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STUDY DESIGN.: Mechanical and microstructural assessment of nucleus-annulus integration. OBJECTIVE.: To investigate the existence of structural integration between the nucleus and the inner annulus. SUMMARY OF BACKGROUND DATA.: The nucleus is often viewed as a hydrostatically functioning entity that is largely separate from its surroundings. The boundary between nucleus and annulus is acknowledged as difficult to define. METHODS.: Ten-millimeter-thick sagittal slabs were cut from the central region of ovine lumbar discs. The annulus-nucleus transition region was isolated and the resulting samples subjected to transverse tensile loading up to failure. Similar samples were stretched to about 4 to 5 times their original separation and then subjected to microstructural examination to investigate structural integration across the inner annulus-nucleus region. RESULTS.: The annulus-nucleus boundary could support an average load of 5.7 N (range, 2-11.5 N). Tensile loading causes the fibrous structure of the nucleus to be drawn into an approximate alignment in the transverse stretch direction with an associated reverse inpulling of the inner annular layers. At high magnification, the horizontally aligned nucleus fibers can be seen to branch and blend with the inner annular structure. CONCLUSION.: The nucleus contains a convoluted but highly structured network of fibers of varying length, which appear to integrate with the inner annulus and confer a significant degree of transverse interconnectivity that can be demonstrated mechanically. This new experimental evidence, together with that from a previous study demonstrating nucleus-endplate connectivity, makes it clear that the nucleus cannot be considered as a separate entity in the disc. We propose that this structural integration provides the nucleus with a form of tethered mobility that supports physiological functions distinct from the primary strength requirements of the motion segment. [ABSTRACT FROM AUTHOR]

The disc nucleus is commonly thought of as a largely unstructured gel. However, exactly how the nucleus integrates structurally with the endplates remains somewhat ambiguous. The purpose of this study was to investigate whether a substantial level of structural/mechanical cohesion does, in fact, exist across the nucleus-endplate junction. Vertebra-nucleus-vertebra samples were obtained from mature ovine lumbar motion segments and subjected to a novel technique involving circumferential transverse severing (i.e. ring-severing) of the annulus fibrosus designed to eliminate its strain-limiting influence. These samples were loaded in tension and then chemically fixed in order to preserve the stretched nucleus material. Structural continuity across the nucleus-endplate junctions was sufficient for the samples to support, on average, 20 N before tensile failure occurred. Microscopic examination revealed nucleus fibres inserting into the endplates and the significant level of load carried by the nucleus material indicates that there is some form of structural continuity from vertebra to vertebra in the central nucleus region. [ABSTRACT FROM AUTHOR]

A simple and environmentally friendly approach toward the thermoplastic processing of rapidly degradable plastic-enzyme composites using three-dimensional (3D) printing techniques is described. Polycaprolactone/Amano lipase (PCL/AL) composite films (10 mm × 10 mm; height [ h ] = ∼400 μm) with an AL loading of 0.1, 1.0, and 5.0% were prepared via 3D printing techniques that entail direct mixing in the solid state and thermal layer-by-layer extrusion. It was found that AL can tolerate in situ processing temperatures up to 130 °C in the solid-state for 60 min without loss of enzymatic activity. The composites were degraded in phosphate buffer (8 mg/mL, composite to buffer) for 7 days at 37 °C and the resulting average percent total weight loss (WL avg % ) was found to be 5.2, 92.9, and 100%, for the 0.1, 1.0, and 5.0% films, respectively. The degradation rates of PCL/AL composites were found to be faster than AL applied externally in the buffer. Thicker PCL/AL 1.0% films (10 mm × 10 mm; h = ∼500 μm) were also degraded over a 7 day period to examine how the weight loss occurs over time with 3.0, 18.1, 36.4, 46.4, and 70.2% weight loss for days 1, 2, 3, 4, and 7, respectively. Differential scanning calorimetry (DSC) analysis shows that the film's percent crystallinity ( D xtal% ) increases over time with D xtal% = 46.5 for day 0 and 53.1% for day 7. Scanning electron microscopy (SEM) analysis found that film erosion begins at the surface and that water can penetrate the interior via surface pores activating the enzymes embedded in the film. Controlled release experiments utilizing dye-loaded PCL/AL/dye (AL = 1.0%; dye = 0.1%) composites were degraded over a 7 day period with the bulk of the dye released by the fourth day. The PCL/AL multimaterial objects containing AL-resistant polylactic acid (PLA) were also printed and degraded to demonstrate the application of this material on more complex structures.

The aging western society is heavily afflicted with intervertebral disc (IVD) degeneration. Replacement or repair of the degenerated IVD with an artificial bio-mimetic construct is one of the challenges of future research due to its complex structure and unique biomechanical function. Herein, biocomposite laminates made of long collagen fibers in unidirectional (-1.3 ± 2.1°) and angle-plied ± 30° orientations (30.4 ± 6.4 and -29.8 ± 4.5), embedded in alginate hydrogel, were fabricated to mimic the form of single annulus fibrosus (AF) lamella and the circumferential AF, respectively. The mechanical behavior of the composites was measured and compared with in vitro existing data of the human native AF as well as with new data obtained from ovine and bovine specimens. The mechanical behavior was found to reproduce the full stress- strain behavior of the human AF single lamella in several regions of the AF and the Young's modulus was 28.3 ± 8.6 MPa. Moreover, the modulus of the angle-plied laminates was 16.8 ± 2.9 MPa, which is approximately 5% less than the in vitro data. The full stress-strain behavior was also compared with bovine and ovine circumferential AF samples and found to be very similar, with a difference in the modulus of 4.1% and 19.7%, respectively. Moreover, an FE model of the L3-L4 functional spinal unit (FSU) was developed and calibrated to evaluate the mechanical ability of the biocomposite to be used as an AF substitute under physiological IVD loading modes. The biocomposite demonstrated a good ability to mimic the stiffness of the native tissue under physiologic loading modes as flexion, extension, lateral bending and compression, but was too flexible under torsion. It was found that the proposed biomimetics AF design resulted in a compatible function in several mechanical levels, which holds great potential to be used as a viable AF replacement towards full IVD engineering., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning β-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (E a ) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other β-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation. IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning β-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large β-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species., (Copyright © 2019 Wade et al.)

The annulus fibrosus (AF) of the intervertebral disc (IVD) consists of a set of concentric layers composed of a primary circumferential collagen fibers arranged in an alternating oblique orientation. Moreover, there exists an additional secondary set of radial translamellar collagen fibers which connects the concentric layers, creating an interconnected fiber network. The aim of this study was to investigate the mechanical role of the radial fiber network. Toward that goal, a three-dimensional (3D) finite element model of the L3-L4 spinal segment was generated and calibrated to axial compression and pure moment loading. The AF model explicitly recognizes the two heterogeneous networks of fibers. The presence of radial fibers demonstrated a pronounced effect on the local disc responses under lateral bending, flexion, and extension modes. In these modes, the radial fibers were in a tensile state in the disc region that subjected to compression. In addition, the circumferential fibers, on the opposite side of the IVD, were also under tension. The local stress in the matrix was decreased in up to 9% in the radial fibers presence. This implies an active fiber network acting collectively to reduce the stresses and strains in the AF lamellae. Moreover, a reduction of 26.6% in the matrix sideways expansion was seen in the presence of the radial fibers near the neutral bending axis of the disc. The proposed biomechanical model provided a new insight into the mechanical role of the radial collagen fibers in the AF structure. This model can assist in the design of future IVD substitutes., (Copyright © 2019 by ASME.)

Background: The intervertebral disc (IVD) is a complex organ that acts as a flexible coupling between two adjacent vertebral bodies and must therefore accommodate compression, bending, and torsion. It consists of three main components, which are elegantly structured to allow this: the annulus fibrosus (AF), the nucleus pulposus (NP), and the end-plates (EP)., Purpose: Thus far, it has not been possible to examine the microarchitecture of the disc directly in three dimensions in its unaltered state and thus knowledge of the overall architecture of the disc has been inferred from a range of imaging sources, or by using destructive methods., Study Design: A nondestructive ultrahigh field Magnetic Resonance Imaging (MRI) of 11.7 T was used together with image analysis to visualize the ovine IVDs., Methods: Three-dimensional image stacks from eight IVDs harvested from sheep, half of which were 4 to 5 years old and the others approximately 2 years old were reconstructed and examined, and their microstructure were imaged. The overall structure of the disc, including the average of 14 AF lamellae (9-28), NP, and EP was then visualized with particular attention given to integrating elements as radial translamellar cross-links, AF-NP transition zone EP-AF integration and EP-NP insertion nodes (ie the connecting junctions between the EP and NP). Moreover, collagen fiber orientation was determined at different depths and locations throughout the annulus., Results: It was found that there was a clearer demarcation in the AF-NP transition zone of the younger discs compared with the older ones. This difference was reflected in the visibility of AF-NP and EP-AF integration. It was also possible to view the fiber architecture of the AF-NP integration in greater depth than was possible previously with histological techniques. These fibers were mainly observed in the younger discs and their length was measured to be of 2.6 ± 0.2 mm., Conclusions: The present results provide a substantial advance in visualization of the three-dimensional architecture of an intact IVD and the integration of its components., (Copyright © 2018. Published by Elsevier Inc.)

Study Design: Microstructural investigation of compression-induced herniation of a lumbar disc held in a concordant complex posture., Objective: To explore the significance of loading rate in a highly asymmetric concordant posture, comparing the mechanisms of failure to an earlier study using a nonconcordant complex posture., Summary of Background Data: A recent study with a nonconcordant complex posture (turning in the opposite direction to that which the load is applied) demonstrated the vulnerability of the disc to loading that is borne by one set of oblique-counter oblique fiber sets in the alternating lamellae of the annulus, and aggravated by an elevated loading rate. Given the strain rate-dependent properties of the disc it might be expected that the outcome differs if the posture is reversed., Methods: Forty-one motion segments from ovine 16 spines were split into two cohorts; adopting the previously employed low rate (40 mm/min) and surprise rate (400 mm/min) of loading. Both groups of damaged discs were then analyzed microstructurally., Results: With the lower rate loading the concordant posture significantly reduced the load required to cause disc failure than earlier described for nonconcordant posture (6.9 vs. 8.4 kN), with more direct tears and alternate lamella damage extending to the anterior disc. Contrary to this result, with a surprise rate, the load at failure was significantly increased with the concordant posture (8.08 vs. 6.96 kN), although remaining significantly less than that from a simple flexed posture (9.6 kN). Analysis of the damage modes and postures suggest facet engagement plays a significant role., Conclusion: This study confirms that adding shear to the posture lowers the load at failure, and causes alternate lamella rupture. Load at failure in a complex posture is not determined by loading rate alone. Rather, the strain rate-dependent properties of the disc influence which elements of the system are brought into play., Level of Evidence: N/A.

Study Design: Microstructural investigation of low frequency cyclic loading and flexing of the lumbar disc., Objective: To explore micro-level structural damage in motion segments subjected to low frequency repetitive loading and flexing at sub-acute loads., Summary of Background Data: Cumulative exposure to mechanical load has been implicated in low back pain and injury. The mechanical pathways by which cyclic loading physically affects spine tissues remain unclear, in part due to the absence of high quality microstructural evidence., Methods: The study utilized seven intact ovine lumbar spines and from each spine one motion segment was used as a control, two others were cyclically loaded. Ten motion segments were subjected to 5000 cycles at 0.5 Hz with a peak load corresponding to ∼30% of that required to achieve failure. An additional small group of segments subjected to 10,000 or 30,000 cycles was similarly analyzed. Following chemical fixation and decalcification samples were cryosectioned along one of the oblique fiber angles and imaged in their fully hydrated state using differential interference contrast optical microscopy. Structural damage obtained from the images was organized into an algebraic shell for analysis., Results: At 5000 cycles the disc damage was limited to inner wall distortions, evidence of stress concentrations at bridging-lamellae attachments, and small delaminations. The high-cycle discs tested exhibited significant mid-wall damage. There was no evidence of nuclear material being displaced., Conclusion: At this low frequency and without the application of sustained loading or a more severe loading regime, or maintaining a constant flexion with repetitive loading, it seems unlikely that actual nuclear migration occurs. It is possible that the inner-annular damage shown in the low dose group could disrupt pathways for nutrient diffusion leading to earlier cell death and matrix degradation, thus contributing to a cascade of degeneration., Level of Evidence: N/A.

Purpose: Part I of this study explored mechanisms of disc failure in a complex posture incorporating physiological amounts of flexion and shear at a loading rate considerably lower than likely to occur in a typical in vivo manual handling situation. Given the strain-rate-dependent mechanical properties of the heavily hydrated disc, loading rate will likely influence the mechanisms of disc failure. Part II investigates the mechanisms of failure in healthy discs subjected to surprise-rate compression while held in the same complex posture., Methods: 37 motion segments from 13 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object at a displacement rate of 400 mm/min. Seven of the 37 samples reached the predetermined displacement prior to a reduction in load and were classified as early stage failures, providing insight to initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy., Results: The average failure load under high rate complex loading was 6.96 kN (STD 1.48 kN), significantly lower statistically than for low rate complex loading [8.42 kN (STD 1.22 kN)]. Also, unlike simple flexion or low rate complex loading, direct radial ruptures and non-continuous mid-wall tearing in the posterior and posterolateral regions were commonly accompanied by disruption extending to the lateral and anterior disc., Conclusion: This study has again shown that multiple modes of damage are common when compressing a segment in a complex posture, and the load bearing ability, already less than in a neutral or flexed posture, is further compromised with high rate complex loading.

Purpose: To date, the mechanisms of disc failure have been explored at a microstructural level in relatively simple postures. However, in vivo the disc is known to be subjected to complex loading in compression, bending and shear, and the influence of these factors on the mechanisms of disc failure is yet to be described at a microstructural level. The purpose of this study was to provide a microstructural analysis of the mechanisms of failure in healthy discs subjected to compression while held in a complex posture incorporating physiological amounts of flexion and facet-constrained shear., Methods: 30 motion segments from 10 healthy mature ovine lumbar spines were compressed in a complex posture intended to simulate the situation arising when bending and twisting while lifting a heavy object, and at a displacement rate of 40 mm/min. Nine of the 30 samples reached the predetermined displacement prior to a reduction in load and were classified as early-stage failures, providing insight into initial areas of disc disruption. Both groups of damaged discs were then analysed microstructurally using light microscopy., Results: Complex postures significantly reduced the load required to cause disc failure than earlier described for flexed postures [8.42 kN (STD 1.22 kN) compared to 9.69 kN (STD 2.56 kN)] and resulted in a very different failure morphology to that observed in either simple flexion or direct compression, involving infiltration of nucleus material in a circuitous path to the annular periphery., Conclusion: The complex posture as used in this study significantly reduced the load required to cause disc failure, providing further evidence that asymmetric postures while lifting should be avoided if possible.

Objectives: To determine service utilization and identify patient characteristics associated with service utilization in youth with obesity presenting for structured weight management, and to explore weight-related outcomes associated with service utilization., Study Design: In this retrospective study conducted between January 2008 and December 2013, we examined variables associated with the care of 2089 patients aged 2-18 years presenting for an initial visit to 2 tertiary care-based, multidisciplinary structured weight management clinics., Results: Only 53% of patients returned for a second visit, 29% returned for a third visit, and virtually none (0.5%) completed the recommended 6 visits within 6 months. Patients who were Hispanic, government-insured, and whose parent/s spoke Spanish were more likely to return to clinic. Of those patients who returned for at least a second visit, 70% demonstrated a reduction in or maintenance of body mass index z-score., Conclusions: Patient retention remains a significant barrier to effective pediatric weight management. Structured weight management programs should increase their efforts to engage patients and families at the initial visit and identify and address barriers to follow up., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Study Design: Microstructural investigation of vibration-induced disruption of the flexed lumbar disc., Objective: The aim of the study was to explore micro-level structural damage in motion segments subjected to vibration at subcritical peak loads., Summary of Background Data: Epidemiological evidence suggests that cumulative whole body vibration may damage the disc and thus play an important role in low back pain. In vitro investigations have produced herniations via cyclic loading (and cyclic with added vibrations as an exacerbating exposure), but offered only limited microstructural analysis., Methods: Twenty-nine healthy mature ovine lumbar motion segments flexed 7° and subjected to vibration loading (1300 ± 500 N) in a sinusoidal waveform at 5 Hz to simulate moderately severe physiologic exposure. Discs were tested either in the range of 20,000 to 48,000 cycles (medium dose) or 70,000 to 120,000 cycles (high dose). Damaged discs were analyzed microstructurally., Results: There was no large drop in displacement over the duration of both vibration doses indicating an absence of catastrophic failure in all tests. The tested discs experienced internal damage that included delamination and disruption to the inner and mid-annular layers as well as diffuse tracking of nucleus material, and involved both the posterior and anterior regions. Less frequent tearing between the inner disc and endplate was also observed. Annular distortions also progressed into a more severe form of damage, which included intralamellar tearing and buckling and obvious strain distortion around the bridging elements within the annular wall., Conclusion: Vibration loading causes delamination and disruption of the inner and mid-annular layers and limited diffuse tracking of nucleus material. These subtle levels of disruption could play a significant role in initiating the degenerative cascade via micro-level disruption leading to cell death and altered nutrient pathways., Level of Evidence: 5.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world's leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

