Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Targeting MCL-1 and BCL-2 with polatuzumab vedotin and venetoclax overcomes treatment resistance in R/R non-Hodgkin lymphoma: Results from preclinical models and a Phase Ib study.

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.

An investigation of range of motion preservation in fusionless anterior double screw and cord constructs for scoliosis correction.

PURPOSE: To evaluate the motion-preserving properties of vertebral body tethering with varying cord/screw constructs and cord thicknesses in cadaveric thoracolumbar spines. METHODS: In vitro flexibility tests were performed on six fresh-frozen human cadaveric spines (T1-L5) (2 M, 4F) with a median age of 63 (59-to-80). An ± 8 Nm load was applied to determine range of motion (ROM) in flexion-extension (FE), lateral bending (LB), and axial rotation (AR) in the thoracic and lumbar spine. Specimens were tested with screws (T5-L4) and without cords. Single (4.0 mm and 5.0 mm) and double (4.0 mm) cord constructs were sequentially tensioned to 100 N and tested: (1) Single 4.0 mm and (2) 5.0 mm cords (T5-T12); (3) Double 4.0 mm cords (T5-12); (4) Single 4.0 mm and (5) 5.0 mm cord (T12-L4); (6) Double 4.0 mm cords (T12-L4). RESULTS: In the thoracic spine (T5-T12), 4.0-5.0 mm single-cord constructs showed slight reductions in FE and 27-33% reductions in LB compared to intact, while double-cord constructs showed reductions of 24% and 40%, respectively. In the lumbar spine (T12-L4), double-cord constructs had greater reductions in FE (24%), LB (74%), and AR (25%) compared to intact, while single-cord constructs exhibited reductions of 2-4%, 68-69%, and 19-20%, respectively. CONCLUSIONS: The present biomechanical study found similar motion for 4.0-5.0 mm single-cord constructs and the least motion for double-cord constructs in the thoracic and lumbar spine suggesting that larger diameter 5.0 mm cords may be a more promising motion-preserving option, due to their increased durability compared to smaller cords. Future clinical studies are necessary to determine the impact of these findings on patient outcomes.

Traumatic Lingual Hematoma Resulting in Bilateral Temporal Mandibular Joint Dislocations.

BACKGROUND: Lingual hematoma (LH) is a relatively uncommon entity seen after both medical and traumatic etiologies. Regardless of the cause, the feared complication is acute airway obstruction. CASE REPORT: Our case involves a 39-year-old man who presented to the Emergency Department via emergency medical services with an enlarging LH after an unwitnessed fall, suspected to be an alcohol withdrawal seizure. The bleeding was likely exacerbated by previously undiagnosed thrombocytopenia. Airway stabilization was rapidly established via nasotracheal intubation after standard intubation techniques were deemed unfeasible. Despite correction of the coagulopathy, the LH continued to expand, resulting in bilateral tympanomandibular joint (TMJ) dislocations. To our knowledge, this complication has not been previously reported as a complication of LH. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Despite being a relatively uncommon condition, LH has the potential to result in life-threatening airway obstruction with limited airway options. Prompt airway stabilization should be the first priority upon diagnosis. A rapidly evolving LH can limit standard orotracheal rapid sequence intubation options, and may require alternative airway procedures. Additionally, ongoing lingual swelling after airway stabilization has now been shown in our case to result in bilateral TMJ dislocations. Concurrent management of reversible coagulopathy may help prevent this complication or reduce its severity.

Effective treatment of HER2-amplified breast cancer by targeting HER3 and ß1 integrin.

