Role of CD44 in Chemotherapy Treatment Outcome: A Scoping Review of Clinical Studies.

Cluster of differentiation 44 (CD44), a cell surface adhesion molecule overexpressed in cancer stem cells, has been implicated in chemoresistance. This scoping review, following PRISMA-ScR guidelines, systematically identified and evaluated clinical studies on the impact of CD44 expression on chemotherapy treatment outcomes across various cancer types. The search encompassed PubMed (1985-2023) and SCOPUS (1936-2023) databases, yielding a total of 12,659 articles, of which 40 met the inclusion criteria and were included in the qualitative synthesis using a predefined data extraction table. Data collected included the cancer type, sample size, interventions, control, treatment outcome, study type, expression of CD44 variants and isoforms, and effect of CD44 on chemotherapy outcome. Most of the studies demonstrated an association between increased CD44 expression and negative chemotherapeutic outcomes such as shorter overall survival, increased tumor recurrence, and resistance to chemotherapy, indicating a potential role of CD44 upregulation in chemoresistance in cancer patients. However, a subset of studies also reported non-significant relationships or conflicting results. In summary, this scoping review highlighted the breadth of the available literature investigating the clinical association between CD44 and chemotherapeutic outcomes. Further research is required to elucidate this relationship to aid clinicians in managing CD44-positive cancer patients.

Damage patterns in polyethylene fixed bearings of retrieved total ankle replacements.

INTRODUCTION: Poor long-term outcomes continue to hinder the universal adoption of total ankle replacements (TAR) for end stage arthritis. In the present study, polyethylene inserts of TARs retrieved at revision surgery were analyzed for burnishing, scratching, mechanical damage, pitting, and embedded particles. METHODS: Fourteen retrieved polyethylene inserts from a fixed bearing total ankle replacement design currently in clinical use were analyzed. Duration of time in vivo was between 11.5 months and 120.1 months. Three investigators independently graded each articular surface in quadrants for five features of damage: burnishing, scratching, mechanical damage, pitting, and embedded particles. RESULTS: No correlation was found for burnishing between the anterior and posterior aspects (p = 0.47); however, scratching and pitting were significantly higher on the posterior aspect compared to the anterior aspect (p < 0.03). There was a high correlation between burnishing and in vivo duration of the implant (anterior: R = 0.67, p = 0.01, posterior: R = 0.68, p = 0.01). CONCLUSION: The higher concentration of posterior damage on these polyethylene inserts suggested that prosthesis-related (design) or surgeon-related (technique) factors might restrict the articulation of the implant. The resulting higher stresses in the posterior articular surfaces may have contributed to the failure of retrieved implants Keywords: Retrieval, Polyethylene Damage, Total Ankle Replacement.

Preclinical biomechanical testing models for the tibiotalar joint and its replacements: A systematic review.

In recent years, total ankle replacements have gained increasing popularity as an alternative to fusion. Preclinical testing of TARs requires reliable in vitro models which, in turn, need thorough knowledge of the kinematics of the tibiotalar joint. Surprisingly few studies have been published to simulate the in vivo kinematics of the tibiotalar joint. Among these studies, there is a wide range of methods and magnitudes of applied loads. The purpose of the present review was to summarize the applied loads, positions that were tested during static simulations, and ranges of motion simulated that have been used in human cadaveric models of the tibiotalar joint. Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, PubMed and Google Scholar were searched for studies pertaining to cadaveric tibiotalar joint kinematics. Our search yielded 12 appropriate articles that were included in the systematic review. While it is well known that loads at the tibiotalar joint are frequently as high as 5 times bodyweight [1], these studies reported applied loads varying from 200N-750N, below average bodyweight. Three studies used dynamic loading of custom apparatuses to drive cadaver limbs along predetermined paths to simulate gait. Conversely, the other nine studies applied static loads (∼300N), performed at discreet points during the stance phase, considerably lower than physiological conditions. The present systematic review calls for an urgent need to establish a consensus for preclinical evaluation of TARs for biomechanical function.

Biomechanical comparison of fixation stability using a Lisfranc plate versus transarticular screws.

BACKGROUND: To obtain adequate fixation in treating Lisfranc soft tissue injuries, the joint is commonly stabilized using multiple transarticular screws; however iatrogenic injury is a concern. Alternatively, two parallel, longitudinally placed plates, can be used to stabilize the 1st and 2nd tarsometatarsal joints; however this may not provide adequate stability along the Lisfranc ligament. Several biomechanical studies have compared earlier methods of fixation using plates to the standard transarticular screw fixation method, highlighting the potential issue of transverse stability using plates. A novel dorsal plate is introduced, intended to provide transverse and longitudinal stability, without injury to the articular cartilage. METHODS: A biomechanical cadaver model was developed to compare the fixation stability of a novel Lisfranc plate to that of traditional fixation, using transarticular screws. Thirteen pairs of cadaveric specimens were tested intact, after a simulated Lisfranc injury, and then following implant fixation, using one method of fixation randomly assigned, on either side of each pair. Optical motion tracking was used to measure the motion between each of the following four bones: 1st metatarsal, 2nd metatarsal, 1st cuneiform, and 2nd cuneiform. Testing included both cyclic abduction loading and cyclic axial loading. RESULTS: Both the Lisfranc plate and screw fixation method provided stability such that the average 3D motions across the Lisfranc joint (between 2nd metatarsal and 1st cuneiform), were between 0.2 and 0.4mm under cyclic abduction loading, and between 0.4 and 0.5mm under cyclic axial loading. Comparing the stability of fixation between the Lisfranc plate and the screws, the differences in motion were all 0.3mm or lower, with no clinically significant differences (p>0.16). CONCLUSIONS: Diastasis at the Lisfranc joint following fixation with a novel plate or transarticular screw fixation were comparable. Therefore, the Lisfranc plate may provide adequate support without risk of iatrogenic injury to the articular cartilage.

Adherent-invasive Escherichia coli blocks interferon-γ-induced signal transducer and activator of transcription (STAT)-1 in human intestinal epithelial cells.

Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with Crohn disease. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. The aim of this study was to determine whether activation of STAT1 by IFNγ was prevented by AIEC infection, and to define the mechanisms used. Human epithelial cells were infected with three different AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ, and STAT1 activation was monitored by immunoblotting. Our data show that live AIEC with active protein synthesis machinery is able to prevent IFNγ-mediated STAT1 phosphorylation, and that a secreted factor may be involved. We conclude that the suppression of epithelial cell STAT1 signal transduction by AIEC strains isolated from patients with Crohn disease represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Natural Killer Cell­Derived Extracellular Vesicles as Potential Anti­Viral Nanomaterials.

In viral infections, natural killer (NK) cells exhibit anti-viral activity by inducing apoptosis in infected host cells and impeding viral replication through heightened cytokine release. Extracellular vesicles derived from NK cells (NK-EVs) also contain the membrane composition, homing capabilities, and cargo that enable anti-viral activity. These characteristics, and their biocompatibility and low immunogenicity, give NK-EVs the potential to be a viable therapeutic platform. This study characterizes the size, EV-specific protein expression, cell internalization, biocompatibility, and anti-viral miRNA cargo to evaluate the anti-viral properties of NK-EVs. After 48 h of NK-EV incubation in inflamed A549 lung epithelial cells, or conditions that mimic lung viral infections such as during COVID-19, cells treated with NK-EVs exhibit upregulated anti-viral miRNA cargo (miR-27a, miR-27b, miR-369-3p, miR-491-5p) compared to the non-treated controls and cells treated with control EVs derived from lung epithelial cells. Additionally, NK-EVs effectively reduce expression of viral RNA and pro-inflammatory cytokine (TNF-α, IL-8) levels in SARS-CoV-2 infected Vero E6 kidney epithelial cells and in infected mice without causing tissue damage while significantly decreasing pro-inflammatory cytokine compared to non-treated controls. Herein, this work elucidates the potential of NK-EVs as safe, anti-viral nanomaterials, offering a promising alternative to conventional NK cell and anti-viral therapies.

Postfracture survival in a population-based study of adults aged ≥66 yr: a call to action at hospital discharge.