Pore-forming proteins (PFPs) encompass a broad family of proteins that are used for virulence or immune defense. Members of the cholesterol-dependent cytolysins (CDCs) and membrane attack complex/perforin (MACPF) family of PFPs form large β-barrel pores in the membrane. The CDC/MACPF proteins contain a characteristic four-stranded β-sheet that is flanked by two α-helical bundles, which unfold to form two transmembrane β-hairpins. Apicomplexan eukaryotic parasites express CDC/MACPFs termed perforin-like proteins (PLPs). Here we review recent studies that provide key insights into the assembly and regulation of the Apicomplexan PLP (ApiMACPF) molecular pore-forming mechanisms, which are necessary for the osmotically driven rupture of the parasitophorous vacuole and host cell membrane, and cell traversal by these parasites., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Study Design: Investigation of the elastic network in disc annulus and its function., Objective: To investigate the involvement of the elastic network in the structural interconnectivity of the annulus and to examine its possible mechanical role., Summary of Background Data: The lamellae of the disc are now known to consist of bundles of collagen fibers organized into compartments. There is strong interconnectivity between adjacent compartments and between adjacent lamellae, possibly aided by a translamellar bridging network, containing blood vessels. An elastic network exists across the disc annulus and is particularly dense between the lamellae, and forms crossing bridges within the lamellae., Methods: Blocks of annulus taken from bovine caudal discs were studied in either their unloaded or radially stretched state then fixed and sectioned, and their structure analyzed optically using immunohistology., Results: An elastic network enclosed the collagen compartments, connecting the compartments with each other and with the elastic network of adjacent lamellae, formed an integrated network across the annulus, linking it together. Stretching experiments demonstrated the mechanical interconnectivities of the elastic fibers and the collagen compartments., Conclusion: The annulus can be viewed as a modular structure organized into compartments of collagen bundles enclosed by an elastic sheath. The elastic network of these sheaths is interconnected mechanically across the entire annulus. This organization is also seen in the modular structure of tendon and muscle. The results provide a new understanding annulus structure and its interconnectivity, and contribute to fundamental structural information relevant to disc tissue engineering and mechanical modeling., Level of Evidence: N/A.

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of β-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the β-barrel pore., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Study Design: Microstructural investigation of compression-induced herniation of the flexed lumbar disc., Objective: To provide a microstructural analysis of the mechanisms of annular wall failure in healthy discs subjected to flexion and a rate of compression comparable with the maximum rate at which the muscles of the spinal column can generate a force., Summary of Background Data: Clinical evidence indicates the involvement of the endplate in herniation. It is known that both an elevated rate of compression and a flexed posture are necessary to cause disc failure either within the midspan of the annulus or at the annular-endplate interface. However, the question of what effect a sudden or "surprise" loading might have on the mode of failure is, as yet, unanswered., Methods: Twenty-four healthy mature ovine lumbar motion segments were compressed to failure in high physiological flexion (10º). This occurred over approximately 5 mm of crosshead displacement in 0.75 seconds that resulted in a displacement rate of 400 mm/min (defined as a "surprise" rate) and was intended to simulate the maximum rate at which the muscles of the spinal column can generate a force. The damaged discs were then analyzed microstructurally., Results: Fifty-eight percent of discs suffered annular-endplate junction rupture, 25% suffered midspan annular rupture, and the balance of 17% endplate fracture. Microstructural analysis indicated that annular rupture initiated at the endplate apical ridge in the mid-to-outer region of the annulus in both annular-endplate and midspan annulus rupture., Conclusion: Motion segments subjected to a "surprise" loading rate are likely to fail via some form of annular rupture. Failure under such sudden loading occurs mostly via rupture of the annular-endplate junction and is thought to arise from a rate-induced mechanostructural imbalance between the annulus and the endplate., Level of Evidence: N/A.

The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250-300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis.

β-Barrel pore-forming toxins (βPFTs) form an obligatory oligomeric prepore intermediate before the formation of the β-barrel pore. The molecular components that control the critical prepore-to-pore transition remain unknown for βPFTs. Using the archetype βPFT perfringolysin O, we show that E183 of each monomer within the prepore complex forms an intermolecular electrostatic interaction with K336 of the adjacent monomer on completion of the prepore complex. The signal generated throughout the prepore complex by this interaction irrevocably commits it to the formation of the membrane-inserted giant β-barrel pore. This interaction supplies the free energy to overcome the energy barrier (determined here to be ∼ 19 kcal/mol) to the prepore-to-pore transition by the coordinated disruption of a critical interface within each monomer. These studies provide the first insight to our knowledge into the molecular mechanism that controls the prepore-to-pore transition for a βPFT.

The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant β-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophysical, and biochemical analyses has revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their β-barrel pore in eukaryotic membranes. Our knowledge of the molecular details of these dramatic structural changes in CDCs has transformed our understanding of how giant pore complexes are assembled and has been critical to our understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs are employed in pore-independent processes.