The central role of HER2 as the disease driver and HER3 as its essential partner has made them rational targets for the treatment of HER2-amplifed breast cancers, and there is considerable interest in developing highly effective treatment regimens for this disease that consist of targeted therapies alone. Much of these efforts are focused on dual targeting approaches, particularly dual targeting of the HER2-HER3 tumor driver complex itself, or vertical combinations that target downstream PI3K or Akt in addition to HER2. There is also potential in lateral combinations based on evidence implicating cross-talk with other membrane receptor systems, particularly integrins, and such lateral combinations can potentially involve either HER2 or HER3. We established a preclinical model of targeting HER3 using doxycycline-inducible shRNA and determined the efficacy of a ß1 integrin inhibitor in combination with targeting HER3. We report that targeting HER3 and ß1 integrin provides a particularly effective combination therapy approach for HER2-amplified cancers, surpassing the combination of HER2 and ß1 integrin targeting, and evading some of the safety concerns associated with direct HER2-targeting. This further validates HER3 as a major hub mediating the tumorigenic functions of HER2 and identifies it as a high value target for lateral combination therapy strategies.

Effect of potting technique on the measurement of six degree-of-freedom viscoelastic properties of human lumbar spine segments.

Polymethyl methacrylate (PMMA) and Wood's Metal are fixation media for biomechanical testing; however, the effect of each potting medium on the measured six degree-of-freedom (DOF) mechanical properties of human lumbar intervertebral discs is unknown. The first aim of this study was to compare the measured 6DOF elastic and viscoelastic properties of the disc when embedded in PMMA compared to repotting in Wood's Metal. The second aim was to compare the surface temperature of the disc when potted with PMMA and Wood's Metal. Six human lumbar functional spinal units (FSUs) were first potted in PMMA, and subjected to overnight preload in a saline bath at 37 °C followed by five haversine loading cycles at 0.1 Hz in each of 6DOF loading directions (compression, left/right lateral bending, flexion, extension, left/right axial rotation, anterior/posterior, and lateral shear). Each specimen was then repotted in Wood's Metal and subjected to a 2-h re-equilibrating preload followed by repeating the same 6DOF tests. Outcome measures of stiffness and phase angle were calculated from the final loading cycle in each DOF and were expressed as normalized percentages relative to PMMA (100%). Disc surface temperatures (anterior, left/right lateral) were measured during potting. Paired t-tests (with alpha adjusted for multiple DOF) were conducted to compare the differences in each outcome parameter between PMMA and Wood's Metal. No significant differences in stiffness or phase angle were found between PMMA and Wood's Metal. On average, the largest trending differences were found in the shear DOFs for both stiffness (approximately 35% greater for Wood's Metal compared to PMMA) and phase angle (approximately 15% greater for Wood's Metal). A significant difference in disc temperature was found at the anterior surface after potting with Wood's Metal compared to PMMA, which did not exceed 26 °C. Wood's Metal is linear elastic, stiffer than PMMA and may reduce measurement artifact of potting medium, particularly in the shear directions. Furthermore, it is easier to remove than PMMA, reuseable, and cost effective.

HER3 signalling is regulated through a multitude of redundant mechanisms in HER2-driven tumour cells.

HER2 (human epidermal growth factor receptor-2)-amplified tumours are characterized by constitutive signalling via the HER2-HER3 co-receptor complex. Although phosphorylation activity is driven entirely by the HER2 kinase, signal volume generated by the complex is under the control of HER3, and a large capacity to increase its signalling output accounts for the resiliency of the HER2-HER3 tumour driver and accounts for the limited efficacies of anti-cancer drugs designed to target it. In the present paper we describe deeper insights into the dynamic nature of HER3 signalling. Signalling output by HER3 is under several modes of regulation, including transcriptional, post-transcriptional, translational, post-translational and localizational control. These redundant mechanisms can each increase HER3 signalling output and are engaged in various degrees depending on how the HER3/PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signalling network is disturbed. The highly dynamic nature of HER3 expression and signalling, and the plurality of downstream elements and redundant mechanisms that function to ensure HER3 signalling throughput identify HER3 as a major signalling hub in HER2-amplified cancers and a highly resourceful guardian of tumorigenic signalling in these tumours.

Robotic-Assisted Navigation for Stereotactic Neurosurgery: A Cadaveric Investigation of Accuracy, Time, and Radiation.

