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The scientific process is nonlinear, unpredictable, and ongoing. Assessing the nature of science is difficult with methods that rely on Likert-scale or multiple-choice questions. This study evaluated conceptions about the scientific process using student-created visual representations that we term "flowcharts." The methodology, Scientific Process Flowchart Assessment (SPFA), consisted of a prompt and rubric that was designed to assess students' understanding of the scientific process. Forty flowcharts representing a multidisciplinary group without intervention and 26 flowcharts representing pre- and postinstruction were evaluated over five dimensions: connections, experimental design, reasons for doing science, nature of science, and interconnectivity. Pre to post flowcharts showed a statistically significant improvement in the number of items and ratings for the dimensions. Comparison of the terms used and connections between terms on student flowcharts revealed an enhanced and more nuanced understanding of the scientific process, especially in the areas of application to society and communication within the scientific community. We propose that SPFA can be used in a variety of circumstances, including in the determination of what curricula or interventions would be useful in a course or program, in the assessment of curriculum, or in the evaluation of students performing research projects., (© 2016 K. J. Wilson and B. Rigakos. CBE—Life Sciences Education © 2016 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Purpose: Counteracting posterior translation of the tibia with an anterior force on the posterior proximal tibia has been demonstrated clinically to improve posterior knee laxity following posterior cruciate ligament (PCL) injury. This study quantified forces applied to the posterior proximal tibia by two knee braces designed for treatment of PCL injuries., Methods: The forces applied by two knee braces to the posterior proximal tibia and in vivo three-dimensional knee kinematics of six adult, male, healthy volunteer subjects (mean ± standard deviation: height, 182.5 ± 5.2 cm; body mass, 83.2 ± 9.3 kg; body mass index, 24.9 ± 1.5 kg/m(2); age, 25.8 ± 2.9 years) were measured using a custom pressure mapping technique and traditional surface marker motion capture techniques, while subjects performed three functional activities. The activities included seated unloaded knee flexion, squatting, and stair descent in a new generation dynamic force (DF) PCL brace and a static force (SF) PCL brace., Results: During unloaded flexion at the lowest force level setting, the force applied by the DF brace increased as a function of flexion angle (slope = 0.7 N/°; p < 0.001) compared to the SF brace effect. Force applied by the SF brace did not significantly change as a function of flexion angle (slope = 0.0 N/°; n.s.). By 45° of flexion, the average force applied by the DF brace (48.1 N) was significantly larger (p < 0.001) than the average force applied by the SF brace (25.0 N). The difference in force continued to increase as flexion angle increased. During stair descent, average force (mean ± standard deviation) at toe off was significantly higher (p = 0.013) for the DF brace (78.7 ± 21.6 N) than the SF brace (37.3 ± 7.2 N). Similar trends were observed for squatting and for the higher force level settings., Conclusions: The DF brace applied forces to the posterior proximal tibia that dynamically increased with increased flexion angle. Additionally, the DF brace applied significantly larger forces at higher flexion angles compared to the SF brace where the PCL is known to experience larger in situ forces. Clinical studies are necessary to determine whether the loading characteristics of the DF brace, which more closely replicated the in situ loading profile of the native PCL, results in long-term improved posterior knee laxity following PCL injury., Level of Evidence: II.

A key facet of the Patient Protection and Affordable Care Act (PPACA) is the expansion of health insurance coverage. However, even with the PPACA, an estimated 11.2 million undocumented immigrants will remain uncovered. The majority of the remaining uncovered immigrant population is of Mexican origin. We assess the long-term benefits and short-term costs of providing coverage to male migrants from Mexico, employing data from the 2007-2011 Mexican Migration Project (MMP) and the 2009 Medical Expenditures Panel (MEPS) survey. Our results show that health status prior to migration, age at time of interview, emigrating from Central Mexico, and use of health services in the U.S. all predict declines in health at a significant level. We also find that having spent more than 10 cumulative years in the U.S. has borderline significance in predicting health decline (p=.052). Estimated coverage costs for health insurance for largely undocumented immigrants increase over time, but remain lower than those of comparable U.S.-born individuals. We conclude with several policy implications.

Purpose: To determine whether reaming of anterior cruciate ligament (ACL) reconstruction tibial tunnels with a 10-mm-diameter reamer would result in injury to the anterior roots of the medial and lateral menisci in an in vitro model when using a tibial aiming device at two settings (40° and 60°)., Methods: Three-dimensional footprints of the ACL and the anterior roots of the menisci were measured for 12 human cadaveric tibias. Measurements were taken before and after attempted reaming of an ACL tibial reconstruction tunnel in the calculated ACL centre using a tibial aiming device set at two angles (40° and 60°)., Results: Iatrogenic injury to the anterior root of the medial meniscus caused by overlap with the reamed tibial tunnel was found in 3/6 specimens in Group 1 (40°) and 0/6 specimens in Group 2 (60°). The average area of iatrogenic injury in Group 1 was 9.6 mm(2) (8.6 % of the root). There was iatrogenic injury to the anterior root of the lateral meniscus in 4/6 specimens for both groups. The average area of iatrogenic injury was 20.5 mm(2) (28.8 %) for Group 1 and 16.2 mm(2) (25.9 %) for Group 2., Conclusions: Reaming of ACL tibial reconstruction tunnels carries a risk of iatrogenic injury to the anterior meniscus roots, even when tunnels are reamed based on guide pin placement in the calculated centre of the ACL. Reaming at varying tibial aiming device angle settings can affect the obliquity of the tunnel aperture and cause iatrogenic injury to the anterior meniscal roots. Caution should be exercised clinically to avoid iatrogenic injury to both the anterior meniscal roots while reaming tibial tunnels during ACL reconstructions.

Predictions from theories of the processes of word reading acquisition have rarely been tested against evidence from exceptionally early readers. The theories of Ehri, Share, and Byrne, and an alternative, Knowledge Sources theory, were so tested. The former three theories postulate that full development of context-free letter sounds and awareness of phonemes are required for normal acquisition, while the claim of the alternative is that with or without such, children can use sublexical information from their emerging reading vocabularies to acquire word reading. Results from two independent samples of children aged 3-5, and 5 years, with mean word reading levels of 7 and 9 years respectively, showed underdevelopment of their context-free letter sounds and phoneme awareness, relative to their word reading levels and normal comparison samples. Despite such underdevelopment, these exceptional readers engaged in a form of phonological recoding that enabled pseudoword reading, at the level of older-age normal controls matched on word reading level. Moreover, in the 5-year-old sample further experiments showed that, relative to normal controls, they had a bias toward use of sublexical information from their reading vocabularies for phonological recoding of heterophonic pseudowords with irregular consistent spelling, and were superior in accessing word meanings independently of phonology, although only if the readers were without exposure to explicit phonics. The three theories were less satisfactory than the alternative theory in accounting for the learning of the exceptionally early readers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Introduction: The objective of our study was to evaluate the cost-effectiveness of a community-based intervention designed to improve physical activity levels and dietary intake and to reduce diabetes risk in a largely Hispanic population residing along the U.S.-Mexico border., Method: We forecasted disease outcomes, quality-adjusted life-years (QALYs) gained, and lifetime costs associated with actual and projected attainment of 2% and 5% weight loss taking a societal cost perspective. We extrapolated changes in beverage calorie consumption between baseline and 6-month follow-up to attain projected weight loss measures. Outcomes were projected 5, 10, and 20 years into the future and discounted at a 3.0% rate., Results: The incremental cost-effectiveness ratio was $57,430 and $61,893, respectively, per QALY gained when compared with usual care for the 2% and 5% weight loss scenarios. The intervention was particularly cost-effective for morbidly obese participants. Cost-effectiveness improves when using 3-year weight loss projections based on changes in sugar-sweetened beverage caloric consumption to $49,478 and $24,092 for the 2% and 5% weight loss scenarios., Conclusions: This analysis demonstrates that a culturally sensitive community-based weight loss and maintenance intervention can be cost-effective even when healthy weight individuals participate., (© 2014 Society for Public Health Education.)

Background: Revision anterior cruciate ligament (ACL) reconstruction requires a precise evaluation of previous tunnel locations and diameters. Enlargement of the tunnels, despite not usually affecting primary reconstruction outcomes, plays an important role in revision ACL management. Three dimensional (3D) computed tomography (CT) models are reported to be the most accurate method for identifying the tunnel position and possible conflicts with a revision tunnel placement. However, the ability of 3D CT to measure the tunnel size is still not proven. The goal of this study was to evaluate the ability of measuring the size of the bone tunnels in ACL reconstructed knees with 3D CT compared to the traditional two dimensional (2D) CT method., Methods: Twenty-four patients had CT scans performed immediately following ACL reconstruction surgery. Their femoral tunnels size were measured by a standard 2D CT measurement and then compared with three novel 3D CT measuring methods: the best transverse section method, the best fit cylinder method and the wall thickness method. The drill size used during surgery was used as a control measure for the tunnel width. Intra-class correlation coefficients were obtained., Results: The intra-class correlation coefficient and respective 95% confidence interval range (ICC [95%CI]) for the three methods compared with the drill sizes were 0.899 [0.811-0.947] for the best transverse section method, 0.745 [0.553-0.862] for the best fit cylinder method, -0.004 [-0.081 to -0.12] for the wall thickness method and 0.922 [0.713-0.97] for the 2D CT method. The mean differences compared to the drill size were 0.02 mm for the best fit transverse section method, 0.01 mm for the best fit cylinder diameter method, 3.34 mm for the wall thickness method and 0.29 mm for the 2D CT method. The intra-rater agreement (ICC [95%CI]) was excellent for the best transverse section method 0.999 [0.998-0.999] and the 2D CT method 0.969 [0.941-0.984]., Conclusions: The 3D best transverse section method presented a high correlation to the drill sizes and high intra-rater agreement, and was the best method for ACL tunnel evaluation in a 3D CT based model.

Background: The quantitative anatomical relationships of the main ligamentous, tendinous, and osseous structures of the sternoclavicular joint have not been widely investigated. The purpose of this study was to provide a quantitative description of the sternoclavicular joint in relation to relevant surgical landmarks., Methods: We dissected eleven nonpaired, fresh-frozen cadaveric sternoclavicular joints from four men and seven women (mean age at death, fifty-three years; range, thirty-three to sixty-four years) and measured the ligaments, musculature, and osseous landmarks with use of a three-dimensional coordinate-measuring device., Results: The clavicular pectoralis ridge, located at the 9:30 clock-face position on a right clavicle, served as a reliable osseous landmark for reference to the soft-tissue attachments around the sternoclavicular joint. The costoclavicular ligament was the largest ligament of the sternoclavicular joint, with 80% greater footprint area than that of the posterior sternoclavicular ligament. Articular cartilage covered 67% of the medial end of the clavicle and was located anteroinferiorly. The sternohyoid muscle inserted directly over the posterior sternoclavicular joint and the medial end of the clavicle, whereas the sternothyroid muscle inserted 9.5 mm inferior to the posterior-superior articular margin of the manubrium and coursed 19.8 mm laterally along the first rib. An avascular plane that can serve as a "safe zone" for posterior dissection was observed in each specimen, posterior to the sternoclavicular joint and anterior to the sternohyoid and sternothyroid muscles., Conclusions: The clavicular pectoralis ridge can be used as an intraoperative guide for clavicle orientation and tunnel placement in sternoclavicular ligament reconstruction. Sternoclavicular joint resection arthroplasty should avoid injuring the costoclavicular ligament, which is the largest sternoclavicular joint ligament. Resection of only the anteroinferior aspect of the medial end of the clavicle may provide adequate decompression while preserving the stability of the clavicle. The location of the sternohyoid and sternothyroid musculotendinous insertions appear to provide a "safe zone" for posterior clavicle and manubrial dissection., (Copyright © 2014 by The Journal of Bone and Joint Surgery, Incorporated.)

Objective: A standardized definition of normative T2 values across the articular surface of the hip must be defined in order to fully understand T2 values for detecting early degeneration. Therefore, in this article, we seek to lay foundational methodology for reproducible quantitative evaluation of hip cartilage damage using T2 mapping to determine the normative T2 values in asymptomatic individuals., Design: Nineteen prospectively enrolled asymptomatic volunteers (age 18-35 years, males 10, females 9, alpha angle 49.3º ± 7.2º) were evaluated with a sagittal T2 mapping sequence at 3.0 T magnetic resonance imaging. Acetabular and femoral cartilage was manually segmented directly on the second echo of the T2 mapping sequence by 3 raters, twice. Segmentations were divided into 12 subregions modified from the geographic zone method. Median T2 values within each subregion were compiled for further analysis and interrater and intrarater reliability was assessed., Results: In the femur, the posterior-superior subregion was significantly higher (P ≤ 0.05) than those in the posterior-inferior and anterior-inferior subregions. In the acetabulum, the anterior-inferior subregion was significantly higher (P ≤ 0.001) than in the anterior-superior, middle, and posterior-inferior subregions. T2 values of the posterior-superior subregion were significantly higher (P ≤ 0.05) than the anterior-superior, middle, and posterior-inferior subregions. Interrater agreement was generally fair to good.

