Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors.

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)µM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality.

Vascular challenges in renal transplantation.

BACKGROUND: The increasing demand for transplantable organs, especially kidneys, has led to expanded criteria for renal transplant donors. As a result of the expanded criteria, more organs with vascular anomalies and/or pathologies are available for transplant. This retrospective study evaluated the impact of vascular repair on the outcome in kidney transplantation in a single center over a 15-year period. MATERIAL AND METHODS: Between January 1997 and May 2012, 1134 deceased donor renal transplantations were performed in the Department for Vascular and Endovascular Surgery of the University of Düsseldorf, Germany. RESULTS: A vascular reconstruction of some type was necessary to repair renal vessels or to prepare the recipient site for transplantation in 374 of 1134 (33.0%) renal transplantations. The iliac artery in 12.3% (139/1134) of cases and the renal artery in 10.1% (115/1134) of cases showed severe atherosclerosis and a thrombo-endarterectomy was required. Organ loss occurred in 13 cases (1.1%) due to vascular failure. The 5-year graft survival for kidneys with reconstructed renal arteries was 84.3% in deceased donor renal transplantations (86.1% without arterial reconstruction). CONCLUSIONS: The demand for renal transplants has led to more marginal-quality organs and older donors and/or recipients waiting for a second or third transplantation. Thus, the expertise of a vascular surgeon is extremely helpful in a transplantation center because it allows for marginal organ transplantation with acceptable 5-year graft survival rates.

Long-term graft outcome after renal arterial reconstruction during living related kidney transplantation.

BACKGROUND/OBJECTIVES: The aim of this retrospective study was to evaluate the repair of vascular variations/pathologies in living donor kidney transplantations in a single centre over a 15-year period. METHODS: Between 01/1997 and 05/2012, 338 living donor renal transplantations were performed in the Department for Endovascular and Vascular Surgery, University of Düsseldorf, Germany. Twenty-four of them showed disorders, like multiple renal arteries (MRA), atherosclerotic stenosis or fibromuscular dysplasia (FMD) needing vascular repair before transplantation. RESULTS: Mean age of donors was 51 ± 11.2, in recipient's 44 ± 13.9 years. In seven transplantations, renal artery (RA) repair was performed because of MRA. Atherosclerotic stenosis of the RA was apparent in 12 cases needing a repair with disobliteration. FMD was the reason in five transplantations for vascular repair. Complications like renal vessel thrombosis, lymphocele, heamorrhage, distal urinary leakage and ureteral obstruction was not significantly associated with RA reconstruction. Comparison of renal function in kidneys with reconstructed RA compared with kidneys without vascular repair showed no significant difference in primary function and serum creatinine up to the first year after transplantation. Mean follow-up was 75.6 ± 48.1 months. The 5-year graft survival rate for kidneys with RA repair was 88.5 vs. 93.4 % without reconstruction. CONCLUSIONS: We could show that RA pathologies, suitable repaired, are not a contraindication for transplantation with acceptable 5-year-graft-survival rates.

Significance of finger forces and kinematics during handwriting in writer's cramp.

Muscular hyperactivity during handwriting, irregular and jerky scripts, as well as awkward and slowed pen movements are the cardinal symptoms of writer's cramp. Accordingly, impaired kinematics and increased force have been reported in writer's cramp. However, the relationship between these symptoms has rarely been investigated. In addition, measurements of finger forces have been restricted to the vertical pen pressure. In the present study, the pen of a graphic tablet was equipped with a force sensor matrix to measure also the grip force produced against the pen barrel despite highly variable pen grips of the patients. Kinematics of writing movements, vertical pen pressure, and grip force were compared in 27 patients with writer's cramp and normal control writers during writing of a test sentence. As expected, all measures revealed a significantly worse writing performance in the patients compared to the control subjects. Exaggerated forces were more frequent than abnormal kinematics, and evidenced by prolonged movement times and reduced writing frequencies. Correlations were found neither between kinematics and force measures nor between the two forces. Interestingly, patients relaxed the grip force during short periods of non-writing by the same relative amount as control subjects. The finding of a large heterogeneity of performances patterns in writer's cramp may reflect the variability of dystonic symptoms as well as the highly variable compensatory strategies of individual patients. Measurements of finger force and in particular of the grip force are valuable and important descriptors of individual impairment characteristics that are independent of writing kinematics.

Effects of modified pen grip and handwriting training on writer's cramp.