BACKGROUND AND OBJECTIVES: Despite frequent use, stereotactic head frames require manual coordinate calculations and manual frame settings that are associated with human error. This study examines freestanding robot-assisted navigation (RAN) as a means to reduce the drawbacks of traditional cranial stereotaxy and improve targeting accuracy. METHODS: Seven cadaveric human torsos with heads were tested with 8 anatomic coordinates selected for lead placement mirrored in each hemisphere. Right and left hemispheres of the brain were randomly assigned to either the traditional stereotactic arc-based (ARC) group or the RAN group. Both target accuracy and trajectory accuracy were measured. Procedural time and the radiation required for registration were also measured. RESULTS: The accuracy of the RAN group was significantly greater than that of the ARC group in both target (1.2 ± 0.5 mm vs 1.7 ± 1.2 mm, P = .005) and trajectory (0.9 ± 0.6 mm vs 1.3 ± 0.9 mm, P = .004) measurements. Total procedural time was also significantly faster for the RAN group than for the ARC group (44.6 ± 7.7 minutes vs 86.0 ± 12.5 minutes, P < .001). The RAN group had significantly reduced time per electrode placement (2.9 ± 0.9 minutes vs 5.8 ± 2.0 minutes, P < .001) and significantly reduced radiation during registration (1.9 ± 1.1 mGy vs 76.2 ± 5.0 mGy, P < .001) compared with the ARC group. CONCLUSION: In this cadaveric study, cranial leads were placed faster and with greater accuracy using RAN than those placed with conventional stereotactic arc-based technique. RAN also required significantly less radiation to register the specimen's coordinate system to the planned trajectories. Clinical testing should be performed to further investigate RAN for stereotactic cranial surgery.

The role of HER3, the unpretentious member of the HER family, in cancer biology and cancer therapeutics.

Many types of human cancer are characterized by deregulation of the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors. In some cancers, genomic events causing overactivity of individual HER family members are etiologically linked with the pathogenesis of these cancers, and constitute the driving signaling function underlying their tumorigenic behavior. HER3 stands out among this family as the only member lacking catalytic kinase function. Cancers with driving HER3 amplifications or mutations have not been found, and studies of its expression in tumors have been only weakly provocative. However, substantial evidence, predominantly from experimental models, now suggest that its non-catalytic functions are critically important in many cancers driven by its' HER family partners. Furthermore, new insights into the mechanism of activation in the HER family has provided clear evidence of functionality in the HER3 kinase domain. The convergence of structural, mechanistic, and experimental evidence highlighting HER3 functions that may be critical in tumorigenesis have now led to renewed efforts towards identification of cancers or subtypes of cancers wherein HER3 function may be important in tumor progression or drug resistance. It appears now that its failure to earn the traditional definition of an oncogene has allowed the tumor promoting functions of HER3 to elude the effects of cancer therapeutics. But experimental science has now unmasked the unpretentious role of HER3 in cancer biology, and the next generation of cancer therapies will undoubtedly perform much better because of it.

Fixation Strength of Modified Iliac Screw Trajectory Compared to Traditional Iliac and S2 Alar-Iliac Trajectories: A Cadaveric Study.

OBJECTIVE: Traditional iliac (TI) screws require extensive dissection, involve offset-connectors, and have prominent screw heads that may cause patient discomfort. S2 alar-iliac (S2AI) screws require less dissection, do not need offset connectors, and are less prominent. However, the biomechanical consequences of S2AI screws crossing the alar-iliac joint is unknown. The present study investigates the fixation strength of a modified iliac (MI) screw, which has a more medial entry point and reduced screw prominence, but does not cross the alar-iliac joint. METHODS: Eighteen sacropelvic spines were divided into 3 groups (n = 6): TI, S2AI, and MI. Each specimen was fixed unilaterally with S1 pedicle screws and pelvic fixation according to its group. Screws were loaded at ±10 Nm at 3Hz for 1000 cycles. Motion of each screw and rod strain above and below the S1 screw was measured. RESULTS: Toggle of the S1 screw was lowest for the TI group, followed by the MI and S2AI groups, but there were no significant differences (P = 0.421). Toggle of the iliac screw relative to the pelvis was also lowest for the TI group, followed by the MI group, and was greatest for the S2AI group, without significant differences (P = 0.179). Rod strain was similar across all groups. CONCLUSIONS: No statistically significant differences were found between the TI, S2AI, and MI techniques with regard to screw toggle or rod strain. Advantages of the MI screw include its lower profile and a medialized starting point eliminating the need for offset-connectors.

