Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylaminopropylcarbamylpentyl-Sepharose CL-4B (C3---C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3---C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 1 of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. O.3 U VIII:C/mg protein). The newly developed process using the specially designed C3---C5 affinity matrix has potential application in the process-scale purification of FVIII

Current issues around the pharmacotherapy of ADHD in children and adults

Background New drugs and new formulations enter the growing market for ADHD medication. The growing awareness of possible persistence of ADHD impairment beyond childhood and adolescence resulting in increased pharmacotherapy of ADHD in adults, is also a good reason for making an inventory of the what is generally known about pharmacotherapy in ADHD. Aim To discuss current issues in the possible pharmacotherapy treatment of ADHD in children, adolescents and adults with respect to the position of pharmacotherapy in ADHD treatment guidelines, the pharmacoepidemiological trends, and current concerns about the drugs used. Methods A search of the literature with an emphasis on the position of pharmacotherapy in ADHD treatment guidelines, the pharmacoepidemiological trends, and current concerns about the drugs used in pharmacotherapy. Results According to the guidelines, the treatment of ADHD in children consists of psychosocial interventions in combination with pharmacotherapy when needed. Stimulants are the first-choice drugs in the pharmacological treatment of ADHD in children despite a number of well known and frequently reported side effects like sleep disorders and loss of appetite. With regard to the treatment of adults, stimulant treatment was recommended as the first-choice pharmacotherapy in the single guideline available. Both in children and adults, there appears to be an additional though limited role for the nonadrenergic drug atomoxetine. The increase of ADHD medication use, in children, adolescents and in adults, can not only be interpreted as a sign of overdiagnosis of ADHD. Despite the frequent use of stimulants, there is still a lack of clarity on the effects of long-term use on growth and nutritional status of children. Cardiovascular effects of both stimulants and atomoxetine are rare but can be severe. The literature suggests that atomoxetine may be associated with suicidal ideation in children. Conclusion Although pharmacotherapy is increasing common in the treatment of ADHD in both children and adults, there are still a lot of questions about side effects and how best to counter them. This suggests an important role for close monitoring of children and adults treated with stimulants or atomoxetine

Interaction Between the a3 Region of Factor VIII and the TIL'E’ Domains of the von Willebrand Factor

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL’E’ domains of VWF. Binding between the FVIII a3 region and VWF TIL’E’ was here observed using NMR spectroscopy, where chemical shift changes were localized to two β-sheet regions on the edge of TIL’E’ upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL’E’ as well as mutants of TIL’E’, which further highlights the importance of the β-sheet region of TIL’E’ for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the β-sheet region of TIL’E’ is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex

Repeated successful surgical rescues of early and delayed multiple ruptures of ventricular septum, right ventricle and aneurysmal left ventricle following massive biventricular infarction

A 58 year old man underwent 6 surgical interventions for various complications of massive biventricular myocardial infarction over a period of 2 years following acute occlusion of a possibly "hyperdominant" left anterior descending coronary artery. These included concomitant repair of apicoanterior post-infarction VSD and right ventricular free wall rupture, repeat repair of recurrent VSD following inferoposterior extension of VSD in the infarcted septum 5 weeks later, repair of delayed right ventricular free wall rupture 4 weeks subsequently, repair of a bleeding left ventricular aneurysm eroding through left chest wall 16 months thereafter, repair of right upper lobe lung tear causing massive anterior mediastinal haemorrhage, mimicking yet another cardiac rupture, 2 months later, followed, at the same admission, 2 weeks later, by sternal reconstruction for dehisced and infected sternum using pedicled myocutaneous latissimus dorsi flap. 5 years after the latissimus myoplasty, the patient remains in NYHA class 1 and is leading a normal life

Lateralized Kinematics of Predation Behavior in a Lake Tanganyika Scale-Eating Cichlid Fish

Behavioral lateralization has been documented in many vertebrates. The scale-eating cichlid fish Perissodus microlepis is well known for exhibiting lateral dimorphism in its mouth morphology and lateralized behavior in robbing scales from prey fish. A previous field study indicated that this mouth asymmetry closely correlates with the side on which prey is attacked, but details of this species' predation behavior have not been previously analyzed because of the rapidity of the movements. Here, we studied scale-eating behavior in cichlids in a tank through high-speed video monitoring and quantitative assessment of behavioral laterality and kinematics. The fish observed showed a clear bias toward striking on one side, which closely correlated with their asymmetric mouth morphologies. Furthermore, the maximum angular velocity and amplitude of body flexion were significantly larger during attacks on the preferred side compared to those on the nonpreferred side, permitting increased predation success. In contrast, no such lateral difference in movement elements was observed in acoustically evoked flexion during the escape response, which is similar to flexion during scale eating and suggests that they share a common motor control pathway. Thus the neuronal circuits controlling body flexion during scale eating may be functionally lateralized upstream of this common motor pathway

A randomised controlled trial among cleaners-Effects on strength, balance and kinesiophobia

<p>Abstract</p> <p>Background</p> <p>Cleaners constitute a job group with poor health and low socioeconomic resources. Therefore, there is a great need for scientifically documented health promoting initiatives for cleaners. However, both workplace initiatives and high quality intervention studies are lacking. The aim of this study was to evaluate the effects of a 3-month workplace trial with interventions to improve physical or cognitive behavioural resources among cleaners.</p> <p>Methods</p> <p>A cluster randomised controlled trial was conducted among 294 female cleaners from 9 workplaces. The participants were allocated to three groups: Physical coordination training (PCT, n = 95), Cognitive behavioural theory-based training (CBTr, n = 99) and Reference group (REF, n = 100). Interventions were conducted during work hours for an average of 1 hour/week. Muscle strength was measured by maximal voluntary contractions in trunk/extension, and shoulder abduction/elevation. Postural balance was measured on a force platform. Kinesiophobia was measured with Tampa Scale for Kinesiophobia. Test and questionnaires were completed at baseline and at 3-month follow-up and analyses followed the intention-to-treat (ITT) principle with last observation carried forward in case of missing data at follow-up. Reports and analyses are given on true observations as well.</p> <p>Results</p> <p>ITT-analyses revealed that PCT improved strength of the trunk (p < .05) and postural balance (p < .05) compared to CBTr and REF. Based on true observations the strength and balance improvements corresponded to ~20% and ~16%, respectively. ITT-analyses showed that CBTr reduced kinesiophobia compared to PCT and REF (p < .05). Based on true observations, the improvement corresponded to a ~16% improvement.</p> <p>Conclusion</p> <p>This workplace-based intervention study including PCT and CBTr among cleaners improved strength and postural balance from PCT, and kinesiophobia from CBTr. The improved strength, postural balance and kinesiophobia may improve the cleaners' tolerance for high physical work demands. Future studies should investigate the potential in the combination of PCT and CBTr in a workplace intervention.</p> <p>Trial registration</p> <p>Current controlled trials <a href="http://www.controlled-trials.com/ISRCTN96241850">ISRCTN96241850</a></p

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins