Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states

Skeletal muscle has the ability to achieve rapid repair in response to injury or disease. Many individuals with Marfan syndrome (MFS), caused by a deficiency of extracellular fibrillin-1, exhibit myopathy and often are unable to increase muscle mass despite physical exercise. Evidence suggests that selected manifestations of MFS reflect excessive signaling by transforming growth factor (TGF)-beta (refs. 2,3). TGF-beta is a known inhibitor of terminal differentiation of cultured myoblasts; however, the functional contribution of TGF-beta signaling to disease pathogenesis in various inherited myopathic states in vivo remains unknown. Here we show that increased TGF-beta activity leads to failed muscle regeneration in fibrillin-1-deficient mice. Systemic antagonism of TGF-beta through administration of TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor blocker losartan normalizes muscle architecture, repair and function in vivo. Moreover, we show TGF-beta-induced failure of muscle regeneration and a similar therapeutic response in a dystrophin-deficient mouse model of Duchenne muscular dystrophy.     

Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion

The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.Flagellum-based motility plays a critical role in the biology and pathogenesis of many bacteria (3, 6, 17, 31). The well-conserved flagellum is commonly divided into three physical parts: the flagellar motor, the helically shaped flagellar filament, and the hook which provides a universal joint between the motor and the filament. In most bacteria, counterclockwise rotation of the flagella results in bundling of the helical flagella and propulsion of the cell through liquid or viscous environments. Clockwise rotation of the flagellar motor results in random turning of the cell with little translational motion (“tumbling”). Bacterial motility is thus a zigzag pattern of runs and tumbles, in which chemotactic signals favor running toward attractants and away from repellents (3).Borrelia burgdorferi and other closely related spirochetes are the causative agents of Lyme disease, which is transmitted to humans via infected Ixodes ticks (40). Spirochetes have a distinctive morphology in that the flagella are enclosed within the outer membrane sheath and are thus called periplasmic flagella (6). The flagellar motors are located at both ends of the cell and are coordinated to rotate in opposite directions during translational motion and in the same direction (i.e., both clockwise or both counterclockwise) during the spirochete equivalent of tumbling, called “flexing” (6, 15). Spirochetes are also capable of reversing translational motion by coordinated reversal of the direction of motor rotation at both ends of the cell. Rotation of the flagella causes a serpentine movement of the entire cell body, allowing B. burgdorferi to efficiently bore its way through tissue and disseminate throughout the mammalian host, resulting in manifestations in the joints, nervous system, and heart (40).The flagellar motor is an extraordinary nanomachine powered by the electrochemical potential of specific ions across the cytoplasmic membrane (3). Current knowledge of the flagellar motor structure and rotational mechanisms is based primarily on studies of Escherichia coli and Salmonella enterica and is summarized in several recent comprehensive reviews (3, 22, 31, 39, 42). The flagellar motor is constructed from at least 20 different kinds of proteins. The approximate location of these flagellar proteins has been determined by a variety of approaches and appears to be relatively consistent in a wide variety of bacteria. It can be divided into several morphological domains: the MS ring (FliF, the base for the flagellar motor); the C ring (FliG, FliM, and FliN, the switch complex regulating motor rotation); the export apparatus (multiple-protein complex located at the cytoplasmic side of the MS ring); the rod (connecting the MS ring and the hook); the L and P rings on the rod (thought to serve as bushings at the outer membrane and at the peptidoglycan layer, respectively); and the stator, which is the motor force generator embedded in the cytoplasmic membrane. Electron microscopy studies of the purified flagellar motor have provided a detailed view of the rotor/C-ring assembly (11, 44). However, there is no structural information on the stator and the export apparatus in these reconstructions, because these membrane-associated structures are not retained following detergent extraction during the extensive basal body purification process. The stator and the export apparatus were visualized by using freeze fracture preparations of cytoplasmic membranes. It appears that 10 to 16 stator units form circular arrays in the membrane (9, 20). Part of the export apparatus is located in the central space of the C ring (18). Recently a 7-nm-resolution structure of the intact flagellar motor in situ was revealed by averaging 20 structures obtained using cryo-electron tomography (cryo-ET) of Treponema primitia cells (32). Further analysis of the intact flagellar motor structure would lead to a better understanding of the motor protein distribution, the rotor-stator interaction, and the mechanism of bacterial motility.Cryo-ET has emerged as a three-dimensional (3-D) imaging technique to bridge the information gap between X-ray crystallographic and optical microscopic methods (24, 30). This process involves rapidly freezing viable cells, collecting a series of electron micrographs at different angles, and computationally combining the resulting images into a 3-D density map. Cryo-ET allows investigation of the structure-function relationship of molecular complexes and supramolecular assemblies in their cellular environments without fixation, dehydration, embedding, or sectioning artifacts. Spirochetes are well suited for cryo-ET analysis because of their narrow cell diameter (typically 0.2 to 0.3 μm). Recently the cellular architecture of Treponema primitia, Treponema denticola, and B. burgdorferi, as well as the configuration of the B. burgdorferi periplasmic flagella, were revealed by cryo-ET (7, 16, 26, 33). In combination with advanced computational methods, cryo-ET is currently the most promising approach for determining the cellular architecture in situ at molecular resolution (30). We have developed novel strategies for capturing and averaging thousands of 3-D images of large macromolecular assemblies to obtain ∼2.0-nm-resolution structures (28, 29).In this study, we present the molecular structures of infectious wild-type (WT) and mutant B. burgdorferi organisms and their flagellar motors in situ using high-throughput cryo-ET and 3-D image analysis. By averaging subvolumes of 1,280 flagellar motors from 322 cells, we obtained a ∼3.5-nm-resolution model of the intact flagellar motor, providing a detailed view of rotor-stator interactions. In addition, detergent treatment of intact cells provided a preliminary identification of the rotor and stator structures. Through the comparison of WT and mutant cells, we have also determined the location of the flgI gene product in the B. burgdorferi flagellar motor.     