Background: Lateral ankle sprains are common sports injuries that may require surgery for chronic lateral ankle instability. Anatomic repair or reconstruction is desired, yet there is a scarcity of quantitative information regarding the origins and insertions of the lateral ligaments related to surgically pertinent osseous landmarks., Methods: Fourteen ankle specimens were dissected to isolate the anterior talofibular ligament, calcaneofibular ligament, posterior talofibular ligament, and cervical ligament. A three-dimensional coordinate measurement device was used to determine the origins, insertions, footprint areas, orientations, and distances from osseous landmarks., Results: A single-banded anterior talofibular ligament was identified in seven of the fourteen specimens, and a double-banded anterior talofibular ligament was identified in the remaining seven. The single-banded anterior talofibular ligament originated an average of 13.8 mm (95% confidence interval [CI], 12.3 to 15.3) from the inferior tip of the lateral malleolus at the anterior fibular border and inserted an average of 17.8 mm (95% CI, 16.3 to 19.3) superior to the apex of the lateral talar process along the anterior border of the talar lateral articular facet. The calcaneofibular ligament originated an average of 5.3 mm (95% CI, 4.2 to 6.5) from the inferior tip of the lateral malleolus at the anterior fibular border and inserted an average of 16.3 mm (95% CI, 14.5 to 18.1) from the posterior point of the peroneal tubercle. The posterior talofibular ligament was the largest ligament and originated an average of 4.8 mm (95% CI, 3.7 to 5.9) superior to the inferior tip of the lateral malleolus in the digital fossa to insert an average of 13.2 mm (95% CI, 11.5 to 14.9) from the talar posterolateral tubercle. The cervical ligament originated on the superior part of the calcaneus and inserted at a point that was approximately 50% of the talar neck anteroposterior distance., Conclusions: Consistent distances from the anterior talofibular ligament, calcaneofibular ligament, posterior talofibular ligament, and cervical ligament footprint centers to osseous landmarks were identified., Clinical Relevance: Footprint center distances from surgically relevant osseous landmarks identified in this study can be used during reconstructive surgery of the lateral ankle ligaments and may result in more anatomically accurate placement of the reconstructed ligaments., (Copyright © 2014 by The Journal of Bone and Joint Surgery, Incorporated.)

Background: The deltoid ligament has both superficial and deep layers and consists of up to six ligamentous bands. The prevalence of the individual bands is variable, and no consensus as to which bands are constant or variable exists. Although other studies have looked at the variance in the deltoid anatomy, none have quantified the distance to relevant osseous landmarks., Methods: The deltoid ligaments from fourteen non-paired, fresh-frozen cadaveric specimens were isolated and the ligamentous bands were identified. The lengths, footprint areas, orientations, and distances from relevant osseous landmarks were measured with a three-dimensional coordinate measurement device., Results: In all specimens, the tibionavicular, tibiospring, and deep posterior tibiotalar ligaments were identified. Three additional bands were variable in our specimen cohort: the tibiocalcaneal, superficial posterior tibiotalar, and deep anterior tibiotalar ligaments. The deep posterior tibiotalar ligament was the largest band of the deltoid ligament. The origins from the distal center of the intercollicular groove were 16.1 mm (95% confidence interval, 14.7 to 17.5 mm) for the tibionavicular ligament, 13.1 mm (95% confidence interval, 11.1 to 15.1 mm) for the tibiospring ligament, and 7.6 mm (95% confidence interval, 6.7 to 8.5 mm) for the deep posterior tibiotalar ligament. Relevant to other pertinent osseous landmarks, the tibionavicular ligament inserted at 9.7 mm (95% confidence interval, 8.4 to 11.0 mm) from the tuberosity of the navicular, the tibiospring inserted at 35% (95% confidence interval, 33.4% to 36.6%) of the spring ligament's posteroanterior distance, and the deep posterior tibiotalar ligament inserted at 17.8 mm (95% confidence interval, 16.3 to 19.3 mm) from the posteromedial talar tubercle., Conclusions: The tibionavicular, tibiospring, and deep posterior tibiotalar ligament bands were constant components of the deltoid ligament. The deep posterior tibiotalar ligament was the largest band of the deltoid ligament., Clinical Relevance: The anatomical data regarding the deltoid ligament bands in this study will help to guide anatomical placement of repairs and reconstructions for deltoid ligament injury or instability.

Bone metastases are a clinically significant problem that arises in approximately 70% of metastatic breast cancer patients. Once established in the bone, tumor cells induce changes in the bone microenvironment that lead to bone destruction, pain, and significant morbidity. While much is known about the later stages of bone disease, less is known about the earlier stages or the changes in protein expression in the tumor micro-environment. Due to promising results of combining magnetic resonance imaging (MRI) and Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry (MALDI IMS) ion images in the brain, we developed methods for applying these modalities to models of tumor-induced bone disease in order to better understand the changes in protein expression that occur within the tumor-bone microenvironment. Specifically, we integrated 3-dimensional-volume reconstructions of spatially resolved MALDI IMS with high-resolution anatomical and diffusion weighted MRI data and histology in an intratibial model of breast tumor-induced bone disease. This approach enables us to analyze proteomic profiles from MALDI IMS data with corresponding in vivo imaging and ex vivo histology data. To the best of our knowledge, this is the first time that these three modalities have been rigorously registered in the bone. The MALDI mass-to-charge ratio peaks indicate differential expression of calcyclin, ubiquitin, and other proteins within the tumor cells, while peaks corresponding to hemoglobin A and calgranulin A provided molecular information that aided in the identification of areas rich in red and white blood cells, respectively. This multi-modality approach will allow us to comprehensively understand the bone-tumor microenvironment and thus may allow us to better develop and test approaches for inhibiting bone metastases., (Published by Elsevier Inc.)