Streptococcus pneumoniae produces the pore-forming toxin pneumolysin (PLY), which is a member of the cholesterol-dependent cytolysin (CDC) family of toxins. The CDCs recognize and bind the 3β-hydroxyl group of cholesterol at the cell surface, which initiates membrane pore formation. The cholesterol transport lipoproteins, which carry cholesterol in their outer monolayer, are potential off-pathway binding targets for the CDCs and are present at significant levels in the serum and the interstitial spaces of cells. Herein we show that cholesterol carried specifically by the ApoB-100-containing lipoprotein particles (CH-ApoB-100) in the mouse, but not that carried by human or guinea pig particles, is a potent inhibitor of the PLY pore-forming mechanism. Cholesterol present in the outer monolayer of mouse ApoB-100 particles is recognized and bound by PLY, which stimulates premature assembly of the PLY oligomeric complex thereby inactivating PLY. These studies further suggest that the vast difference in the inhibitory capacity of mouse CH-ApoB-100 and that of the human and the guinea pig is due to differences in the presentation of cholesterol in the outer monolayer of their ApoB-100 particles. Therefore mouse CH-ApoB-100 represents a significant innate CDC inhibitor that is absent in humans, which may underestimate the contribution of CDCs to human disease when utilizing mouse models of disease.

Study Design: Microstructural investigation of compression-induced disruption of the flexed lumbar disc., Objective: To provide a microstructural analysis of the mechanisms of annular wall failure in healthy discs subjected to flexion and an elevated rate of compression., Summary of Background Data: At the level of the motion segment failure of the disc in compression has been extensively studied. However, at the microstructural level the exact mechanisms of disc failure are still poorly understood, especially in relation to loading posture and rate., Methods: Seventy-two healthy mature ovine lumbar motion segments were compressed to failure in either a neutral posture or in high physiological flexion (10°) at a displacement rate of either 2 mm/min (low) or 40 mm/min (high). Testing at the high rate was terminated at stages ranging from initial wall tearing through to facet fracture so as to capture the evolution of failure up to full herniation. The damaged discs were then analyzed microstructurally., Results: Approximately, 50% of the motion segments compressed in flexion at the high rate experienced annulus or annulus-endplate junction failure, the remainder failed via endplate fracture with no detectable wall damage. The average load to induce disc failure in flexion was 18% lower (P < 0.05) than that required to induce endplate fracture. Microstructural analysis indicated that wall rupture occurred first in the posterior mid-then-outer annulus., Conclusion: Disc wall failure in healthy motion segments requires both flexion and an elevated rate of compression. Damage is initiated in the mid-then-outer annular fibers, this a likely consequence of the higher strain burden in these same fibers arising from endplate curvature. Given the similarity in geometry between ovine and human endplates, it is proposed that comparable mechanisms of damage initiation and herniation occur in human lumbar discs., Level of Evidence: N/A.

Purpose: Recent investigations using an ovine spine model have established that the disc nucleus contains a highly convoluted fibre network with endplate-to-endplate connectivity, this connectivity being achieved via distinctive nodal attachment points. The purpose of this study was to investigate how this nodal anchoring system might be influenced by maturation., Methods: Lumbar motion segments were dissected from newborn, 3, 12 months and fully mature ovine animals, subjected to a novel annular ring-severing procedure to remove the strain-limiting influence of the annulus, then either mechanically tested to destruction or examined microstructurally and ultrastructurally. The morphology of the nodes and their linear density within the relatively thin section planes were analysed to provide a basis for comparison between the four age groups., Results: Mechanical testing following ring severing revealed that the remaining nuclear material in all samples, irrespective of maturity, had the ability to transmit a substantial load from endplate to endplate. Imaging of the ring-severed samples from all age groups in their stretched, but unruptured state revealed the presence of axially aligned fibrosity in the nucleus region consistent with endplate-to-endplate connectivity. Endplate insertion nodes were observed in all age groups. Ultrastructural examination revealed that the fibrillar architecture of these nodes in the newborn discs was similar to that observed in the nodes of mature discs. However, there was a rapid increase in their linear density between birth and 3 months, after which this remained constant., Conclusions: The nodal attachment points identified previously in mature ovine discs are also present in newborn, and 3- and 12-month-old animals with an initial rapid increase in their linear density between birth and 3 months, after which it remained constant. The size and morphology of the attachment points were similar for all ages. Our study suggests that the increase in nodal density in the ovine disc endplate is part of an adaptive response to the loading environment that the disc is exposed to from birth to maturity.