Comprehensive Evaluation of Accessory Rod Position, Rod Material and Diameter, Use of Cross-connectors, and Anterior Column Support in a Pedicle Subtraction Osteotomy Model: Part I: Effects on Apical Rod Strain: An In Vitro and In Silico Biomechanical Study.

STUDY DESIGN: In silico finite element study. OBJECTIVE: The aim of this study was to evaluate the effect of six construct factors on apical rod strain in an in silico pedicle subtraction osteotomy (PSO) model: traditional inline and alternative Ames-Deviren-Gupta (ADG) multi-rod techniques, number of accessory rods (three- vs. four-rod), rod material (cobalt-chrome [CoCr] or stainless steel [SS] vs. titanium [Ti]), rod diameter (5.5 vs. 6.35 mm), and use of cross-connectors (CC), or anterior column support (ACS). SUMMARY OF BACKGROUND DATA: Rod fracture following lumbar PSO is frequently reported. Clinicians may modulate reconstructs with multiple rods, rod position, rod material and diameter, and with CC or ACS to reduce mechanical demand or rod contouring. A comprehensive evaluation of these features on rod strain is lacking. METHODS: A finite element model (T12-S1) with intervertebral discs and ligaments was created and validated with cadaveric motion data. Apical rod strain of primary and accessory rods was collected for 96 constructs across all six construct factors, and normalized to the Ti two-rod control. RESULTS: Regardless of construct features, CoCr and SS material reduced strain across all rods by 49.1% and 38.1%, respectively; increasing rod diameter from 5.5 mm to 6.35 mm rods reduced strain by 32.0%. Use of CC or lumbosacral ACS minimally affected apical rod strain (<2% difference from constructs without CC or ACS). Compared to the ADG technique, traditional inline reconstruction reduced primary rod strain by 32.2%; however, ADG primary rod required 14.2° less rod contouring. The inline technique produced asymmetrical loading between left and right rods, only when three rods were used. CONCLUSION: The number of rods and position of accessory rods affected strain distribution on posterior fixation. Increasing rod diameter and using CoCr rods was most effective in reducing rod strain. Neither CC nor lumbosacral ACS affected apical rod strain. LEVEL OF EVIDENCE: N/A.

Comprehensive In Silico Evaluation of Accessory Rod Position, Rod Material and Diameter, Use of Cross-connectors, and Anterior Column Support in a Pedicle Subtraction Osteotomy Model: Part II: Effects on Lumbosacral Rod and Screw Strain.