Objective: Before quantitative imaging techniques can become clinically valuable, the method, and more specifically, the regions of locating and reporting these values should be standardized toward reproducibility comparisons across centers and longitudinal follow-up of individual patients. The purpose of this technical note is to describe a rigorous and reproducible method of locating, analyzing, and reporting quantitative MRI values in hip articular cartilage with an approach that is consistent with current orthopedic literature., Design: To demonstrate this localization and documentation, 3 patients (age, 23 ± 5.1 years; 2 males, 1 female) who presented with symptomatic mixed-type femoroacetabular impingement (α angle, 63.3° ± 2.1°; center edge angle, 39° ± 4.2°) were evaluated with T2-mapping at 3 T MRI prior to hip arthroscopy. Manual segmentation was performed and cartilage of the acetabulum and femur was divided into 12 subregions adapted from the geographic zone method. Bone landmarks in the acetabulum and femur, identifiable both in arthroscopy and MR images, were manually selected and the coordinates exported for division of cartilage., Results: Mean T2 values in each zone are presented., Conclusions: The current work outlines a standardized system to locate and describe quantitative mapping values that could aid in surgical decision making, planning, and the noninvasive longitudinal follow-up of implemented cartilage preservation and restoration techniques.

Typically, full Bayesian estimation of correlated event rates can be computationally challenging since estimators are intractable. When estimation of event rates represents one activity within a larger modeling process, there is an incentive to develop more efficient inference than provided by a full Bayesian model. We develop a new subjective inference method for correlated event rates based on a Bayes linear Bayes model under the assumption that events are generated from a homogeneous Poisson process. To reduce the elicitation burden we introduce homogenization factors to the model and, as an alternative to a subjective prior, an empirical method using the method of moments is developed. Inference under the new method is compared against estimates obtained under a full Bayesian model, which takes a multivariate gamma prior, where the predictive and posterior distributions are derived in terms of well-known functions. The mathematical properties of both models are presented. A simulation study shows that the Bayes linear Bayes inference method and the full Bayesian model provide equally reliable estimates. An illustrative example, motivated by a problem of estimating correlated event rates across different users in a simple supply chain, shows how ignoring the correlation leads to biased estimation of event rates., (© 2013 Society for Risk Analysis.)

Structure-activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC50 0.7 nM, FMS cell IC50 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate whether semi-quantitative analysis of high temporal resolution dynamic contrast-enhanced MRI (DCE-MRI) acquired early in treatment can predict the response of locally advanced breast cancer (LABC) to neoadjuvant chemotherapy (NAC)., Materials and Methods: As part of an IRB-approved prospective study, 21 patients with LABC provided informed consent and underwent high temporal resolution 3T DCE-MRI before and after 1cycle of NAC. Using measurements performed by two radiologists, the following parameters were extracted for lesions at both examinations: lesion size (short and long axes, in both early and late phases of enhancement), radiologist's subjective assessment of lesion enhancement, and percentages of voxels within the lesion demonstrating progressive, plateau, or washout kinetics. The latter data were calculated using two filters, one selecting for voxels enhancing ≥50% over baseline and one for voxels enhancing ≥100% over baseline. Pretreatment imaging parameters and parameter changes following cycle 1 of NAC were evaluated for their ability to discriminate patients with an eventual pathological complete response (pCR)., Results: All 21 patients completed NAC followed by surgery, with 9 patients achieving a pCR. No pretreatment imaging parameters were predictive of pCR. However, change after cycle 1 of NAC in percentage of voxels demonstrating washout kinetics with a 100% enhancement filter discriminated patients with an eventual pCR with an area under the receiver operating characteristic curve (AUC) of 0.77. Changes in other parameters, including lesion size, did not predict pCR., Conclusion: Semi-quantitative analysis of high temporal resolution DCE-MRI in patients with LABC can discriminate patients with an eventual pCR after one cycle of NAC., (© 2013 Elsevier Inc. All rights reserved.)

Determining biophysical sensitivity and specificity of quantitative magnetic resonance imaging is essential to develop effective imaging metrics of neurodegeneration. Among these metrics, apparent pool size ratio (PSR) from quantitative magnetization transfer (qMT) imaging and radial diffusivity (RD) from diffusion tensor imaging (DTI) are both known to relate to histological measure of myelin density and integrity. However their relative sensitivities towards quantitative myelin detection are unknown. In this study, we correlated high-resolution quantitative magnetic resonance imaging measures of subvoxel tissue structures with corresponding quantitative myelin histology in a lipopolysaccharide (LPS) mediated animal model of MS. Specifically, we acquired quantitative magnetization transfer (qMT) and diffusion tensor imaging (DTI) metrics (on the same tissue sample) in an animal model system of type III oligodendrogliopathy which lacked prominent lymphocytic infiltration, a system that had not been previously examined with quantitative MRI. We find that the qMT measured apparent pool size ratio (PSR) showed the strongest correlation with a histological measure of myelin content. DTI measured RD showed the next strongest correlation, and other DTI and relaxation parameters (such as the longitudinal relaxation rate (R1f) or fractional anisotropy (FA)) showed considerably weaker correlations with myelin content., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: Hepatoblastomas often require neoadjuvant chemotherapy to facilitate partial hepatectomy, which necessitates freedom of tumor borders from the confluence of hepatic veins (COHV), portal vein bifurcation (PVB), and retrohepatic inferior vena cava (IVC). This study aimed to clarify the effect of incremental neoadjuvant cycles on the AHEP0731 protocol criteria of hepatoblastoma resectability., Methods: Hepatoblastoma responses to neoadjuvant chemotherapy were analyzed among patients (n=23) treated at two children's hospitals between 1996 and 2010. Using digital imaging data, ellipsoid and point-based models were created to measure tumor volume regression and respective distances from tumor borders nearest to the COHV, PVB, and IVC., Results: Hepatoblastoma volumes regressed with incremental neoadjuvant chemotherapy cycles (p<0.001). Although tumor borders regressed away from the COHV (p=0.008), on average only 1.1mm was gained. No change from tumor borders to the PVB was detected (p=0.102). Distances from tumor borders to the IVC remained stable at one hospital (p=0.612), but increased only 0.15 mm every 10 days of therapy at the other (p=0.002). Neoadjuvant chemotherapy induced slightly more tumors to meet the threshold vascular margin of 1cm (baseline to completion): COHV, 11 (47.8%) to 17 (73.9%; p=0.058); PVB, 11 (47.8%) to 15 (65.2%; p=0.157); and IVC, 4 (17.4%) to 10 (43.5%; p=0.034). No differences were detected in demographic or disease-specific characteristics between patients who did or did not achieve this 1-cm margin after conclusion of chemotherapy., Conclusion: Hepatoblastoma volumes regress significantly with increasing neoadjuvant chemotherapy cycles. However, tumors often remain anchored to the major hepatic vasculature, showing marginal improvement in resectability criteria., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This research aimed to validate the specificity of the newly developed nanobeacon for imaging the Thomsen-Friedenreich (TF) antigen, a potential biomarker of colorectal cancer. The imaging agent is comprised of a submicron-sized polystyrene nanosphere encapsulated with a Coumarin 6 dye. The surface of the nanosphere was modified with peanut agglutinin (PNA) and poly(N-vinylacetamide (PNVA) moieties. The former binds to Gal-β(1-3)GalNAc with high affinity while the latter enhances the specificity of PNA for the carbohydrates. The specificity of the nanobeacon was evaluated in human colorectal cancer cells and specimens, and the data were compared with immunohistochemical staining and flow cytometric analysis. Additionally, distribution of the nanobeacon in vivo was assessed using an "intestinal loop" mouse model. Quantitative analysis of the data indicated that approximately 2 μg of PNA were detected for each milligram of the nanobeacon. The nanobeacon specifically reported colorectal tumors by recognizing the tumor-specific antigen through the surface-immobilized PNA. Removal of TF from human colorectal cancer cells and tissues resulted in a loss of fluorescence signal, which suggests the specificity of the probe. Most importantly, the probe was not absorbed systematically in the large intestine upon topical application. As a result, no registered toxicity was associated with the probe. These data demonstrate the potential use of this novel nanobeacon for imaging the TF antigen as a biomarker for the early detection and prediction of the progression of colorectal cancer at the molecular level., (Copyright © 2012 UICC.)

