The influence of limb alignment and transfemoral amputation technique on muscle capacity during gait

Many factors influence successful outcomes following transfemoral amputation. One factor is surgical technique. In this study, the influence of limb alignment and surgical technique on a muscle’s capacity to generate force was examined using musculoskeletal modeling. Non-amputee and transfemoral amputee models were analyzed while hip adduction, femur length, and reattached muscle wrap position, tension and stabilization technique were systematically varied. With muscle tension preserved, wrap position and femur length had little influence on muscle capacity. However, limb alignment, muscle tension and stabilization technique notably influenced muscle capacity. Overall, myodesis stabilization provided greater muscle balance and function than myoplasty stabilization.     

Mechanistic Basis for Type 2 Long QT Syndrome Caused by KCNH2 Mutations that Disrupt Conserved Arginine Residues in the Voltage Sensor

KCNH2 encodes the Kv11.1 channel, which conducts the rapidly activating delayed rectifier K⁺ current (I _Kr) in the heart. KCNH2 mutations cause type 2 long QT syndrome (LQT2), which increases the risk for life-threatening ventricular arrhythmias. LQT2 mutations are predicted to prolong the cardiac action potential (AP) by reducing I _Kr during repolarization. Kv11.1 contains several conserved basic amino acids in the fourth transmembrane segment (S4) of the voltage sensor that are important for normal channel trafficking and gating. This study sought to determine the mechanism(s) by which LQT2 mutations at conserved arginine residues in S4 (R531Q, R531W or R534L) alter Kv11.1 function. Western blot analyses of HEK293 cells transiently expressing R531Q, R531W or R534L suggested that only R534L inhibited Kv11.1 trafficking. Voltage-clamping experiments showed that R531Q or R531W dramatically altered Kv11.1 current (I _Kv11.1) activation, inactivation, recovery from inactivation and deactivation. Coexpression of wild type (to mimic the patients’ genotypes) mostly corrected the changes in I _Kv11.1 activation and inactivation, but deactivation kinetics were still faster. Computational simulations using a human ventricular AP model showed that accelerating deactivation rates was sufficient to prolong the AP, but these effects were minimal compared to simply reducing I _Kr. These are the first data to demonstrate that coexpressing wild type can correct activation and inactivation dysfunction caused by mutations at a critical voltage-sensing residue in Kv11.1. We conclude that some Kv11.1 mutations might accelerate deactivation to cause LQT2 but that the ventricular AP duration is much more sensitive to mutations that decrease I _Kr. This likely explains why most LQT2 mutations are nonsense or trafficking-deficient.     

Impact in quarantine of the galling weevil Lepidapion argentatum on shoot growth of French broom (Genista monspessulana), an invasive weed in the western U.S.

In weed biocontrol, there is a need for pre-release efficacy assessments for potential agents. Genista monspessulana ((L.) L. A. S. Johnson (Fabaceae), French broom) is an invasive perennial shrub in the western U.S. The galling weevil Lepidapion argentatum Gerstaecker is a potential biocontrol agent. The impact of increasing weevil density on galling damage, plant height, width, leaf damage, and relative growth rate (RGR) was assessed in greenhouse experiments on two to three-month-old seedlings infested with either one or three weevils. Infestation by three female weevils caused 48% more galls producing 27% more larvae than did infestation with one female while causing only 1% leaf damage and no difference in total leaf area. Infestation with multiple weevils caused a 55% and 29% decrease in plant height and canopy width respectively, while single-weevil infestation decreased height by 32% and width to the same degree as for multiple weevils. The RGR of seedlings infested with three weevils was three times slower than the controls, while growth was reduced 2-fold by single-weevil infestation. Reductions in plant size and growth rate induced by weevil galling could reduce plant competitive survival to reproduction and also plant population dispersal as seedlings. Our results suggest that L. argentatum has the potential to cause impact to French broom seedlings if released in the invasive range.     

The Robinson Protocol: a treadmill anaerobic performance test

The current investigation was designed to further examine the reliability of the Robinson protocol, which is a run-to-exhaustion treadmill test. Robinson (10) originally examined this protocol with 5 subjects. The significance of the initial exploratory study was the impetus for expanding the study to examine the reliability of the protocol with a larger sample. Fifteen male subjects participated in 3 trial runs on the treadmill. The first trial was a modified McConnell (7) test to determine the aerobic capacity of each subject. The second and third trials were identical Robinson protocols (10). The first trial run mean, in seconds (262.04 +/- 74.50), was not significantly different from the second trial run mean (257.30 +/- 72.65), p = 0.526 (2 tailed). As expected, trial 1 and trial 2 were highly correlated (intraclass) (r = 0.927, p < 0.001). These results provide additional support for the hypothesis that the Robinson protocol with a greater subject pool is a reliable protocol that can be used in research studies interested in examining various physiological interventions or anaerobic training.     

