive and non-processive kinesins. A chimera with the motor domain of Kinesin-1 and the dimerization domain of a non-processive Kinesin-3 motor behaves qualitatively as conventional kinesin and moves processively in TIRF and bead motility assays, suggesting that spatial proximity of two Kinein-1 motor domains is sufficient for processive behavior. In the reverse chimera, the non-processive motor domains are unable to step along microtubules, despite the presence of the Kinesin-1 neck coiled coil. Still, ATP-binding to one head of these chimera induces ADP-release from the partner head, a characteristic feature of alternating site catalysis. These results show that processive movement of kinesin dimers requires elements in the motor head that respond to ADP-release and induce stepping, in addition to a proper spacing of the motor heads via the neck coiled coil

Myocardial CT perfusion imaging for the detection of obstructive coronary artery disease: multisegment reconstruction does not improve diagnostic performance

Background: Multisegment reconstruction (MSR) was introduced to shorten the temporal reconstruction window of computed tomography (CT) and thereby reduce motion artefacts. We investigated whether MSR of myocardial CT perfusion (CTP) can improve diagnostic performance in detecting obstructive coronary artery disease (CAD) compared with halfscan reconstruction (HSR). Methods: A total of 134 patients (median age 65.7 years) with clinical indication for invasive coronary angiography and without cardiac surgery prospectively underwent static CTP. In 93 patients with multisegment acquisition, we retrospectively performed both MSR and HSR and searched both reconstructions for perfusion defects. Subgroups with known (n = 68) or suspected CAD (n = 25) and high heart rate (n = 30) were analysed. The area under the curve (AUC) was compared applying DeLong approach using >= 50% stenosis on invasive coronary angiography as reference standard. Results: Per-patient analysis revealed the overall AUC of MSR (0.65 [95% confidence interval 0.53, 0.78]) to be inferior to that of HSR (0.79 [0.69, 0.88]; p = 0.011). AUCs of MSR and HSR were similar in all subgroups analysed (known CAD 0.62 [0.45, 0.79] versus 0.72 [0.57, 0.86]; p = 0.157; suspected CAD 0.80 [0.63, 0.97] versus 0.89 [0.77, 1.00]; p = 0.243; high heart rate 0.46 [0.19, 0.73] versus 0.55 [0.33, 0.77]; p = 0.389). Median stress radiation dose was higher for MSR than for HSR (6.67 mSv versus 3.64 mSv, p < 0.001). Conclusions: MSR did not improve diagnostic performance of myocardial CTP imaging while increasing radiation dose compared with HSR

Aortic aneurysm and aortic graft infection related to Mycobacterium bovis after intravesical Bacille Calmette–Guérin therapy—a case series

Background: So called "mycotic" aortic aneurysms account for only 0.7 to 1.3% of all aortic aneurysms and are commonly caused by Staphylococcus aureus and Salmonella species. Bacillus Calmette-Guerin (BCG), a live attenuated strain of Mycobacterium bovis, is part of the therapy of non-muscle-invasive bladder cancer (NMIBC). Case presentation: We report a case series of three patients with a mycobacterial graft infection related to BCG after surgical treatment of a presumed mycotic aortic aneurysm as an extremely rare complication after NMIBC treatment. All three patients developed aortic aneurysm after BCG instillation and subsequent mycobacterial graft infection. Conclusion: Diagnosis requires a high degree of suspicion because of its nonspecific symptoms and imaging. The pathogen is not detected by standard microbiological testing. Treatment includes triple antimycobacterial therapy and radical surgical interventions. Graft preservation may be considered if no anastomosis is involved

Auto- and cross-power spectral analysis of dual trap optical tweezer experiments using Bayesian inference