As industrialized countries around the world encounter rising health care costs with little to show in quality and health outcome improvements, they increasingly are turning to pay-for-performance mechanisms to align provider action with quality, equity, and efficiency goals. The primary objective of most pay-for-performance schemes is to improve quality of care, and the logic of linking financial rewards to quality and performance metrics makes sense. However, despite broad international experience with pay-for-performance, evidence of its impact is limited, frequently conflicting, focuses largely on improvements in the provision and structure of care rather than health outcomes, and tends to generate more questions than it does answers.

The ubiquitin proteasome system is involved in degradation of old or damaged sarcomeric proteins. Most E3 ubiquitin ligases are associated with cullins, which function as scaffolds for assembly of the protein degradation machinery. Cullin 3 uses an adaptor to link to substrates; in Caenorhabditis elegans, one of these adaptors is the BTB-domain protein MEL-26 (maternal effect lethal). Here we show that MEL-26 interacts with the giant sarcomeric protein UNC-89 (obscurin). MEL-26 and UNC-89 partially colocalize at sarcomeric M-lines. Loss of function or gain of function of mel-26 results in disorganization of myosin thick filaments similar to that found in unc-89 mutants. It had been reported that in early C. elegans embryos, a target of the CUL-3/MEL-26 ubiquitylation complex is the microtubule-severing enzyme katanin (MEI-1). Loss of function or gain of function of mei-1 also results in disorganization of thick filaments similar to unc-89 mutants. Genetic data indicate that at least some of the mel-26 loss-of-function phenotype in muscle can be attributed to increased microtubule-severing activity of MEI-1. The level of MEI-1 protein is reduced in an unc-89 mutant, suggesting that the normal role of UNC-89 is to inhibit the CUL-3/MEL-26 complex toward MEI-1.

Systemic bacterial infection is characterized by a robust whole-organism inflammatory response. Analysis of the immune response to infection involves technologies that typically focus on single organ systems and lack spatial information. Additionally, the analysis of individual inflammatory proteins requires antibodies specific to the protein of interest, limiting the panel of proteins that can be analyzed. Herein we describe the application of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) to mice systemically infected with Staphylococcus aureus to identify inflammatory protein masses that respond to infection throughout an entire infected animal. Integrating the resolution afforded by magnetic resonance imaging (MRI) with the sensitivity of MALDI IMS provides three-dimensional spatially resolved information regarding the distribution of innate immune proteins during systemic infection, allowing comparisons to in vivo structural information and soft-tissue contrast via MRI. Thus, integrating MALDI IMS with MRI provides a systems-biology approach to study inflammation during infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Epidermal growth factor (EGF) family peptides are ligands for the EGF receptor (EGFR). Here, we elucidate functional differences among EGFR ligands and mechanisms underlying these distinctions. In 32D/EGFR myeloid and MCF10A breast cells, soluble amphiregulin (AR), transforming growth factor alpha (TGFα), neuregulin 2 beta, and epigen stimulate greater EGFR coupling to cell proliferation and DNA synthesis than do EGF, betacellulin, heparin-binding EGF-like growth factor, and epiregulin. EGF competitively antagonizes AR, indicating that its functional differences reflect dissimilar intrinsic activity at EGFR. EGF stimulates much greater phosphorylation of EGFR Tyr1045 than does AR. Moreover, the EGFR Y1045F mutation and z-cbl dominant-negative mutant of the c-cbl ubiquitin ligase potentiate the effect of EGF but not of AR. Both EGF and AR stimulate phosphorylation of EGFR Tyr992. However, the EGFR Y992F mutation and phospholipase C gamma inhibitor U73122 reduce the effect of AR much more than that of EGF. Expression of TGFα in 32D/EGFR cells causes greater EGFR coupling to cell proliferation than does expression of EGF. Moreover, expression of EGF in 32D/EGFR cells causes these cells to be largely refractory to stimulation with soluble EGF. Thus, EGFR ligands are functionally distinct in models of paracrine and autocrine signaling and EGFR coupling to biological responses may be specified by competition among functionally distinct EGFR ligands.

The ErbB4 receptor tyrosine kinase possesses both tumour suppressor and oncogenic activities. Thus pharmacological agents are needed to help elucidate ErbB4 functions. However, limitations of existing ErbB4 agonists and antagonists have led us to seek novel ErbB4 antagonists. The Q43L mutant of the ErbB4 agonist NRG2β (neuregulin 2β) stimulates ErbB4 tyrosine phosphorylation, yet fails to stimulate ErbB4 coupling to cell proliferation. Thus in the present paper we hypothesize that NRG2β/Q43L may be an ErbB4 antagonist. NRG2β/Q43L competitively antagonizes agonist stimulation of ErbB4 coupling to cell proliferation. NRG2β/Q43L stimulates less ErbB4 tyrosine phosphorylation than does NRG2β. In addition, NRG2β stimulation of cell proliferation requires PI3K (phosphoinositide 3-kinase) activity and NRG2β stimulates greater Akt phosphorylation than does NRG2β/Q43L. Moreover, EGFR [EGF (epidermal growth factor) receptor] kinase activity (but not that of ErbB4) is critical for coupling ErbB4 to proliferation. Experiments utilizing ErbB4 splicing isoforms and mutants suggest that NRG2β and NRG2β/Q43L may differentially stimulate ErbB4 coupling to the transcriptional co-regulator YAP (Yes-associated protein). Finally, NRG2β/Q43L competitively antagonizes agonist stimulation of EGFR and ErbB2/ErbB3, indicating that NRG2β/Q43L is a pan-ErbB antagonist. Thus we postulate that NRG2β/Q43L and other antagonistic ligands stimulate ErbB tyrosine phosphorylation on a set of residues distinct from that stimulated by agonists, thus suggesting a novel mechanism of ErbB receptor regulation. Moreover, NRG2β/Q43L and related ligand-based antagonists establish a paradigm for the discovery of anti-ErbB therapeutics.