Postfracture survival rates provide prognostic information but are rarely reported along with other mortality outcomes in adults aged ≥50 yr. The timing of survival change following a fracture also needs to be further elucidated. This population-based, matched-cohort, retrospective database study examined 98 474 patients (73% women) aged ≥66 yr with an index fracture occurring at an osteoporotic site (hip, clinical vertebral, proximal non-hip non-vertebral [pNHNV], and distal non-hip non-vertebral [dNHNV]) from 2011 to 2015, who were matched (1:1) to nonfracture individuals based on sex, age, and comorbidities. All-cause 1- and 5-yr overall survival and relative survival ratios (RSRs) were assessed, and time trends in survival changes were characterized starting immediately after a fracture. In both sexes, overall survival was markedly decreased over 6 yr of follow-up after hip, vertebral, and pNHNV fractures, and as expected, worse survival rates were observed in older patients and males. The lowest 5-yr RSRs were observed after hip fractures in males (66-85 yr, 51.9%-63.9%; ≥86 yr, 34.5%), followed by vertebral fractures in males (66-85 yr, 53.2%-69.4%; ≥86 yr, 35.5%), and hip fractures in females (66-85 yr, 69.8%-79.0%; ≥86 yr, 52.8%). Although RSRs did not decrease as markedly after dNHNV fractures in younger patients, relatively low 5-yr RSRs were observed in females (75.9%) and males (69.5%) aged ≥86 yr. The greatest reduction in survival occurred within the initial month after hip, vertebral, and pNHNV fractures, indicating a high relative impact of short-term factors, with survival-reduction effects persisting over time. Therefore, the most critical period for implementing interventions aimed at improving post-fracture prognosis appears to be immediately after a fracture; however, considering the immediate need for introducing such interventions, primary fracture prevention is also crucial to prevent the occurrence of the initial fracture in high-risk patients.

Evaluation of Topology Optimization Using 3D Printing for Bioresorbable Fusion Cages: A Biomechanical Study in a Porcine Model.

STUDY DESIGN: Preclinical biomechanical study of topology optimization versus standard ring design for bioresorbable poly-ε-caprolactone (PCL) cervical spine fusion cages delivering bone morphogenetic protein-2 (BMP-2) using a porcine model. OBJECTIVE: The aim was to evaluate range of motion (ROM) and bone fusion, as a function of topology optimization and BMP-2 delivery method. SUMMARY OF BACKGROUND DATA: 3D printing technology enables fabrication of topology-optimized cages using bioresorbable materials, offering several advantages including customization, and lower stiffness. Delivery of BMP-2 using topology optimization may enhance the quality of fusion. METHODS: Twenty-two 6-month-old pigs underwent anterior cervical discectomy fusion at one level using 3D printed PCL cages. Experimental groups (N=6 each) included: Group 1: ring design with surface adsorbed BMP-2, Group 2: topology-optimized rectangular design with surface adsorbed BMP-2, and Group 3: ring design with BMP-2 delivery via collagen sponge. Additional specimens, two of each design, were implanted without BMP-2, as controls. Complete cervical segments were harvested six months postoperatively. Nanocomputed tomography was performed to assess complete bony bridging. Pure moment biomechanical testing was conducted in all three planes, separately. Continuous 3D motions were recorded and analyzed. RESULTS: Three subjects suffered early surgical complications and were not evaluated. Overall, ROM for experimental specimens, regardless of design or BMP-2 delivery method, was comparable, with no clinically significant differences among groups. Among experimental specimens at the level of the fusion, ROM was <1.0° in flexion and extension, indicative of fusion, based on clinically applied criteria for fusion of <2 to 4°. Despite the measured biomechanical stability, using computed tomography evaluation, complete bony bridging was observed in 40% of the specimens in Group 1, 50% of Group 2, 100% of Group 3, and none of the control specimens. CONCLUSION: A topology-optimized PCL cage with BMP-2 is capable of resulting in an intervertebral fusion, similar to a conventional ring-based design of the same bioresorbable material.

Nanoparticles-based technologies for cholera detection and therapy.