Background:  Pneumococcus, meningococcus, and Haemophilus influenzae cause a similar spectrum of infections in the ear, lung, blood, and brain. They share cross-reactive antigens that bind to the laminin receptor of the blood-brain barrier as a molecular basis for neurotropism, and this step in pathogenesis was addressed in vaccine design., Methods:  Biologically active peptides derived from choline-binding protein A (CbpA) of pneumococcus were identified and then genetically fused to L460D pneumolysoid. The fusion construct was tested for vaccine efficacy in mouse models of nasopharyngeal carriage, otitis media, pneumonia, sepsis, and meningitis., Results:  The CbpA peptide-L460D pneumolysoid fusion protein was more broadly immunogenic than pneumolysoid alone, and antibodies were active in vitro against Streptococcus pneumoniae, Neisseria meningitidis, and H. influenzae. Passive and active immunization protected mice from pneumococcal carriage, otitis media, pneumonia, bacteremia, meningitis, and meningococcal sepsis., Conclusions:  The CbpA peptide-L460D pneumolysoid fusion protein was broadly protective against pneumococcal infection, with the potential for additional protection against other meningeal pathogens.

The intervertebral disc nucleus has traditionally been viewed as a largely unstructured amorphous gel having little obvious integration with the cartilaginous endplates (CEPs). However, recent work by the present authors has provided clear evidence of structural cohesion across the nucleus-endplate junction via a distinctive microanatomical feature termed insertion nodes. The aim of this study was to explore the nature of these insertion nodes at the fibrillar level. Specially prepared vertebra-nucleus-vertebra composite samples from ovine lumbar motion segments were extended axially and chemically fixed in this stretched state, and then decalcified. Sections taken from the samples were prepared for examination by scanning electron microscopy. A close morphological correlation was obtained between previously published optical microscopic images of the nodes and those seen using low magnification SEM. Progressively high magnifications provided insight into the fibrillar-level modes of structural integration across the nucleus-endplate junction. The closely packed fibrils of the CEP were largely parallel to the vertebral endplate and formed a dense, multi-layer substrate within which the nodal fibrils appeared to be anchored. Our idealised structural model proposes a mechanism by which this integration is achieved. The nodal fibrils, in curving into the CEP, are locked in place within its close-packed layers of transversely aligned fibrils, and probably at multiple levels. Secondly, there appears to be a subtle interweaving of the strongly aligned nodal fibrils with the multi-directional endplate fibrils. It is suggested that this structural integration provides the nucleus with a form of tethered mobility that supports physiological functions quite distinct from the primary strength requirements of the disc., (© 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.)

Background: Traditional management of pyloric stenosis has consisted of open pyloromyotomy during which the surgeon is able to palpate and determine whether the hypertrophied pylorus has been completely divided. During the last decade, laparoscopic pyloromyotomy has become an increasingly popular approach for this condition. The purpose of this study was to determine whether there is an effective pyloromyotomy length that will allow the surgeon to feel confident that a complete pyloromyotomy was performed with the laparoscopic approach., Methods: All infants undergoing laparoscopic pyloromyotomy from October 1999 through October 2003 at a single institution were retrospectively studied. Clinical variables collected included the patient's age, gender, electrolyte status on admission, the elapsed time from admission to operation, ultrasonographic dimensions of the hypertrophied pylorus, operative time, the length of the pyloromyotomy performed, the time to initial and to full feedings, and the duration of the postoperative hospitalization., Results: One hundred seventy-one patients comprised the study group. The age (mean +/- standard deviation) at the time of operation was 5.2 +/- 2.8 weeks. The mean preoperative ultrasonic measurements for both pyloric thickness and pyloric length were 4.3 +/- 0.7 mm and 19.5 +/- 2.8 mm, respectively. The average pyloromyotomy incision length for this entire group was 1.9 +/- 0.21 cm. The mean operative time was 23.5 +/- 8.3 minutes. There were no mucosal perforations, no conversions to an open procedure, and no evidence for an incomplete pyloromyotomy., Conclusions: Laparoscopic pyloromyotomy is a safe and effective technique for infants with pyloric stenosis. A pyloromyotomy incision length of approximately 2 cm appears to be an effective measure of a complete pyloromyotomy.

Recurrent mutations in vivo in T-lymphocytes identify clonally restricted genomic instabilities in some individuals. Cell-based assays allow initial recognition of clones with mutator phenotypes, but genotypic selection is required to determine frequencies and temporal sequences of potentially independent mutational events isolated only as complex changes in the same allele. The present work illustrates how two single-base insertions in the HPRT gene recovered only as a double event in a cell-based assay were shown to arise as separate in vivo mutations, being individually present at frequencies of < or =10(-4) and < or =10(-5), respectively, in peripheral blood. Full characterizations of mutator clones will allow elucidation of the earliest events in the emergence of genomic instability in human somatic cells.