The behavior of glucose oxidase (GOx) on gold nanoparticles (NPs) was investigated as a function of (1) NP surface chemistry, (2) stabilizing protein additives, and (3) protein microenvironment. GOx secondary structure and unfolding was probed by circular dichroism (CD) spectroscopy and fluorescence, and GOx enzymatic activity was measured by a colorimetric assay. We also examined the activity and structure of GOx after displacement from the NP surface. Generally, GOx behavior was negatively impacted by conjugation to the NP, and conjugation conditions could vary the influence of the NP. Surface chemistry and protein microenvironment could improve behavior, but addition of stabilizing proteins negatively influenced activity. After displacement from the NPs, GOx tended to remain unfolded, indicating that the interactions with the NP were irreversible.

Caenorhabditis elegans is a premier model genetic system for discovering new information about the assembly and maintenance of striated muscle. The localization of a protein within a nematode muscle cell can reveal important clues to its function. In C. elegans, proteins can be localized by two different methods at the light microscopy level: GFP tagged proteins and indirect immunofluorescence. Although there are advantages and disadvantages of each method, antibodies can be used to localize proteins expressed at endogenous levels and without tags that might interfere with function. Immunolocalization requires efficient and effective methods of fixation. Here, we describe in detail two different methods for fixation of adult worms, the Nonet method and the Constant Spring method. We also discuss the advantages and the disadvantages of each, and how to choose between them. These methods are also useful for localizing proteins expressed in other cell types.

Introduction: The objective of our study was to estimate the long-term cost-effectiveness of a lifestyle modification program led by community health workers (CHWs) for low-income Hispanic adults with type 2 diabetes., Methods: We forecasted disease outcomes, quality-adjusted life years (QALYs) gained, and lifetime costs associated with attaining different hemoglobin A1c (A1c) levels. Outcomes were projected 20 years into the future and discounted at a 3.0% rate. Sensitivity analyses were conducted to assess the extent to which our results were dependent on assumptions related to program effectiveness, projected years, discount rates, and costs., Results: The incremental cost-effectiveness ratio of the intervention ranged from $10,995 to $33,319 per QALY gained when compared with usual care. The intervention was particularly cost-effective for adults with high glycemic levels (A1c > 9%). The results are robust to changes in multiple parameters., Conclusion: The CHW program was cost-effective. This study adds to the evidence that culturally sensitive lifestyle modification programs to control diabetes can be a cost-effective way to improve health among Hispanics with diabetes, particularly among those with high A1c levels.

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC(50) = 0.69 nM, cell assay IC(50) = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

Objectives: Aboriginal people are under-represented in epidemiological research, largely due to past failures to engage and recruit Aboriginal communities, research fatigue and the use of culturally inappropriate methods. A qualitative study was undertaken in rural and urban Aboriginal communities in north-eastern and south-western Ontario to identify culturally congruent public health research methodologies., Study Design: A qualitative participatory research study using focus group discussions., Methods: This study employed a participatory research framework to elicit methodological suggestions for conducting public health research with Aboriginal communities during focus groups with healthcare providers from six diverse Aboriginal health organizations in Ontario, Canada., Results: Continuing requests for participation in health research studies have led to community exhaustion. Discussions explored appropriate methods to obtain community approval and support for a study, the need for cultural sensitivity training for researchers, the value of conducting studies of interest and benefit to the community, advantages and disadvantages of qualitative and quantitative studies, the benefit of both Aboriginal and non-Aboriginal ethics reviews, the importance of safeguarding trusted information, types of incentives that may enhance study participation, suggestions to improve the collection of questionnaire information and biological specimens, how to resolve contentious issues and dissemination of study results., Conclusion: In order to successfully engage Aboriginal people in health studies, researchers need to build rapport with communities, have a community presence, be respectful and collaborative, utilize incentives, and employ flexible and adaptive methodologies of reasonable length. Oral interviews are preferred to self-completed information. The use of more mixed methods methodologies was suggested when quantitative data collection is necessary. Communities expect presentations about research findings., (Copyright © 2011 The Royal Society of Public Health. Published by Elsevier Ltd. All rights reserved.)

The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

The camel racing industry would have added value in being able to assign parentage with high certainty. This study was aimed at assessing and applying microsatellite multiplexes to construct a parentage testing system for camels. An efficient system of 17 loci from 700 camel samples was used to construct a database of unrelated adults. Based on this, we estimated measures of polymorphism among the markers. In three multiplex reactions, we detected a total of 224 alleles, with 5–23 alleles/locus (mean = 13.18 ± 6.95 SD) and an average heterozygosity (HE) of 0.54 (range 0.032–0.905). The total parentage exclusion probability was 0.99999 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.9999 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. We used 15 juveniles for parentage testing, as well as 17 sires (bull camels) and 21 dams (cows). In the case of parentage assignment, the microsatellite panel assigned all 15 offspring parentage with high confidence. Overall, these findings offer a set of microsatellite markers that are easy, simple and highly informative for parentage testing in camels.

During efforts to improve the bioavailability of FMS kinase inhibitors 1 and 2, a series of saturated and aromatic 4-heterocycles of reduced basicity were prepared and evaluated in an attempt to also improve the cardiovascular safety profile over lead arylamide 1, which possessed ion channel activity. The resultant compounds retained excellent potency and exhibited diminished ion channel activity., (2010 Elsevier Ltd. All rights reserved.)