Cholera is a waterborne disease caused by Vibrio cholerae bacteria generally transmitted through contaminated food or water sources. Although it has been eradicated in most Western countries, cholera continues to be a highly transmitted and lethal disease in several African and Southeast Asian countries. Unfortunately, current diagnostic methods for cholera have challenges including high cost or delayed diagnoses that can lead to increased disease transmission during pandemics, while current treatments such as therapeutic drugs and vaccines have limited efficacy against drug-resistant serogroups of Vibrio cholerae. As such, new solutions that can treat cholera in an efficient manner that avoids Vibrio cholerae's adaptive immunity are needed. Nanoparticles (NPs) are a suitable platform for enhancing current theranostic tools because of their biocompatibility and ability to improve drug circulation and targeting. Nanoparticle surfaces can also be modified with various protein receptors targeting cholera toxins produced by Vibrio cholerae. This review will address recent developments in diagnostics, therapeutics, and prevention against cholera particularly focusing on the use of metal-based nanoparticles and organic nanoparticles. We will then discuss future directions regarding nanoparticle research for cholera.

Enterohemorrhagic Escherichia coli O157:H7 Shiga toxins inhibit gamma interferon-mediated cellular activation.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

Taper Material Loss in Total Hip Replacements: Is It Affected by Joint Friction?

BACKGROUND: Metal debris and corrosion products generated from the taper junctions of modular joint replacements have been recognized as contributors to failure. Therefore, understanding the factors associated with increased taper wear and corrosion is fundamental to improving implant performance. METHODS: A cohort of 85 large-diameter metal-on-metal heads and cups retrieved at revision surgery, after 10 to 96 months of service, was evaluated. First, metrology was conducted to quantify head taper material loss and implant articular surface wear. Then, joint frictional moments for each retrieved head-and-cup pair were measured during 10 cycles of simulated physiological gait in a biomechanical model. Taper material loss was evaluated for correlations with frictional moments, articular wear, head diameter, head-cup clearance, and time in vivo. RESULTS: Peak resultant frictional moments ranged from 9.1 to 26.3 Nm, averaging 17.3 ± 2.7 Nm. Fretting and corrosion damage during in vivo service resulted in material loss from the head tapers ranging between 0.04 and 25.57 mm3, compared with combined head and cup articular wear of 0.80 to 351.75 mm3 in this cohort. Taper material loss was not correlated with higher frictional moments (R = -0.20 to 0.11, p = 0.07 to 0.81). Higher frictional moments from axial rotation were correlated with higher head and cup wear (R = 0.33, p < 0.01). The correlation between taper material loss and head diameter was weak and did not reach statistical significance (R = 0.20, p = 0.07). Taper material loss was not correlated with nominal head-cup clearance (R = 0.06, p = 0.6). Finally, taper material loss increased significantly over time (R = 0.34, p < 0.01). CONCLUSIONS: Despite serious concerns regarding trunnionosis, volumes of head taper wear were generally lower than those of articular surface wear. There was no statistical correlation between taper wear and frictional moments. Therefore, the results suggest that high friction in metal-on-metal implants does not contribute to higher material loss at the head taper, despite high bending moments. CLINICAL RELEVANCE: The amount of metal debris and corrosion products from taper junctions of the joint arthroplasties, widely recognized as an insidious cause of failure, was not correlated with joint frictional moments. Multiple factors affect taper wear: implant design, material, size, surface finish, and patient weight and activity level. However, in the present cohort, high friction of metal-on-metal total hip replacements likely did not contribute to increased volume of material loss at taper interfaces, despite increased moments at the locations of taper material loss.

The insidious risk of periprosthetic fracture in clinically functional total hip arthroplasties: A biomechanical study of willed joints.

Femoral bone quality is a major risk factor of periprosthetic fracture after total hip arthroplasty (THA), which has mortality similar to native hip fractures but higher short-term morbidity. The goal of this study was to quantify cortical strains at the site of expected Vancouver Type-B periprosthetic fracture as a function of bone mineral density, femoral stem material, and fixation method using a series of 29 autopsy-retrieved, clinically asymptomatic hip joints with THA. Periprosthetic bone mineral content and density was assessed using dual-energy X-ray absorptiometry by Gruen Zone. Specimens then underwent combined cyclic axial and torsional loading, increasing incrementally from 100 N and ±1 Nm to peaks of 700 N and ±5 Nm. All specimens experienced significantly higher strains on the lateral surface than on the anterior surface, indicating that the bending loads in the frontal plane, rather than axial/torsional loads, had the predominant effect. Multiple significant relationships (p = 0.04, p = 0.02) were found between predicted periprosthetic strains calculated from radiographic measurements and observed principal strains. Though THA in the present study were in successful clinical service, the produced results indicated that some femurs with rigid cemented or noncemented implants were potentially at high risk for Vancouver Type-B fractures, which may be predicted radiographically.