STUDY DESIGN: In silico finite element study. OBJECTIVE: The aim of this study was to evaluate effects of six construct factors on rod and screw strain at the lumbosacral junction in an in silico pedicle subtraction osteotomy (PSO) model: traditional inline and alternative Ames-Deviren-Gupta (ADG) multi-rod techniques, number of accessory rods (three-rod vs. four-rod), rod material (cobalt-chrome [CoCr] or stainless steel [SS] vs. titanium [Ti]), rod diameter (5.5 vs. 6.35 mm), and use of cross-connectors (CC), or anterior column support (ACS). SUMMARY OF BACKGROUND DATA: Implant failure and pseudoarthrosis at the lumbosacral junction following PSO are frequently reported. Clinicians may modulate reconstructs with multiple rods, rod position, rod material, and diameter, and with CC or ACS to reduce mechanical demand. An evaluation of these features' effects on rod and screw strains is lacking. METHODS: A finite element model (T12-S1) with intervertebral discs and ligaments was created and validated with cadaveric motion data. Lumbosacral rod and screw strain data were collected for 96 constructs across all six construct factors and normalized to the Ti 2-Rod control. RESULTS: The inline technique resulted in 12.5% to 51.3% more rod strain and decreased screw strain (88.3% to 95%) compared to ADG at the lumbosacral junction. An asymmetrical strain distribution was observed in the three-rod inline technique in comparison to four-rod, which was more evenly distributed. Regardless of construct features, rod strain was significantly decreased by rod material (CoCr > SS > Ti), and increasing rod diameter from 5.5 mm to 6.35 mm reduced strain by 9.9% to 22.1%. ACS resulted in significant reduction of rod (37.8%-59.8%) and screw strains (23.2%-65.8%). CONCLUSION: Increasing rod diameter, using CoCr rods, and ACS were the most effective methods in reducing rod strain at the lumbosacral junction. The inline technique decreased screw strain and increased rod strain compared to ADG. LEVEL OF EVIDENCE: N/A.

Defining "county": A mixed-methods inquiry of county emergency medicine residency programs.

INTRODUCTION: There is no clear unified definition of "county programs" in emergency medicine (EM). Key residency directories are varied in designation, despite it being one of the most important match factors for applicants. The Council of Residency Directors EM County Program Community of Practice consists of residency program leadership from a unified collective of programs that identify as "county." This paper's framework was spurred from numerous group discussions to better understand unifying themes that define county programs. METHODOLOGY: This institutional review board-exempt work provides qualitative descriptive results via a mixed-methods inquiry utilizing survey data and quantitative data from programs that self-designate as county. UNIQUE TREATMENT ANALYSIS AND CRITIQUE: Most respondents work, identify, and trained at a county program. The majority defined county programs by commitment to care for the underserved, funding from the city or state, low-resourced, and urban setting. Major qualitative themes included mission, clinical environment, research, training, and applicant recommendations. Comparing the attributes of programs by self-described type of training environment, county programs are typically larger, older, in central metro areas, and more likely to be 4 years in duration and have higher patient volumes when compared to community or university programs. When comparing hospital-level attributes of primary training sites county programs are more likely to be owned and operated by local governments or governmental hospital districts and authorities and see more disproportionate-share hospital patients. IMPLICATIONS FOR EDUCATION AND TRAINING IN EM: To be considered a county program we recommend some or most of the following attributes be present: a shared mission to medically underserved and vulnerable patients, an urban location with city or county funding, an ED with high patient volumes, supportive of resident autonomy, and research expertise focusing on underserved populations.

Mechanisms of Failure Following Simulated Repetitive Lifting: A Clinically Relevant Biomechanical Cadaveric Study.

STUDY DESIGN: A biomechanical analysis correlating internal disc strains and tissue damage during simulated repetitive lifting. OBJECTIVE: To understand the failure modes during simulated safe and unsafe repetitive lifting. SUMMARY OF BACKGROUND DATA: Repetitive lifting has been shown to lead to lumbar disc herniation (LDH). In vitro studies have developed a qualitative understanding of the effect of repetitive loading on LDH. However, no studies have measured internal disc strains and subsequently correlated these with disc damage. METHODS: Thirty human cadaver lumbar functional spinal units were subjected to an equivalent of 1 year of simulated repetitive lifting under safe and unsafe levels of compression, in combination with flexion (13-15°), and right axial rotation (2°) for 20,000 cycles or until failure. Safe or unsafe lifting were applied as a compressive load to mimic holding a 20 kg weight either close to, or at arm's length, from the body, respectively. Maximum shear strains (MSS) were measured, and disc damage scores were determined in nine regions from axial post-test magnetic resonance imaging (MRI) and macroscopic images. RESULTS: Twenty percent of specimens in the safe lifting group failed before 20,000 cycles due to endplate failure, compared with 67% in the unsafe group. Over half of the specimens in the safe lifting group failed via either disc protrusion or LDH, compared with only 20% via protrusion in the unsafe group. Significant positive correlations were found between MRI and macroscopic damage scores in all regions (rs > 0.385, P < 0.049). A significant positive correlation was observed in the left lateral region for MSS versus macroscopic damage score (rs = 0.486, P < 0.037) and MSS versus failure mode (rs = 0.724, P = 0.018, only specimens with disc failure). Pfirrmann Grade 3 discs were strongly associated with subsequent LDH (P = 0.003). CONCLUSION: Increased shear strains were observed in the contralateral side to the applied rotation as disc injury progressed from protrusion to LDH. Larger compressive loads applied to simulate unsafe lifting led to frequent early failure of the endplate, however, smaller compressive loads at similar flexion angles applied under safe lifting led to more loading cycles before failure, where the site of failure was more likely to be the disc. Our study demonstrated that unsafe lifting leads to greater risk of injury compared with safe lifting, and LDH and disc protrusion were more common in the posterior/posterolateral regions. LEVEL OF EVIDENCE: N/A.