Diffusion tensor imaging (DTI)-based muscle fiber tracking enables the measurement of muscle architectural parameters, such as pennation angle (theta) and fiber tract length (L(ft)), throughout the entire muscle. Little is known, however, about the repeatability of either the muscle architectural measures or the underlying diffusion measures. Therefore, the goal of this study was to investigate the repeatability of DTI fiber tracking-based measurements and theta and L(ft). Four DTI acquisitions were performed on two days that allowed for between acquisition, within day, and between day analyses. The eigenvalues and fractional anisotropy were calculated at the maximum cross-sectional area of, and fiber tracking was performed in, the tibialis anterior muscle of nine healthy subjects. The between acquisitions condition had the highest repeatability for the DTI indices and the architectural parameters. The overall inter class correlation coefficients (ICC's) were greater than 0.6 for both theta and L(ft) and the repeatability coefficients were theta < 10.2 degrees and L(ft) < 50 mm. In conclusion, under the experimental and data analysis conditions used, the repeatability of the diffusion measures is very good and repeatability of the architectural measurements is acceptable. Therefore, this study demonstrates the feasibility for longitudinal studies of alterations in muscle architecture using DTI-based fiber tracking, under similar noise conditions and with similar diffusion characteristics., (2010 John Wiley & Sons, Ltd.)

Objective: Quantifying vasospasm has traditionally been performed manually, a method prone to imprecision and user bias. An alternative approach is to use computerized image analysis techniques to define and quantify the diameter of a vessel. The goal of this article is to demonstrate a novel automated vessel measurement algorithm specific to the needs of vasospasm studies and to compare it with traditional manual measurements in an animal model of vasospasm., Methods: A total of 576 arterial diameter measurements were collected by 4 independent, blinded examiners from 24 angiograms in a rabbit subarachnoid hemorrhage (SAH) model. Measurements were taken from 3 segments of the basilar artery in anteroposterior and lateral projections, both before SAH and after SAH-induced vasospasm. Means and standard deviations of 288 manual measurements were compared with 288 automated measurements., Results: The precision of automated measurements was significantly improved compared with standardized manual measurements (85.7% decrease in variation; P < .001). When using automated measurements, the precision was not affected by vessel size, but when using manual measurements, smaller arteries were less precise (P = .04). There was no significant difference in precision between 2 different contrast concentrations (P = .32)., Conclusion: Automated measurements of basilar artery diameters are more precise than manual measurements, both before and after SAH-induced vasospasm. The variability in the manual group worsens when the artery is smaller secondary to vasospasm, indicating a need for the use of this segmentation method.

There is increasing evidence that tumor-associated macrophages promote the malignancy of some cancers. Colony-stimulating factor-1 (CSF-1) is expressed by many tumors and is a growth factor for macrophages and mediates osteoclast differentiation. Herein, we report the efficacy of a novel orally active CSF-1 receptor (CSF-1R) kinase inhibitor, JNJ-28312141, in proof of concept studies of solid tumor growth and tumor-induced bone erosion. H460 lung adenocarcinoma cells did not express CSF-1R and were not growth inhibited by JNJ-28312141 in vitro. Nevertheless, daily p.o. administration of JNJ-28312141 caused dose-dependent suppression of H460 tumor growth in nude mice that correlated with marked reductions in F4/80(+) tumor-associated macrophages and with increased plasma CSF-1, a possible biomarker of CSF-1R inhibition. Furthermore, the tumor microvasculature was reduced in JNJ-28312141-treated mice, consistent with a role for macrophages in tumor angiogenesis. In separate studies, JNJ-28312141 was compared with zoledronate in a model in which MRMT-1 mammary carcinoma cells inoculated into the tibias of rats led to severe cortical and trabecular bone lesions. Both agents reduced tumor growth and preserved bone. However, JNJ-28312141 reduced the number of tumor-associated osteoclasts superior to zoledronate. JNJ-28312141 exhibited additional activity against FMS-related receptor tyrosine kinase-3 (FLT3). To more fully define the therapeutic potential of this new agent, JNJ-28312141 was evaluated in a FLT3-dependent acute myeloid leukemia tumor xenograft model and caused tumor regression. In summary, this novel CSF-1R/FLT3 inhibitor represents a new agent with potential therapeutic activity in acute myeloid leukemia and in settings where CSF-1-dependent macrophages and osteoclasts contribute to tumor growth and skeletal events.

The efficient Suzuki cross-coupling of pyrazoline nonaflates with organoboron reagents was achieved to afford diverse 3-substituted-2-pyrazolines in excellent yield. The nonaflates displayed improved reactivity over the corresponding triflates and smoothly coupled to a variety of aryl- and heteroarylboronic acids. This process and its broad scope constitute a rapid, divergent strategy for the synthesis of elaborated 2-pyrazolines that are not readily obtained via conventional methods.

Breast, prostate, pancreatic, colorectal, lung, and head and neck cancers exploit deregulated signaling by ErbB family receptors and their ligands, EGF family peptide growth factors. EGF family members that bind the same receptor are able to stimulate divergent biological responses both in cell culture and in vivo. This is analogous to the functional selectivity exhibited by ligands for G-protein coupled receptors. Here we review this literature and propose that this functional selectivity of EGF family members is due to distinctions in the conformation of the liganded receptor and subsequent differences in the sites of receptor tyrosine phosphorylation and receptor coupling to signaling effectors. We also discuss the roles of divergent ligand activity in establishing and maintaining malignant phenotypes. Finally, we discuss the potential of mutant EGF family ligands as cancer chemotherapeutics targeted to ErbB receptors.

Diffusion tensor imaging-based fiber tracking in skeletal muscle has been used to reconstruct and quantify muscle architecture. In addition, the consistent pattern of muscle fiber geometry enables a quantitative assessment of the fiber tracking. This work describes a method to determine the accuracy of individual muscle fiber tracts based on the location at which the fibers terminate, the fiber path, and similarity to the neighboring fibers. In addition, the effect of different stop criteria settings on this quantitative assessment was investigated. Fiber tracking was performed on the tibialis anterior muscle of nine healthy subjects. Complete fiber tracts covered 89.4 +/- 9.6% and 75.0 +/- 15.2% of the aponeurosis area in the superficial and deep compartments, respectively. Applications of the method include the exclusion of erroneous fiber-tracking results, quantitative assessment of data set quality, and the assessment of fiber-tracking stop criteria., (Copyright 2009 Wiley-Liss, Inc.)

Recent applications of robotics in the field of prostate brachytherapy are seeding the future and could potentially lead to a fully automated prostate brachytherapy surgery. Currently, a typical prostate brachytherapy surgery involves the implantation of upwards of 100 radioactive I-125 seeds by a surgeon. This review supplies background information on prostate biology, brachytherapy of the prostate, robotic brachytherapy, and transrectal ultrasound. Subsequently, it examines the physics involved in ultrasound, radiation from an I-125 source, dosimetry, and robotics. A current semi-automated robotic brachytherapy system is examined in detail and a discussion on future improvements is outlined. Finally, future work to improve prostate brachytherapy is postulated, most notably, phantom optimization using polyvinyl alcohol cryogel. The future of robotic brachytherapy lies in the advent of more sophisticated robotics. This review will give the reader a superior understanding of brachytherapy and its recent robotic advancements. Hopefully, this review will generate new ideas needed to advance prostate brachytherapy procedures leading to more accurate dosimetry, faster procedure time, less ionizing radiation received by surgery staff, more rapid patient recovery, and an overall safer procedure.

An anti-inflammatory 1,2,4-phenylenetriamine-containing series of FMS inhibitors with a potential to form reactive metabolites was transformed into a series with equivalent potency by incorporation of carbon-based replacement groups. Structure-based modeling provided the framework to efficiently effect this transformation and restore potencies to previous levels. This optimization removed a risk factor for potential idiosyncratic drug reactions.

An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.

A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-yl)-2-quinolone-6-carbonitrile 21b, has an IC(50) of 2.5 nM in an in vitro assay and 5.0 nM in a bone marrow-derived macrophage cellular assay. Inhibition of FMS signaling in vivo was also demonstrated in a mouse pharmacodynamic model.

The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET., (Copyright 2007 Wiley-Liss, Inc.)

The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC(50)=0.0008 microM) served as a proof-of-concept candidate in a collagen-induced model of arthritis in mice.

A class of biaryl benzamides was identified and optimized as selective HDAC1&2 inhibitors (SHI-1:2). These agents exhibit selectivity over class II HDACs 4-7, as well as class I HDACs 3 and 8; providing examples of selective HDAC inhibitors for the HDAC isoforms most closely associated with cancer. The hypothesis for the increased selectivity is the binding of a pendant aromatic group in the internal cavity of the HDAC1&2 enzymes. SAR development based on an initial lead led to a series of potent and selective inhibitors with reduced off-target activity and tumor growth inhibition activity in a HCT-116 xenograft model.

Altered patterns of gene expression and the imprinted status of genes have a profound effect on cell physiology and can markedly alter embryonic and fetal development. Failure to maintain correct imprinting patterns can lead to abnormal growth and behavioural problems, or to early pregnancy loss. Recently, it has been reported that the Igf2R and Grb10 genes are biallelically expressed in sheep blastocysts, but monoallelically expressed at Day 21 of development. The present study investigated the imprinting status of 17 genes in in vivo, parthenogenetic and androgenetic bovine blastocysts in order to determine the prevalence of this unique phenomenon. Specifically, the putatively imprinted genes Ata3, Impact, L3Mbtl, Magel2, Mkrn3, Peg3, Snrpn, Ube3a and Zac1 were investigated for the first time in bovine in vitro fertilised embryos. Ata3 was the only gene not detected. The results of the present study revealed that all genes, except Xist, failed to display monoallelic expression patterns in bovine embryos and support recent results reported for ovine embryos. Collectively, the data suggest that monoallelic expression may not be required for most imprinted genes during preimplantation development, especially in ruminants. The research also suggests that monoallelic expression of genes may develop in a gene- and time-dependent manner.

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis., ((c) 2007 Wiley-Liss, Inc.)

The EGF family hormone NRG2beta potently stimulates ErbB4 tyrosine phosphorylation and coupling to IL3 independence. In contrast, the NRG2alpha splicing isoform has lower affinity for ErbB4, does not potently stimulate ErbB4 phosphorylation, and fails to stimulate ErbB4 coupling. Here we investigate these differences. The NRG2beta Q43L mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. This failure to stimulate ErbB4 coupling is not due to differential ligand purity, glycosylation, or stability. The NRG2alpha K45F mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to IL3 independence. Thus, this failure to stimulate ErbB4 coupling is not due to inadequate affinity for ErbB4. In contrast, the NRG2alpha L43Q/K45F mutant stimulates ErbB4 coupling, even though it does not have greater affinity for ErbB4 than does NRG2alpha/K45F. Collectively, these data indicate that Gln43 of NRG2beta is both necessary and sufficient for NRG2 stimulation of ErbB4 coupling to IL3 independence.

The health of aquatic species is dependent on interactions between the environment, pathogens and the host. Under intensive shrimp aquaculture, environmental conditions can degrade, causing significant stress to the cultured organisms. To investigate the effect of environmental stress on shrimp hemocyte gene expression profiles, we applied suppression subtractive hybridization (SSH) in juvenile Penaeus monodon exposed to hyperthermal, hypoxic or hyposmotic conditions. Random sequencing of 258 clones from the SSH revealed 176 distinct sequences of which 58 shared high similarity to sequences in the public databases. The three most common groups of identifiable unique sequences in the SSH libraries were the POL region of non-LTR retrotransposons (31%), genes with immune or potential immune functions (30%), and genes involved in protein synthesis and processing (18%). Stress-regulated differential expression was further verified by quantitative qRT-PCR, with seven out of eight randomly selected genes showing qRT-PCR profiles that conformed to the patterns predicted by SSH. Hence this work provides a list of genes which appear to be up- or down-regulated in response to stress, providing a basis for studying the genetic response of shrimp to environmental stress.

Cultured shrimp are continuously exposed to variable environmental conditions that have been associated with stress and subsequent outbreaks of disease. To investigate the effect of environmental stress on Penaeus monodon gene expression, a 3,853 random cDNA microarray chip was generated with clones originating from six stress-enriched hemocyte libraries generated by suppression subtractive hybridization and a normal hemocyte cDNA library. Changes in temporal gene expression were analyzed from shrimp exposed to hypoxic, hyperthermic, and hypoosmotic conditions; 3.1% of the cDNAs were differentially expressed in response to at least one of the environmental stressors, and 72% of the differentially expressed clones had no significant sequence similarity to previously known genes. Among those genes with high identity to known sequences, the most common functional groups were immune-related genes and non-long terminal repeat retrotransposons. Hierarchical clustering revealed a set of cDNAs with temporal and stress-specific gene expression profiles as well as a set of cDNAs indicating a common stress response between stressors. Hypoxic and hyperthermic stressors induced the most severe short-term response in terms of gene regulation, and the osmotic stress had the least variation in expression profiles relative to the control. These expression data agree with observed differences in shrimp physical appearance and behavior following exposure to stress conditions.