A novel intramedullary nail to control interfragmentary motion in diaphyseal tibial fractures.

Numerous animal and human studies have demonstrated the benefit of controlled interfragmentary motion on fracture healing. In this study, we quantified interfragmentary motion and load transfer in tibial fractures fixed using a novel intramedullary nail (IMN) that allows controlled axial motion. Fifty composite tibias with various fracture patterns were utilized. For all test conditions, two interlocking screws were used to fix the nail in the proximal metaphysis, and two interlocking screws through the distal metaphysis. The nail allowed either no motion (static mode) or 1 mm (dynamic mode) of cyclic axial motion between the two fracture fragments for every fracture pattern tested. As expected, strain shielding was more prominent under static nail conditions. In contrast, specimens tested under dynamic nail conditions transferred axial load between the fracture fragments such that strains near the fracture site were generally similar to those measured on an intact tibia. Maximum shear strains proximal to the fracture were significantly lower in specimens with oblique or butterfly fracture patterns (p < 0.01) compared to intact specimens. This decrease in shear strain indicates that strain shielding effects were likely present due to the implant. However, strain shielding appeared to be reduced in tensile and compressive principal strains. In summary, the novel IMN allowed controlled axial motion between the fragments in a variety of common diaphyseal tibial fracture patterns. Clinical Significance: The present in vitro biomechanical study investigated a novel intramedullary nail capable of controlled axial interfragmentary motion which may potentially enhance fracture healing.

Enterohaemorrhagic, but not enteropathogenic, Escherichia coli infection of epithelial cells disrupts signalling responses to tumour necrosis factor-alpha.

Enterohaemorrhagic Escherichia coli (EHEC), serotype O157 : H7 is a non-invasive, pathogenic bacterium that employs a type III secretion system (T3SS) to inject effector proteins into infected cells. In this study, we demonstrate that EHEC blocks tumour necrosis factor-alpha (TNFα)-induced NF-κB signalling in infected epithelial cells. HEK293T and INT407 epithelial cells were challenged with EHEC prior to stimulation with TNFα. Using complementary techniques, stimulation with TNFα caused activation of NF-κB, as determined by luciferase reporter assay (increase in gene expression), Western blotting (phosphorylation of IκBα), immunofluorescence (p65 nuclear translocation) and immunoassay (CXCL-8 secretion), and each was blocked by EHEC O157 : H7 infection. In contrast, subversion of host cell signalling was not observed following exposure to either enteropathogenic E. coli, strain E2348/69 (O127 : H6) or the laboratory E. coli strain HB101. Heat-killed EHEC had no effect on NF-κB activation by TNFα. Inhibition was mediated, at least in part, by Shiga toxins and by the O157 plasmid, but not by the T3SS or flagellin, as demonstrated by using isogenic mutant strains. These findings indicate the potential for developing novel therapeutic targets to interrupt the infectious process.

Is load control necessary to produce physiological AP displacement and axial rotation in wear testing of TAR?

The International Standard Organization, ISO 22622, specifies two options for joint wear simulator evaluation of total ankle replacements (TARs): load-controlled and displacement-controlled. In the present study, the load-controlled testing parameters were applied to cadaveric specimens to quantify and compare the observed sagittal translations and axial rotations to those specified under the displacement-controlled option. Twelve cadaveric specimens were stripped of extraneous tissues, keeping surrounding ankle ligaments. A halo was used to produce plantarflexion and dorsiflexion of the talus through two screws, while a baseplate resisted axial loads. The axial force and torque were applied to the tibia and fibula under force and torque feedback control. An anterior-posterior force was applied to the tibia. Plantarflexion-dorsiflexion were applied using rotation control. To protect the cadaveric specimens, loads were applied at 50% of the specified load profile while plantarflexion-dorsiflexion rotation was applied as specified. There was variation among specimens in magnitudes of anterior-posterior displacement with peaks ranging from 3.3 mm posteriorly to 3.0 mm anteriorly. Likewise, there was variation among specimens in magnitude of axial rotation, with peaks ranging from 11° external rotation to 4.5° internal rotation. However, the mean magnitudes of AP displacement and axial rotation did not exceed those specified by ISO 22622.