Effectiveness of novel fabrics to resist punctures and lacerations from white shark (Carcharodon carcharias): Implications to reduce injuries from shark bites.

Increases in the number of shark bites, along with increased media attention on shark-human interactions has led to growing interest in preventing injuries from shark bites through the use of personal mitigation measures. The leading cause of fatality from shark bite victims is blood loss; thus reducing haemorrhaging may provide additional time for a shark bite victim to be attended to by emergency services. Despite previous shark-proof suits being bulky and cumbersome, new technological advances in fabric has allowed the development of lightweight alternatives that can be incorporated onto traditional wetsuits. The ability for these fabrics to withstand shark bites has not been scientifically tested. In this report, we compared two types of recently developed protective fabrics that incorporated ultra-high molecular weight polyethylene (UHMWPE) fibre onto neoprene (SharkStop and ActionTX) and compared them to standard neoprene alternatives. We tested nine different fabric variants using three different tests, laboratory-based puncture and laceration tests, along with field-based trials involving white sharks Carcharodon carcharias. Field-based trials consisted of measuring C. carcharias bite force and quantifying damages to the new fabrics following a bite from 3-4 m total length C. carcharias. We found that SharkStop and ActionTX fabric variants were more resistant to puncture, laceration, and bites from C. carcharias. More force was required to puncture the new fabrics compared to control fabrics (laboratory-based tests), and cuts made to the new fabrics were smaller and shallower than those on standard neoprene for both types of test, i.e. laboratory and field tests. Our results showed that UHMWPE fibre increased the resistance of neoprene to shark bites. Although the use of UHMWPE fibre (e.g. SharkStop and ActionTX) may therefore reduce blood loss resulting from a shark bite, research is needed to assess if the reduction in damages to the fabrics extends to human tissues and decreased injuries.

Off-axis response due to mechanical coupling across all six degrees of freedom in the human disc.

The kinematics of the intervertebral disc are defined by six degrees of freedom (DOF): three translations (Tz: axial compression, Tx: lateral shear, and Ty: anterior-posterior shear) and three rotations (Rz: torsion, Rx: flexion-extension, and Ry: lateral bending). There is some evidence that the six DOFs are mechanically coupled, such that loading in one DOF affects the mechanics of the other five "off-axis" DOFs, however, most studies have not controlled and/or measured all six DOFs simultaneously. Additionally, the relationships between disc geometry and disc mechanics are important for evaluation of data from different sized donor and patient discs. The objectives of this study were to quantify the mechanical behavior of the intervertebral disc in all six degrees of freedom (DOFs), measure the coupling between the applied motion in each DOF with the resulting off-axis motions, and test the hypothesis that disc geometry influences these mechanical behaviors. All off-axis displacements and rotations were significantly correlated with the applied DOF and were of similar magnitude as physiologically relevant motion, confirming that off-axis coupling is an important mechanical response. Interestingly, there were pairs of DOFs that were especially strongly coupled: lateral shear (Tx) and lateral bending (Ry), anterior-posterior shear (Ty) and flexion-extension (Rx), and compression (Tz) and torsion (Rz). Large off-axis shears may contribute to injury risk in bending and flexion. In addition, the disc responded to shear (Tx, Ty) and rotational loading (Rx, Ry, and Rz) by increasing in disc height in order to maintain the applied compressive load. Quantifying these mechanical behaviors across all six DOF are critical for designing and testing disc therapies, such as implants and tissue engineered constructs, and also for validating finite element models.

