Analysis of Altered Baseline Brain Activity in Drug-Naive Adult Patients with Social Anxiety Disorder Using Resting-State Functional MRI

Objective

We hypothesize that the amplitude of low-frequency fluctuations (ALFF) is involved in the altered regional baseline brain function in social anxiety disorder (SAD). The aim of the study was to analyze the altered baseline brain activity in drug-naive adult patients with SAD.

Methods

We investigated spontaneous and baseline brain activities by obtaining the resting-state functional magnetic resonance imaging data of 20 drug-naïve adult SAD patients and 19 healthy controls. Voxels were used to analyze the ALFF values using one- and two-sample t-tests. A post-hoc correlation of clinical symptoms was also performed.

Results

Our findings show decreased ALFF in the bilateral insula, left medial superior frontal gyrus, left precuneus, left middle temporal gyrus, right middle temporal pole, and left fusiform gyrus of the SAD group. The SAD patients exhibited significantly increased ALFF in the right inferior temporal gyrus, right middle temporal gyrus, bilateral middle occipital gyrus, orbital superior frontal gyrus, right fusiform gyrus, right medial superior frontal gyrus, and left parahippocampal gyrus. Moreover, the Liebowitz Social Anxiety Scale results for the SAD patients were positively correlated with the mean Z values of the right middle occipital and right inferior occipital but showed a negative correlation with the mean Z values of the right superior temporal gyrus and right medial superior frontal gyrus.

Conclusion

These results of the altered regional baseline brain function in SAD suggest that the regions with abnormal spontaneous activities are involved in the underlying pathophysiology of SAD patients.     

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

The ion motive force (IMF) across the cell membrane is one of the most important sources of biological energy in any cell. The IMF is utilized for many essential biological activities, such as ATP synthesis, solute transport, nutrient uptake, protein secretion, flagella-driven motility, and so on (1). The IMF is the sum of the electrical (Δψ) and chemical (ΔpI) potential differences of ions such as protons (H⁺) (the proton motive force [PMF]) and sodium ions (Na⁺) (the sodium motive force [SMF]) across the membrane and is defined by Eq. 1:IMF=Vm+kBTq ln[ion]in[ion]ex,[1]where V_m is Δψ; [ion]_in and [ion]_ex are the internal and external ion concentrations, respectively; k_B is Boltzmann’s constant; T is the absolute temperature (in kelvins); and q is the charge of the ion. The Δψ corresponds to the membrane voltage (2).The flagellum of the enteric bacterium Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella) is a supramolecular motility machine consisting of the basal body, which acts as a bidirectional rotary motor; the hook, which functions as a universal joint; and the filament, which works as a helical propeller. The Salmonella flagellar motor is powered by a PMF across the cytoplasmic membrane. The motor consists of a rotor and multiple stator units, each of which acts as a transmembrane proton channel complex. The stator unit converts the proton influx through the channel into the force for high-speed rotation of the long helical filament (3, 4).For construction of the hook and filament structures at the cell exterior, a specialized protein transporter utilizes the PMF to transport flagellar building blocks to the distal end of the growing flagellar structure. The flagellar protein transporter consists of a PMF-driven export gate complex made of five transmembrane proteins, FlhA, FlhB, FliP, FliQ, and FliR, and an ATPase ring complex consisting of three cytoplasmic proteins, FliH, FliI, and FliJ (SI Appendix, Fig. S1) (5, 6). These proteins are evolutionarily related to those of the virulence-associated type III secretion systems of pathogenic bacteria, which inject effector proteins into eukaryotic host cells for invasion (7). Furthermore, the entire structure of the ATPase ring complex is structurally similar to the cytoplasmic F₁ part of F_OF₁-ATP synthase, which utilizes the PMF for ATP synthesis (8–10).FliI forms a homo-hexamer that hydrolyzes ATP at an interface between neighboring FliI subunits (10–12). FliJ binds to the central pore of the FliI ring (9). ATP hydrolysis by the FliI ATPase not only activates the transmembrane export gate complex through an interaction between FliJ and the C-terminal cytoplasmic domain of FlhA (FlhA_C) (13, 14) but also opens the entrance gate of the polypeptide channel through an interaction between FliI and the C-terminal cytoplasmic domain of FlhB (FlhB_C) (15). As a result, the export gate complex becomes an active proton/protein antiporter that couples an inward-directed H⁺ flow with an outward-directed protein export (SI Appendix, Fig. S1) (16). When the cytoplasmic ATPase complex becomes nonfunctional, the FlgN chaperone activates the Na⁺-driven export engine of the export gate complex over a wide range of external pH, allowing the export gate complex to drive Na⁺-coupled protein export (17, 18). The transmembrane domain of FlhA (FlhA_TM) acts as a transmembrane ion channel for the transit of both H⁺ and Na⁺ across the cytoplasmic membrane (17).A chemical potential gradient of either H⁺ (ΔpH) or Na⁺ (ΔpNa) is required for efficient inward-directed translocation of H⁺ or Na⁺ when FliH and FliI are absent (13, 17). Although the Δψ component is critical for flagellar protein export by the wild-type export gate complex (19), it remains unknown when and how Δψ is used for the flagellar protein export process. To clarify this question, we used the Salmonella MMHI0117 [ΔfliH-fliI flhB(P28T)] strain (hereafter referred to as ΔHI B*; Table 1) (20), in which the export gate complex uses both Δψ and ΔpNa at different steps of the flagellar protein export process (13, 17). We show that an increase in Δψ generated by an upward shift of the external pH from 7.0 to 8.5 activates flagellar protein export by this mutant even in the absence of ΔpNa, suggesting the presence of a Δψ-dependent activation mechanism for proton-coupled protein secretion by the export gate complex. We also show that an increased Δψ facilitates efficient docking of FliJ to FlhA_C.Table 1.Summary for flagellar protein export properties of Salmonella strains used in this study