The background frequency of mutations in human tissues is an important issue in cancer susceptibility and genotoxic exposure determinations. Here we report the detection of rare mutant leukocytes containing oncogenic base substitutions of the Harvey-ras, N-ras, and p53 genes by the Needle-in-a-Haystack mutation assay with a sensitivity of one cell in a million. Altogether, we detected and identified 17 independent mutations of 66 separate base site analyses of peripheral blood specimens obtained from 19 apparently normal individuals. Two individuals harbored a substantially increased frequency of mutant cells, representing 9 of the 17 independent mutations found. These results suggest that up to 1 in 10 normal individuals may harbor a significant frequency of oncogenic mutations in circulating leukocytes.

Background and induced germline mutagenesis and other genotoxicity studies have been hampered by the lack of a sufficiently sensitive technique for detecting mutations in a small cluster of cells or a single cell in a tissue sample composed of millions of cells. The most frequent type of genetic alteration is intragenic. The vast majority of oncogenic mutations in human and mammalian cancer involves only single base substitutions. We have developed universally applicable techniques that not only provide the necessary sensitivity and specificity for site specific mutagenesis studies, but also identify the point mutation. The exponential amplification procedures of polymerase chain reaction (PCR) and ligase chain reaction (LCR) have been combined with restriction endonuclease (RE) digestion to enable the selective enrichment and detection of single base substitution mutations in human oncogenic loci at a sensitivity of one mutant in more than 10(7) wild type alleles. These PCR/RE/LCR procedures have been successfully designed and used for codons 12 and 248 of the Ha-ras and p53 genes, respectively, both of which contain a natural MspI restriction endonuclease recognition sequence. These procedures have also been adapted for the detection and identification of mutations in oncogenic loci that do not contain a natural restriction endonuclease recognition sequence. Using PCR techniques, a HphI site was incorporated into the codons 12/13 region of the human N-ras gene, which was then used for the selective enrichment of mutants at this oncogenic locus. These PCR/RE/LCR procedures for base substitution mutations in codon 12 of the N-ras gene were found to have the sensitivity of detection of at least one mutant allele in the presence of the DNA equivalent of 10(6) wild type cells. Only one peripheral blood leukocyte DNA specimen out of nine normal individuals displayed an observable Ha-ras mutation that was present at frequency between 10(-5) and 10(-6). These PCR/RE/LCR techniques for detecting and identifying base substitution mutations are universally applicable to almost any locus or base site within the human or animal genome. With the added advantage of the adjustability of both the amount of DNA (number of genomes) to be tested and the sensitivity (10(-2) to 10(-7)) of the assay selection or enrichment procedures, these PCR/RE/LCR techniques will be useful in addressing a broad range of important questions in mutagenesis and carcinogenesis.

New multidentate ligands with structures similar to N,N'-bis[2-hydroxybenzyl]ethylenediamine-N,N'-diacetic acid (HBED) and N,N'-bis(pyridoxyl)ethylenediamine-N,N'-diacetic acid (PLED) were synthesized. The in vitro lipophilicity, electrophoretic behavior, and the in vitro biodistribution were studied for the 111In- and 67, 68Ga-complexes of N,N'-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine-N,N'-diacetic acid (Me4HBED); N,N'-bis(5-t-butyl-2-hydroxy-3-methylbenzyl)ethylenediamine-N,N'-diac eti c acid (t-butyl HBED); N,N'-bis[2-hydroxy-5-sulfobenzyl)ethylenediamine-N,N'-diacetic acid (SHBED); N,N'-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine-N-(2-hydroxyethyl) -N'-acetic acid (HBMA); and N,N'-bis(5-deoxypyridoxyl)ethylenediamine-N,N'-diacetic acid (DPLED). The biodistribution of the radiometal complexes were carried out in rats and an imaging study was performed in a non-human primate. The rapid clearance of the lipophilic complexes from blood and through the hepatobiliary system was easily demonstrated; as well, the hydrophilic complexes were cleared rapidly through the urinary tract. Positron emission tomographic images were generated from a study in a primate after administration of 68Ga-t-butyl HBED. These images well demonstrate the efficient liver accumulation and rapid hepatobiliary clearance (less than 1 h) and well differentiate images of the liver and gall bladder.