Personalised 3D knee compliance from clinically viable knee laxity measurements: A proof of concept ex vivo experiment.

Personalised information of knee mechanics is increasingly used for guiding knee reconstruction surgery. We explored use of uniaxial knee laxity tests mimicking Lachman and Pivot-shift tests for quantifying 3D knee compliance in healthy and injured knees. Two healthy knee specimens (males, 60 and 88 years of age) were tested. Six-degree-of-freedom tibiofemoral displacements were applied to each specimen at 5 intermediate angles between 0° and 90° knee flexion. The force response was recorded. Six-degree-of-freedom and uniaxial tests were repeated after sequential resection of the anterior cruciate, posterior cruciate and lateral collateral ligament. 3D knee compliance (C6DOF) was calculated using the six-degrees-of-freedom measurements for both the healthy and ligament-deficient knees and validated using a leave-one-out cross-validation. 3D knee compliance (CCT) was also calculated using uniaxial measurements for Lachman and Pivot-shift tests both conjointly and separately. C6DOF and CCT matrices were compared component-by-component and using principal axes decomposition. Bland-Altman plots, median and 40-60th percentile range were used as measurements of bias and dispersion. The error on tibiofemoral displacements predicted using C6DOF was < 9.6% for every loading direction and after release of each ligament. Overall, there was good agreement between C6DOF and CCT components for both the component-by-component and principal component comparison. The dispersion of principal components (compliance coefficients, positions and pitches) based on both uniaxial tests was lower than that based on single uniaxial tests. Uniaxial tests may provide personalised information of 3D knee compliance.

Targeting EGFR activity in blood vessels is sufficient to inhibit tumor growth and is accompanied by an increase in VEGFR-2 dependence in tumor endothelial cells.

Epidermal growth factor receptor (EGFR) targeting agents such as kinase inhibitors reduce tumor growth and progression. We have previously reported that EGFR is not only expressed by the tumor cells but by the tumor endothelial cells (EC) as well (Amin, D. N., Hida, K., Bielenberg, D. R., Klagsbrun, M., 2006. Tumor endothelial cells express epidermal growth factor receptor (EGFR) but not ErbB3 and are responsive to EGF and to EGFR kinase inhibitors. Cancer Res. 66, 2173-80). Thus, targeting tumor blood vessel EGFR may be a viable strategy for tumor growth inhibition. We describe here a melanoma xenograft model where the tumor cells express very little or no EGFR but the tumor blood vessels express activated EGFR. The EGFR kinase inhibitor, gefitinib (Iressa), retarded tumor growth with a size decrease of 38% compared to control mice, ostensibly due to targeting of the blood vessels. EC were isolated from tumors of gefitinib-treated mice. These EC were unable to proliferate in response to EGF and displayed relatively weaker activation of MAPK and AKT signaling in response to EGF compared to tumor EC isolated from vehicle-treated mice. In contrast, the tumor EC from gefitinib-treated mice expressed higher levels of VEGFR-2 both at the mRNA and protein level. In addition, these cells were less sensitive to EGFR kinase inhibitors in vitro but more sensitive to a VEGFR-2 kinase inhibitor. These results suggest that in tumor EC from gefitinib-treated mice there is a switch from dependence on EGFR activity to signaling via VEGFR-2. Our data provide a molecular rationale for combination therapies targeting both EGF and VEGF signaling on the tumor vasculature.