A Systematic Review of Unsystematic Total Ankle Replacement Wear Evaluations.

BACKGROUND: Numerous studies have reported the use of laboratory multistation joint simulators to successfully predict wear performance and functionality of hip and knee replacements. In contrast, few studies in the peer-reviewed literature have used joint simulation to quantify the wear performance and functionality of ankle replacements. We performed a systematic review of the literature on joint simulator studies that quantified polyethylene wear in total ankle arthroplasty. In addition to the quantified wear results, the load and motion parameters were identified and compared among the studies. METHODS: A search was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines to identify articles reporting total ankle replacement polyethylene wear using joint simulators. RESULTS: Nine studies that used joint simulators and 1 study that used a computer simulation were found. Although all studies used physiological multidirectional motions (i.e., internal/external rotation, plantar flexion/dorsiflexion, anterior/posterior translation), there was large variability among the studies in the magnitudes of these motions. Among these studies, mean non-cross-linked polyethylene wear ranged from 3.3 ± 0.4 to 25.8 ± 3.1 mm per million cycles. In contrast, mean highly cross-linked polyethylene wear ranged from 2.1 ± 0.3 to 3.3 ± 0.4 mm per million cycles. The wide distribution in wear rates was attributable to the highly inconsistent kinematic parameters and loads applied as well as differences in implant design and materials. CONCLUSIONS: There is a severe lack of clinically applicable data on wear performance of total ankle replacements in the peer-reviewed literature. No universal set of kinematic load parameters has been established. Furthermore, only 2 of the published studies have validated their findings using independently derived data, such as retrieval analysis. These shortcomings make it difficult to compare findings as a function of design parameters and materials, or to draw clinically relevant conclusions from these simulations. More work is required to enhance the predictive capability of in vitro simulations of total ankle replacements. CLINICAL RELEVANCE: The results of joint wear simulator studies may not accurately represent in vivo wear of total ankle replacements. Joint simulator studies should establish that they are accurately replicating in vivo wear, thus enabling use of their predictive capabilities for new materials and designs.

Increasing loads and diminishing returns: a biomechanical study of direct vertebral rotation.

STUDY DESIGN: Biomechanical simulation of DVR and pure-moment testing on thoracic spines. OBJECTIVES: Characterize load-deformation response of thoracic spines under DVR maneuvers until failure, and compare to pure-moment testing of same spines. Despite reports of surgical complications, few studies exist on increase in ROM under DVR torque. Biomechanical models predicting increases from surgical releases have consistently used "pure-moments", a standard established for non-destructive measurement of ROM. Yet, DVR torque is not accurately modeled using pure moments and, moreover, magnitudes of torque applied during DVR maneuvers may be substantially higher than pure-moment testing. METHODS: Cadaveric thoracic spines (N = 11) were imaged, then prepared. Polyaxial pedicle screws were implanted at T7-T10 after surgical releases. Bilateral facetectomies and Ponte osteotomies were completed at T10-T11. A custom apparatus, mounted into an 8-dof MTS load frame, was used to attach to pedicle screws, allowing simulation of surgical DVR maneuvers. Motions of vertebrae were measured using optical motion tracking. Torque was increased until rupture of the T10-T11 disc or fracture at the pedicle screw sites at any level. The torque-rotation behavior was compared to its behavior under pure-moment testing performed prior to the DVR maneuver. RESULTS: Under DVR maneuvers, failure of the T10-T11 discs accompanied in most cases by pedicle screw loosening, occurred at 13.7-54.7 Nm torque, increasing axial rotation by 1.4°-8.9°. In contrast, pure-moment testing (4 Nm) increased axial rotation by only 0.0°-0.9°. CONCLUSIONS: DVR resulted in substantially greater correction potential increases compared to pure-moment testing even at the same torque. These results suggest increased flexibility obtained by osteotomies and facetectomies is underestimated using pure-moment testing, misrepresenting clinical expectations. The present study is an important and necessary step toward the establishment of a more accurate and ultimately surgically applied model. LEVEL OF EVIDENCE: III.