OBJECTIVE: To evaluate the use of a modified pen grip and subsequent handwriting training in patients with writer's cramp (WC). DESIGN: Handwriting performance with normal and modified pen grip was examined once in healthy controls and repeatedly in patients with WC (2 baseline tests before training, directly after training, after a 3-month follow-up). SETTING: Ambulatory care for motor writing disorders. PARTICIPANTS: Patients with WC (n=26) and healthy controls (n=14). INTERVENTION: Seven sessions of handwriting training with various motor exercises were conducted by an occupational therapist. During training, the patients always used a modified pen grip (stabilized between index and middle finger). MAIN OUTCOME MEASURES: Writing frequency and fluency, grip force on the pen, writing pressure, Fahn dystonia scale, visual analog scales for impairment and pain. RESULTS: Patients with WC showed increased writing pressure and grip force before training. Using the modified pen grip caused in both patients with WC and controls a decrease in pressure and grip force. Handwriting training resulted in a further improvement of both parameters in patients with WC. Grip force reduction remained stable over follow-up. CONCLUSIONS: Results suggest that patients with WC benefit from the use of the modified pen grip in combination with handwriting training.

Circadian variations in the kinematics of handwriting and grip strength.

The present study determined whether the motor process of handwriting is influenced by a circadian rhythm during writing tasks of high everyday relevance and analyzed the relationship to the circadian rhythm of grip strength. Ten healthy young male subjects underwent a 40 h sleep-deprivation protocol under constant routine conditions. Starting at 09:00 h, subjects performed three handwriting tasks of increasing perceptual-motor complexity (writing a sentence, writing one's signature, and copying a text for 3 min) and assessed grip strength of both hands every 3 h. Handwriting performance was analyzed by writing speed, writing fluency, script size, break times, and pen pressure. The handwriting tasks revealed a coincident circadian rhythm for the frequency of handwriting as a measure of movement speed, with slowest writing speed at 03:16 h. A weak effect of task complexity was evident for the non-writing episodes: while copying a text, break times were influenced by a circadian rhythm, whereas during sentence writing, the non-writing episodes remained constant. The circadian rhythm of grip strength paralleled the time course of motivation ratings, with least motivation and weakest grip strength around 06:00 h concurrently for both hands. The rate of force production also displayed circadian rhythmicity and sharply decreased with the onset of melatonin secretion. Neither grip strength nor the kinematics of handwriting was influenced by sleep deprivation; only the level of the force rate was decreased the second day. The results show a clear circadian rhythm in the speed of handwriting and grip strength.

Auditory grip force feedback in the treatment of Writer's cramp.

STUDY DESIGN: Pre-post, single-group. INTRODUCTION: Writer's cramp (WC) is a focal dystonia causing impairments in daily life. Behavioral treatment approaches have been shown to improve handwriting performance, though outcomes remain sub-optimal. PURPOSE OF THE STUDY: To examine the effects of the handwriting training and auditory grip force feedback in seven patients with WC. METHODS: Handwriting performance was examined before and after treatment. Writing frequency, fluency, and pressure were recorded with a digitizing tablet and grip forces during handwriting were recorded. Subjective writing performance and pain were rated on visual analog scales. RESULTS: The treatment resulted in significant reductions in writing pressure and pain, while writing performance was significantly improved. CONCLUSIONS: Patients in this study with WC, who exhibit grip force and pressure problems, benefit from feedback-supported handwriting training.

Enzyme-modified field effect transistors based on surface-conductive single-crystalline diamond.

Enzyme-modified field effect transistors (ENFETs) were realized using surface-conductive single-crystalline diamond films. The enzymes penicillinase and acetylcholinesterase were immobilized onto the active area of diamond-based electrolytic solution gated FETs, using different organic linker molecules and cross-linking chemistries. The active area of the devices was patterned to generate enzyme-modified regions next to surface-conductive regions. Penicillinase was chosen as a robust model system, but the main focus of the present paper is on acetylcholinesterase, an enzyme essential for many neuronal signal transduction processes. All the different ENFETs show a clear and specific response to the corresponding substrate, penicillin and acetylcholine. The device response is based on the pH sensitivity of the surface-conductive active area and is enabled by the local pH change induced during the enzymatic reaction. The devices demonstrate promising stability and characteristic variations of the enzymatic activity with measurement conditions. Furthermore, the results from the ENFET measurements were compared with the results of spectrophotometric experiments, carried out with enzymes immobilized on diamond substrates and also with free enzymes in solution. This allows an analysis of the enzyme kinetics, as well as qualitative comparison of the different functionalization methods employed in this study.