The thermal fluctuations of micron-sized beads in dual trap optical tweezer experiments contain complete dynamic information about the viscoelastic properties of the embedding medium and—if present—macromolecular constructs connecting the two beads. To quantitatively interpret the spectral properties of the measured signals, a detailed understanding of the instrumental characteristics is required. To this end, we present a theoretical description of the signal processing in a typical dual trap optical tweezer experiment accounting for polarization crosstalk and instrumental noise and discuss the effect of finite statistics. To infer the unknown parameters from experimental data, a maximum likelihood method based on the statistical properties of the stochastic signals is derived. In a first step, the method can be used for calibration purposes: We propose a scheme involving three consecutive measurements (both traps empty, first one occupied and second empty, and vice versa), by which all instrumental and physical parameters of the setup are determined. We test our approach for a simple model system, namely a pair of unconnected, but hydrodynamically interacting spheres. The comparison to theoretical predictions based on instantaneous as well as retarded hydrodynamics emphasizes the importance of hydrodynamic retardation effects due to vorticity diffusion in the fluid. For more complex experimental scenarios, where macromolecular constructs are tethered between the two beads, the same maximum likelihood method in conjunction with dynamic deconvolution theory will in a second step allow one to determine the viscoelastic properties of the tethered element connecting the two beads

Patient preferences for development in MRI scanner design: a survey of claustrophobic patients in a randomized study

Objective: To investigate which magnetic resonance imaging (MRI) scanner designs claustrophobic patients prefer. Material/methods: We analyzed questionnaires completed by 160 patients at high risk for claustrophobia directly after a scan in either a short-bore or open panoramic scanner as part of a prospective randomized trial Enders et al (BMC Med Imaging 11:4, 2011). Scanner preferences were judged based on schematic drawings of four scanners. Information on the diagnostic performance of the depicted scanners was provided, too. Results: A majority of patients suggested upright open (59/160, 36.9%) and open panoramic (53/160, 33.1%) before short-bore designs (26/160, 16.3%, for all p < 0.001) for future development. When asked about patients' preferred scanner choice for an upcoming examination, information about a better diagnostic performance of a short-bore scanner significantly improved its preference rates (from 6/160 to 49/160 or 3.8 to 30.5%, p < 0.001). Patients with a claustrophobic event preferred open designs significantly more often than patients without a claustrophobic event (p = 0.047). Patients scanned in a short-bore scanner in our trial preferred this design significantly more often (p = 0.003). Noise reduction (51/160, 31.9%), more space over the head (44/160, 27.5%), and overall more space (33/160, 20.6%) were the commonest suggested areas of improvement. Conclusion: Patients at high risk for claustrophobia visually prefer open- over short-bore MRI designs for further development. Education about a better diagnostic performance of a visually less-attractive scanner can increase its acceptance. Noise and space were of most concern for claustrophobic patients. This information can guide individual referral of claustrophobic patients to scanners and future scanner development. Key points: • Patients at high risk for claustrophobia visually favor the further development of open scanners as opposed to short- and closed-bore scanner designs. • Educating claustrophobic patients about a higher diagnostic performance of a short-bore scanner can significantly increase their acceptance of this otherwise visually less-attractive design. • A medical history of earlier claustrophobic events in a given MRI scanner type and focusing on the features "more space" and "noise reduction" can help to guide referral of patients who are at high risk for claustrophobia

A Randomized Controlled Comparison of Image Quality

Background The purpose of the present study was to compare the image quality of spinal magnetic resonance (MR) imaging performed on a high-field horizontal open versus a short-bore MR scanner in a randomized controlled study setup. Methods Altogether, 93 (80% women, mean age 53) consecutive patients underwent spine imaging after random assignement to a 1-T horizontal open MR scanner with a vertical magnetic field or a 1.5-T short-bore MR scanner. This patient subset was part of a larger cohort. Image quality was assessed by determining qualitative parameters, signal-to-noise (SNR) and contrast-to-noise ratios (CNR), and quantitative contour sharpness. Results The image quality parameters were higher for short-bore MR imaging. Regarding all sequences, the relative differences were 39% for the mean overall qualitative image quality, 53% for the mean SNR values, and 34–37% for the quantitative contour sharpness (P<0.0001). The CNR values were also higher for images obtained with the short-bore MR scanner. No sequence was of very poor (nondiagnostic) image quality. Scanning times were significantly longer for examinations performed on the open MR scanner (mean: 32±22 min versus 20±9 min; P<0.0001). Conclusions In this randomized controlled comparison of spinal MR imaging with an open versus a short-bore scanner, short-bore MR imaging revealed considerably higher image quality with shorter scanning times