Functional organization of adult motor cortex is dependent upon continued protein synthesis

The functional organization of adult cerebral cortex is characterized by the presence of highly ordered sensory and motor maps. Despite their archetypical organization, the maps maintain the capacity to rapidly reorganize, suggesting that the neural circuitry underlying cortical representations is inherently plastic. Here we show that the circuitry supporting motor maps is dependent upon continued protein synthesis. Injections of two different protein synthesis inhibitors into adult rat forelimb motor cortex caused an immediate and enduring loss of movement representations. The disappearance of the motor map was accompanied by a significant reduction in synapse number, synapse size, and cortical field potentials and caused skilled forelimb movement impairments. Further, motor skill training led to a reappearance of movement representations. We propose that the circuitry of adult motor cortex is perpetually labile and requires continued protein synthesis in order to maintain its functional organization.     

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.     

Changes in Colonic Bile Acid Composition following Fecal Microbiota Transplantation Are Sufficient to Control Clostridium difficile Germination and Growth

Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (R-CDI), but its mechanisms remain poorly understood. Emerging evidence suggests that gut bile acids have significant influence on the physiology of C. difficile, and therefore on patient susceptibility to recurrent infection. We analyzed spore germination of 10 clinical C. difficile isolates exposed to combinations of bile acids present in patient feces before and after FMT. Bile acids at concentrations found in patients’ feces prior to FMT induced germination of C. difficile, although with variable potency across different strains. However, bile acids at concentrations found in patients after FMT did not induce germination and inhibited vegetative growth of all C. difficile strains. Sequencing of the newly identified germinant receptor in C. difficile, CspC, revealed a possible correspondence of variation in germination responses across isolates with mutations in this receptor. This may be related to interstrain variability in spore germination and vegetative growth in response to bile acids seen in this and other studies. These results support the idea that intra-colonic bile acids play a key mechanistic role in the success of FMT, and suggests that novel therapeutic alternatives for treatment of R-CDI may be developed by targeted manipulation of bile acid composition in the colon.     

Preliminary Assessment of Gastrointestinal Parasites in Two Wild Groups of Endangered Moor Macaques (Macaca maura) from Sulawesi

International Journal of Primatology - Information on parasite biodiversity and abundance can improve our understanding of parasitic infections on endangered wildlife, as parasites can affect host...     

An exploratory study of cardiac function and oxygen uptake during cycle ergometry in overweight children

Objective: Obesity has been proposed to negatively impact cardiac function in overweight (OW) individuals. The relationship between diastolic dysfunction and oxygen uptake (V?o ₂) kinetics is equivocal. This exploratory investigation evaluated the relationship between resting left ventricular function and V?o ₂ kinetics during cycle ergometry in OW and non‐overweight (NO) children and adolescents. Research Methods and Procedures: Fourteen OW (>85 percentile for BMI for age and gender) children, 10 boys and 4 girls (age, 11.7 ± 1.9 years; body mass, 80.6 ± 45.5 kg) and 10 NO children (4 boys, 6 girls) volunteered to participate in the study (age, 12.5 ± 2.1 years; body mass, 45.8 ± 13.8 kg). Resting cardiovascular structure and function were assessed using spectral Doppler echocardiography. All subjects underwent two sub‐maximal exercise stages on a cycle ergometer (3 minutes unloaded and 5 minutes at 50 W, both at a cadence of 50 rpm). Respiratory data were measured on a breath‐by‐breath basis at both workloads and the mean response time (MRT) was calculated. Results: Analysis of the MRT data demonstrated that there were no significant differences between OW and NO (OW, 52.6 ± 11.7 seconds vs. NO, 45.6 ± 7.4 seconds). Significant correlations (p < 0.05) were obtained between MRT V?o ₂ and echocardiographic‐derived mitral valve inflow pressure half‐time (r = 0.55) and between MRT V?o ₂, and mitral valve inflow deceleration time (r = 0.55). Discussion: The evidence from this research suggests a possible link between left ventricular diastolic function at rest and oxygen uptake kinetics during sub‐maximal exercise in OW and NO children and adolescents.