Selectin catch–slip kinetics encode shear threshold adhesive behavior of rolling leukocytes

The selectin family of leukocyte adhesion receptors is principally recognized for mediating transient rolling interactions during the inflammatory response. Recent studies using ultrasensitive force probes to characterize the force–lifetime relationship between P- and L-selectin and their endogenous ligands have underscored the ability of increasing levels of force to initially extend the lifetime of these complexes before disrupting bond integrity. This so-called “catch–slip” transition has provided an appealing explanation for shear threshold phenomena in which increasing levels of shear stress stabilize leukocyte rolling under flow. We recently incorporated catch–slip kinetics into a mechanical model for cell adhesion and corroborated this hypothesis for neutrophils adhering via L-selectin. Here, using adhesive dynamics simulations, we demonstrate that biomembrane force probe measurements of various P- and L-selectin catch bonds faithfully predict differences in cell adhesion patterns that have been described extensively in vitro. Using phenomenological parameters to characterize the dominant features of molecular force spectra, we construct a generalized phase map that reveals that robust shear-threshold behavior is possible only when an applied force very efficiently stabilizes the bound receptor complex. This criteria explains why only a subset of selectin catch bonds exhibit a shear threshold and leads to a quantitative relationship that may be used to predict the magnitude of the shear threshold for families of catch–slip bonds directly from their force spectra. Collectively, our results extend the conceptual framework of adhesive dynamics as a means to translate complex single-molecule biophysics to macroscopic cell behavior.     

Over the wire lead extraction and focused force venoplasty to regain venous access in a totally occluded subclavian vein

Introduction  Venoplasty allows the addition or replacement of leads despite subtotal or total subclavian occlusion. Methods  The threshold of the LV pacing lead implanted for biventricular pacing over a period of 18 months increased to greater than 5 V. A pre implant venogram revealed total subclavian occlusion. Venous access was maintained by extraction of the 4 F LV lead over a wire. Subsequently the sheath would not advance despite 6mm balloon inflation to 30 atm with no residual waist. A wire was placed beside the balloon and the balloon was reinflated. Results  The subclavian obstruction was eliminated without damage to the existing leads. Conclusion  The obstruction formed by the fibrous track around an extracted lead may persist despite what appears to be successful balloon dilation. Inflation with a wire beside the balloon increases the effect eliminating the resistant obstruction without damaging the leads. Acknowledgements of Sources of Financial Support:Dr. Worley receives compensation in various forms from Medtronic, Pressure Products, Guidant, and St Jude Medical. Dr. Gohn receives compensation in various forms from Medtronic. No financial support was provided for the creation of this case report     

Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α₃D using atomic force microscopy (AFM)–based force spectroscopy. α₃D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α₃D’s configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy’s ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α₃D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam–modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 k_BT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Single-molecule force spectroscopy (SMFS) has been remarkably successful across broad classes of biological molecules (RNA, DNA, and proteins) (1–5). A particularly fruitful data acquisition regime probes multiple back-and-forth folding/unfolding transitions at near-equilibrium and equilibrium conditions (6–9). This methodology efficiently yields numerous transitions and therefore a wealth of kinetic data, one-dimensional (1D) free-energy landscape parameters, and even a full 1D projection of the free-energy landscape along the stretching axis (10, 11). The standard SMFS assay has the molecule of interest tethered via a flexible linker to the force probe, such as an optically trapped bead or an atomic force microscopy (AFM) cantilever (Fig. 1A). These micrometer-sized force probes are the primary measurement (x_meas) but have finite response time and are therefore coupled to, but do not precisely track, molecular dynamics (x_prot) (Fig. 1B) (12–14). Additionally, the flexible linker’s compliance modifies this coupling between the molecule and the force probe. Linkers stretched at a finite force (F) can even create an entropic barrier not present in the absence of applied force (15, 16). More generally, there is an expanding set of theoretical and experimental studies (12–30) investigating how such instrumental and assay parameters affect the underlying biomolecular dynamics and whether the measured dynamics are dominated by the instrument used to measure them.Open in a separate windowFig. 1.Probing the folding and unfolding dynamics of a globular protein by SMFS. (A) Cartoon showing a polyprotein consisting of a single copy of α₃D (blue) and two copies of NuG2 (red) stretched with an atomic force microscope. At low forces, the mechanically labile α₃D repeatedly unfolds and refolds as detected by a change in cantilever deflection. (B) A conceptual two-dimensional free-energy landscape shows the underlying protein extension (x_prot) and the experimentally measured extension (x_meas). The macroscopic force probe has finite temporal resolution, and the application of force can introduce an entropic barrier between resolved states. (C) The sum of the equilibrium folding and unfolding rates for α₃D in a strong denaturant (5 to 6 M urea) as a function of pH as determined in a prior smFRET study (37). (D) A conceptual sketch of α₃D’s 1D free-energy landscape deduced by a combination of smFRET and molecular dynamics studies based on Ref. 37. The dramatic increase in α₃D’s kinetics at low pH shown in panel C was explained as increased configurational diffusion along a smooth rather than a rough energy landscape.AFM characterization of proteins is widely used (1–5) and therefore is an important experimental regime to explore, distinct from numerous studies investigating instrumental effects on nucleic acid hairpins measured with optical traps (17, 18, 23, 24, 26, 31). Historically, limited force precision and stability coupled with the slow response of the force probe has made it challenging to perform AFM-based equilibrium and near-equilibrium studies (32) and thereby difficult to quantify the role of instrumental artifacts. Recent work using a standard gold-coated cantilever concluded that the equilibrium dynamics of the fast-folding protein gpW were dominated by the dynamics of the cantilever diffusing on a force-induced entropic barrier (29). Such results raise significant concerns about interpreting rates or landscapes measured in AFM studies of globular protein folding and thereby motivate the following question: How do variations in intrinsic protein folding dynamics manifest in AFM-based studies, particularly in an experimental regime dominated by an instrument-induced entropic barrier?Here, we address this question by directly modulating a globular protein’s underlying folding dynamics without significantly changing the height of the barrier or the free-energy difference between the states. To do so, we studied α₃D using AFM-based force spectroscopy (Fig. 1A). The dynamics and energetics of α₃D, a computationally designed, fast-folding three-helix bundle of 73 amino acids (33, 34), have been studied by traditional ensemble (33) and single-molecule fluorescence resonance energy transfer (smFRET) (35–39) assays. Equilibrium smFRET studies in chemical denaturants showed accelerated folding/unfolding kinetics as pH was reduced (35). A subsequent landmark paper (37) combined state-of-the-art smFRET and microsecond-long, all-atom molecular dynamics simulations to show that this acceleration resulted from suppression of nonnative contacts changing the local roughness of the 1D landscape (Fig. 1 C and D) rather than a change in the height or the overall shape of the barrier between states. In the context of Kramers theory (40), this roughness manifests as a change in D, the conformational diffusion coefficient along the 1D landscape. The authors concluded that most, if not all, of the 14-fold change in folding kinetics came from an increase in D. This pH-dependent change in kinetics serves as a benchmark of α₃D’s dynamics in the absence of the force probe and associated linker. In other words, we will leverage conditions known to modulate the rate of folding along the molecular coordinate (x_prot) while measuring the consequence of that change on the measured coordinate (x_meas) (Fig. 1B).While α₃D provides a conceptually attractive means to modulate intrinsic molecular dynamics, it presents significant experimental challenges. Like gpW (28, 29), α₃D unfolds at a low force (< 15 pN) by AFM standards (2, 3, 41) and exhibits even faster folding kinetics under force. Thus, spatiotemporal resolution is critical, and instrumentation limitations are expected to be even more pronounced. Force drift is also a critical issue, particularly for extended assays (>1 to 100 s) because standard gold-coated cantilevers exhibit significant force drift (42); yet, equilibrium assays of structured RNA (6) and proteins (9) are sensitive to sub-pN changes in F. We therefore used focused ion beam (FIB)–modified cantilevers (32, 43) that combine sub-pN stability over 100 s (43, 44) with a ∼13-fold improvement in spatiotemporal precision compared with the standard cantilever used in the aforementioned AFM study characterizing gpW (29). This stability in conjunction with a newly designed polyprotein construct allowed us to measure an individual α₃D unfold and fold over 5,000 times and for periods up to 1 h using both constant velocity (v) and equilibrium (v = 0) data acquisition protocols. Rates derived from both the equilibrium and dynamic data recapitulated α₃D’s pH-dependent kinetics from smFRET. However, the reconstructed 1D folding-energy landscape was consistent with the predicted entropic barrier and therefore encodes no information about α₃D’s folding landscape beyond ΔG₀, the thermodynamic stability of α₃D. Importantly, rate analysis yielded the expected asymmetric distance to the transition state from the folded and unfolded state and revealed a significant increase in the distance to the unfolding transition state as pH was lowered. These studies demonstrate that AFM-force spectroscopy can track changes in intrinsic protein dynamics with high precision, even in mechanically labile, fast-folding systems.     