Modified pen grip in the treatment of Writer's Cramp.

Writer's Cramp (WC) is a focal, action-related dystonia, which induces hypertonic co-contractions and severely impairs handwriting. One behavioral treatment approach is the handwriting training developed by Mai and Marquardt (1999), [Mai, N., & Marquardt, C. (1999). Schreibtraining in der neurologischen Rehabilitation. In EKN-Materialien für die Rehabilitation. Dortmund: Borgmann] which includes among various motor exercises the use of a modified pen grip (stabilized between index and middle finger). This pen grip has proven particularly successful in clinical practice. The current study aims at elucidating the immediate effects of the modified pen grip on writing in 23 WC patients and 11 healthy controls. All participants wrote a sentence with their usual and also with the modified pen grip. Movement and pressure were recorded with a digitizing tablet. Pressure, movement time for the whole sentence, script size and writing fluency were analyzed. When writing with their usual pen grip, pressure in the WC patients was elevated, and writing speed was decreased compared to healthy controls. Changing over to the modified pen grip reduced the pressure significantly in WC patients and controls, but left other aspects of their writing unaffected. This shows that the use of the modified pen grip is an effective way to normalize pen pressure in WC patients, thereby providing the best conditions for the training of speed and fluency.

Effects of deep brain stimulation on prehensile movements in PD patients are less pronounced when external timing cues are provided.

It has been repeatedly demonstrated that the movements of patients with Parkinson's disease (PD) are less impaired when external timing cues are provided. This suggests that the basal ganglia, which are impaired in PD, are less involved in the control of externally timed movements. In the present study, we tested this hypothesis by contrasting the effect of deep brain stimulation (DBS) in the basal ganglia (more precisely, the internal globus pallidum) on internally versus externally timed movements. Our first movement task was a standard prehensile task involving a reach-to-grasp movement. In the externally-timed condition, the target object was moving rapidly away from the subject; in the internally-timed condition, the target object was stationary. We found, that for most aspects of the prehensile movement the effect of DBS was less pronounced in the externally than in the internally timed condition. A similar reduction of the DBS effects in the externally-timed condition was also found for a second movement task, which required an isolated grasping movement. We conclude that the basal ganglia are significantly less involved in the control of externally timed movements.

Biocytin and biotin uptake into NB2a neuroblastoma and C6 astrocytoma cells.

Uptake of biocytin and biotin was investigated in cultured transformed variants of neuronal (NB2a neuroblastoma) and glial (C6 astrocytoma) CNS cells. NB2a cells took up both compounds but biocytin was transported more efficiently than biotin in the nanomolar concentration range. In NB2a cells a single transport mechanism was found for biocytin with different kinetic parameters in the presence of high extracellular Na+ (Km 0.4 microM, Vmax 20 pmol/min/mg), K+ (Km 1.7 microM, Vmax 32 pmol/min/mg), or choline+ (Km 0.1 microM, Vmax 5 pmol/min/mg). Two transport systems (Km1 17 microM, Vmax1 53 pmol/min/mg; Km2 314 microM, Vmax2 360 pmol/min/mg) were identified for biotin with only system 1 being Na+-dependent. Biocytin uptake was competitively inhibited by excess biotin but not vice versa. Inhibition studies with structural analogs indicated different specificities for biotin and biocytin uptake. Biocytin uptake into C6 cells was hardly detectable whereas biotin was taken up by diffusion (kD 0.6 microl/min/mg) and a single saturable mechanism (Km 70 microM, Vmax 119 pmol/min/mg) at high extracellular Na+. High extracellular K+ enhanced biotin diffusion into C6 cells. Inhibition studies with structural analogs revealed a less specific biotin uptake mechanism in C6 than in NB2a cells. Biocytin normalized deficient biotin-dependent propionyl-CoA carboxylase activity within 4 h in biotin-deficient NB2a cells whereas in C6 cells reactivation was <20% thereby confirming that biocytin is only poorly transported into C6 cells. Specific biocytin uptake into NB2a cells is to our knowledge the first demonstration of a carrier-mediated transport mechanism for this compound. Neuronal biocytin uptake might contribute to the pathogenesis of biotinidase deficiency where biocytin is present in elevated levels.