Secretion of MUC5AC mucin from pancreatic cancer cells in response to forskolin and VIP

Bisphosphonates are potent antiresorptive drugs commonly employed in the treatment of metabolic bone diseases. Despite their frequent use, the mechanisms of bisphosphonates on bone cells have largely remained unclear. Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast formation and activation, whereas osteoprotegerin (OPG) neutralizes RANKL. Various osteotropic drugs have been demonstrated to modulate osteoblastic production of RANKL and OPG. In this study, we assessed the effects of the bisphosphonates pamidronate (PAM) and zoledronic acid (ZOL) on OPG mRNA steady-state levels (by semiquantitative RT-PCR) and protein production (by ELISA) in primary human osteoblasts (hOB). PAM increased OPG mRNA levels and protein secretion by hOB by up to 2- to 3-fold in a dose-dependent fashion with a maximum effect at 10(-6) M (P < 0.001) after 72 h. Similarly, ZOL enhanced OPG gene expression and protein secretion by hOB in a dose-dependent fashion with a maximum effect at 10(-8) M after 72 h, consistent with the higher biological potency of ZOL. Time course experiments indicated a stimulatory effect of PAM and ZOL on osteoblastic OPG protein secretion by 6-fold, respectively (P < 0.001). Pretreatment with PAM and ZOL prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. Analysis of cellular markers of osteoblastic differentiation revealed that PAM and ZOL induced type I collagen secretion and alkaline phosphatase activity by 2- and 4-fold, respectively (P < 0.0001 by ANOVA). In conclusion, our data suggest that bisphosphonates modulate OPG production by normal human osteoblasts, which may contribute to the inhibition of osteoclastic bone resorption. Since, OPG production increases with osteoblastic cell maturation, enhancement of OPG by bisphosphonates could be related to their stimulatory effects on osteoblastic differentiation.     

Fibrillin-2 is dispensable for peripheral nerve development,myelination and regeneration

The extracellular matrix of peripheral nerve is formed from a diverse set of macromolecules, including glycoproteins, collagens and proteoglycans. Recent studies using knockout animal models have demonstrated that individual components of the extracellular matrix play a vital role in peripheral nerve development and regeneration. In this study we identified fibrillin-1 and fibrillin-2, large modular structural glycoproteins, as components of the extracellular matrix of peripheral nerve. Previously it was found that fibrillin-2 null mice display joint contractures, suggesting a possible defect of the peripheral nervous system in these animals. Close examination of the peripheral nerves of fibrillin-2 deficient animals described here revealed some structural abnormalities in the perineurium, while general structure of the nerve and molecular composition of nerve extracellular matrix remained unchanged. We also found that in spite of the obvious motor function impairment, fibrillin-2 null mice failed to display changes of nerve conduction properties or nerve regeneration capacity. Based on the data obtained we can conclude that peripheral neuropathy should be excluded as the cause of the impairment of locomotory function and joint contractures observed in fibrillin-2 deficient animals.     

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets. This is especially the case when a very sensitive assay is required to differentiate often highly potent inhibitors developed by late-stage medicinal chemistry programs. ACK1 is a non-receptor tyrosine kinase that has been shown to be involved in tumorigenesis and metastasis. Here we describe the development of an extremely sensitive high-throughput assay for ACK1 capable of detecting 240 fmol per well of the kinase reaction product employing a BV-tag-based electrochemiluminescence assay. This assay is universally applicable to protein tyrosine kinases using a BV-tag-labeled monoclonal antibody against phosphotyrosine. Furthermore, this assay can be extended to the evaluation of Ser/Thr kinases in those cases where an antibody recognizing the phospho-product is available.     

Classification of the extracellular fields produced by activated neural structures

Background  

Classifying the types of extracellular potentials recorded when neural structures are activated is an important component in understanding nerve pathophysiology. Varying definitions and approaches to understanding the factors that influence the potentials recorded during neural activity have made this issue complex.     

Prevalence of obese patients in a primary care setting

Objective: Previous research has addressed the relationship between BMI and chronic disease in primary care; however, little has been done with regard to the association between obesity and depression in primary care. The purpose of this paper is to assess the relationship between obesity and chronic conditions including depression. Research Methods and Procedures : Data from primary care patients seen at a university‐based family medicine clinic in the southeastern United States were extracted for the time between January 1, 1999 and January 1, 2002. Data extracted included most recent height and weight, age, ethnicity, pregnancy status, number of office visits, blood pressure, cholesterol, hemoglobin A_1C, current diagnoses, and medications. Results : A total of 8197 patients were included in the analysis. Sixty‐nine percent of patients seen in a 3‐year period were either overweight or obese. Comparing blood pressure, cholesterol, diagnoses, and medications between BMI groups found differences in virtually all categories. Diagnoses of high cholesterol, hypertension, diabetes, and depression significantly increased for obese patients. Discussion : Obese patients are over‐represented in primary care, and this over‐representation of obesity correlates with several diagnoses, including depression. Depression is a chronic disease that may interfere with health‐related behaviors and must be addressed within the health care system.     

Synthesis and optimization of substituted furo[2,3-d]-pyrimidin-4-amines and 7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors

Two classes of ACK1 inhibitors, 4,5,6-trisubstituted furo[2,3-d]pyrimidin4-amines and 4,5,6-trisubstituted 7H-pyrrolo[2,3-d]pyrimidin-4-amines, were discovered and evaluated as ACK1 inhibitors. Further structural refinement led to the identification of potent and selective dithiolane inhibitor 37.