Balance and Strength—Estimating the Maximum Prey‐Lifting Potential of the Large Predatory Dinosaur Carcharodontosaurus saharicus

Motivated by the work of palaeo‐art “Double Death (2011),” a biomechanical analysis using three‐dimensional digital models was conducted to assess the potential of a pair of the large, Late Cretaceous theropod dinosaur Carcharodontosaurus saharicus to successfully lift a medium‐sized sauropod and not lose balance. Limaysaurus tessonei from the Late Cretaceous of South America was chosen as the sauropod as it is more completely known, but closely related to the rebbachisaurid sauropods found in the same deposits with C. saharicus. The body models incorporate the details of the low‐density regions associated with lungs, systems of air sacs, and pneumatized axial skeletal regions. These details, along with the surface meshes of the models, were used to estimate the body masses and centers of mass of the two animals. It was found that a 6 t C. saharicus could successfully lift a mass of 2.5 t and not lose balance as the combined center of mass of the body and the load in the jaws would still be over the feet. However, the neck muscles were found to only be capable of producing enough force to hold up the head with an added mass of 424 kg held at the midpoint of the maxillary tooth row. The jaw adductor muscles were more powerful, and could have held a load of 512 kg. The more limiting neck constraint leads to the conclusion that two, adult C. saharicus could successfully lift a L. tessonei with a maximum body mass of 850 kg and a body length of 8.3 m. Anat Rec, 298:1367–1375, 2015. © 2015 Wiley Periodicals, Inc.     

Impact of joint status on contraction steadiness of m. quadriceps femoris in people with severe haemophilia

Impaired contraction steadiness of lower limb muscles affects functional performance and may increase injury risk. We hypothesize that haemophilic arthropathy of the knee and the strength status of quadriceps are relevant factors which compromise a steady contraction. This study addresses the questions if impaired steadiness of the quadriceps is verifiable in people with haemophilia (PWH) and whether a connection between the status of the knee joint and quadriceps strength exists. A total of 157 PWH and 85 controls (C) performed a strength test with a knee extensor device to evaluate their bilateral and unilateral maximal quadriceps strength and steadiness. Isometric steadiness was measured by the coefficient of variation of maximum peak torque (CV‐MVIC in %). For classification of the knee joint status the World Federation of Haemophilia (WFH) score was used. Lower steadiness (higher CV values) was found in PWH compared with C during bilateral [PWH vs. C; 0.63 (0.36/1.13) vs. 0.35 (0.15/0.72), median (Q25/Q75) P < 0.001] and unilateral trials [left leg: 0.70 (0.32/1.64) vs. 0.50 (0.23/1.04), P < 0.05; right leg: 0.68 (0.29/1.51) vs. 0.39 (0.18/0.68), P < 0.001]. PWH with a WFH score difference (≥1) between their extremities showed a less steady contraction in the more affected extremity (P < 0.05). More unsteady contractions have also been found in extremities with lower quadriceps strength compared with the contralateral stronger extremities (P < 0.001), whereby the weaker extremities were associated with a worse joint status (P < 0.001). The results of this study verify an impaired ability to realize a steady contraction of quadriceps in PWH and the influence of joint damage and strength on its manifestation.