Elucidating the formation of 6-deoxyheptose: biochemical characterization of the GDP-D-glycero-d-manno-heptose C6 dehydratase, DmhA, and its associated C4 reductase, DmhB.

6-Deoxyheptose is found within the surface polysaccharides of several bacterial pathogens. In Yersinia pseudotuberculosis, it is important for the barrier function of the O-antigen in vitro and for bacterial dissemination in vivo. The putative C6 dehydratase DmhA and C4 reductase DmhB, that were identified as responsible for 6-deoxyheptose synthesis based on genetics data, represent potential therapeutical targets. Their detailed biochemical characterization is presented herein. The substrate, GDP-D-glycero-D-manno-heptose, was synthesized enzymatically from sedoheptulose 7-phosphate using overexpressed and purified GmhA/B/C/D enzymes from Aneurinibacillus thermoaerophilus. Overexpressed and purified DmhA used this substrate with high efficiency, as indicated by its K(m) of 0.23 mM and k(cat) of 1.1 s(-1). The mass spectrometry (MS) analysis of the reaction product was consistent with a C6 dehydration reaction. DmhB could readily reduce this compound in the presence of NAD(P)H to produce GDP-6-deoxy-D-manno-heptose, as indicated by MS and NMR analyses. DmhA also used GDP-mannose as a substrate with a K(m) of 0.32 mM and a k(cat) of 0.25 min(-1). This kinetic analysis indicates that although the K(m) values for GDP-mannose and GDP-manno-heptose were similar, the genuine substrate for DmhA is GDP-manno-heptose. DmhB was also able to reduce the GDP-4-keto-6-deoxymannose produced by DmhA, although with poor efficiency and exclusively in the presence of NADPH. This study is the first complete biochemical characterization of the 6-deoxyheptose biosynthesis pathway. Also, it allows the screening for inhibitors, the elucidation of substrate specificity determinants, and the synthesis of carbohydrate antigens of therapeutic relevance.

The biosynthesis and biological role of 6-deoxyheptose in the lipopolysaccharide O-antigen of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis O:2a harbours 6-deoxy-d-manno-heptose in its O-antigen. The biological function of 6-deoxyheptose and its role in virulence is unknown and its biosynthetic pathway has not been demonstrated experimentally. Here, we show that dmhA and dmhB are necessary for 6-deoxyheptose biosynthesis in Y. pseudotuberculosis. Their disruption resulted in the lack of 6-deoxyheptose in the O-unit and its replacement by d-glycero-d-manno-heptose, thus indicating relaxed specificity of the glycosyltransferases, polymerase and ligase involved in lipopolysaccharide synthesis. The dmhB mutant exhibited a lower content in ketooctonic acid (Ko)-containing core molecules and reduced ligation and polymerization of the O-unit. We also show that Tyr128 is essential for activity of DmhB, and that DmhB functions as an oligomer, based on the dominant negative effect of overexpression of DmhB Y128F in dmhA. Moreover, we demonstrate that 6-deoxyheptose is important for virulence-related functions of the outer membrane and its appendages in vitro, such as barrier function against bile salts, polymyxin and novobiocin, and flagella-mediated motility. Although both mutants colonized the mouse ceacum as well as the wild type, the dmhB mutant was impaired for colonization of the liver, suggesting that DmhB represents a potential therapeutic target.

Structural studies of the O-antigens of Yersinia pseudotuberculosis O:2a and mutants thereof with impaired 6-deoxy-D-manno-heptose biosynthesis pathway.

The full structure of the long- and short-chain O-antigen of Yersinia pseudotuberculosis O:2a containing two uncommon deoxy sugars, abequose and 6-deoxy-d-manno-heptose (6dmanHep), was established, for the first time, by sugar analysis, NMR spectroscopy, and high-resolution ESIMS. Similar structural studies were also performed on two O:2a mutants with single disruption of 6dmanHep synthesis pathway genes each, which synthesize modified long-chain (dmhA mutant) and short-chain (both dmhA and dmhB mutants) O-antigens with 6dmanHep replaced by its putative biosynthetic precursor, D-glycero-D-manno-heptose.