Protocol for Clinical GLP-1 Receptor PET/CT Imaging with [68Ga]Ga-NODAGA-Exendin-4.

Imaging with radiolabeled exendin enables detection and characterization of glucagon-like peptide 1 receptors (GLP-1Rs) in vivo with high specificity. The novel radiotracer [68Ga]Ga-NODAGA-exendin-4 forms a stable complex after a simple and fast labeling procedure. Beta-cell mass in the islets of Langerhans can be visualized using [68Ga]Ga-NODAGA-exendin-4, which is promising for research into diabetes mellitus (DM) pathophysiology. Furthermore, this radiotracer enables very sensitive detection of insulinomas, resulting from vast overexpression of GLP-1Rs, and seems promising for the detection of focal lesions in congenital hyperinsulinism (CHI). Here, we describe the procedures involved in [68Ga]Ga-NODAGA-exendin-4 positron emission tomography (PET)/computed tomography (CT) imaging including the radiolabeling of the NODAGA-exendin conjugate with 68Ga, quality controls, and PET/CT.

Optimization of acoustic coupling for bottom actuated scattering based subsurface scanning probe microscopy.

The characterization of buried nanoscale structures nondestructively is an important challenge in a number of applications, such as defect detection and metrology in the semiconductor industry. A promising technique is Subsurface Scanning Probe Microscopy (SSPM), which combines ultrasound with Atomic Force Microscopy (AFM). Initially, SSPM was used to measure the viscoelastic contrast between a subsurface feature and its surrounding medium. However, by increasing the ultrasonic frequency to >1 GHz, it has been shown that SSPM can also measure acoustic impedance based contrasts. At these frequencies, it becomes difficult to reliably couple the sound into the sample such that the AFM is able to pick up the scattered sound field. The cause is the existence of strong acoustic resonances in the sample, the transducer, and the coupling layer-the liquid layer used to couple the sound energy from the transducer into the sample-in combination with the nonlinearity of the tip-sample interaction. Thus, it is essential to control and measure the thickness of the coupling layer with nanometer accuracy. Here, we present the design of a mechanical clamp to ensure a stable acoustic coupling. Moreover, an acoustic method is presented to measure the coupling layer thickness in real-time. Stable coupling layers with thicknesses of 700 ± 2 nm were achieved over periods of 2-4 h. Measurements of the downmixed AFM signals showed stable signal intensities for >1 h. The clamp and monitoring method introduced here makes scattering based SSPM practical, robust, and reliable and enables measurement periods of hours.

Cloning and sequence analysis of pituitary cDNA encoding the beta-subunit of Xenopus proteasome.

The proteasome is a multicatalytic proteinase composed of a number of non-identical subunits. A Xenopus pituitary cDNA was isolated and found to code for the beta-subunit of proteasome. The amino acid sequence deduced from the open reading frame consisted of 215 amino acid residues with a calculated molecular weight of 23,979. A comparative structural analysis indicated that the proteasome subunits can be divided into two groups with the same evolutionary origin. One group consists of subunits with an N-terminally blocked residue and includes components C2, C3, C8 and C9, while the second group of non-blocked proteins includes component C5 and the beta-subunit.

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.

Immunocytochemistry and in situ hybridization of neuropeptide Y in the hypothalamus of Xenopus laevis in relation to background adaptation.

The amphibian Xenopus laevis is able to adapt to a dark background by releasing melanophore-stimulating hormone from the pars intermedia of the pituitary gland. The inhibition of melanophore-stimulating hormone release is accomplished by neuropeptide Y-containing axons innervating the pars intermedia. To determine the production site of neuropeptide Y involved in this inhibitory control, the distribution of neuropeptide Y in the brain has been investigated by immunocytochemistry and in situ hybridization. Immunoreactive cell bodies were visualized in, among others, the ventromedial and posterior thalamic nuclei, and the suprachiasmatic and ventral infundibular hypothalamic nuclei. A positive hybridization signal with a Xenopus-specific probe for preproneuropeptide Y-RNA was found in the diencephalic ventromedial thalamic nucleus and in the suprachiasmatic nucleus. With both immunocytochemistry and in situ hybridization, suprachiasmatic neurons appeared to be stained only in animals adapted to a white background; animals adapted to a black background showed no staining. Quantitative image analysis revealed that this effect of background adaptation is specific for suprachiasmatic neurons because no effect could be demonstrated of the background light condition on the ventral infundibular nucleus (immunocytochemistry) or the ventromedial thalamic nucleus (in situ hybridization). These results indicate that neurons in the suprachiasmatic nucleus enable the adaptation of X. laevis to a white background, by producing and releasing neuropeptide Y that inhibits the release of melanophore-stimulating hormone from the melanotrope cells in the pars intermedia of the pituitary gland.

Trunk muscle strength measurements and prediction of low-back pain among workers.

A pilot study was conducted to explore the relationship between the occurrence of low-back pain and parameters of trunk muscle strength. Dynamic strength measurements were performed among 53 male workers without low-back pain and 31 male workers with low-back pain. The average torque of lateral right movement was significantly lower for workers with low-back pain compared to those without. The application of discriminant analysis pointed out that another four strength measures contributed to the discriminant function. These measures were average power of lateral right movement, mean torque and maximum velocity during flexion, and isometric strength during right rotation. The results showed that it was possible to find a linear combination of these discriminating variables that successfully allocated 68% to either the group of workers with low-back pain or the group without. Although this discriminatory power is too small to be of practical significance, the discriminant analysis performed reveals some promising features for further research.

Measurement of trunk bending during work by direct observation and continuous measurement.

An observational method and a continuous measurement technique were simultaneously applied to record bending of the trunk during work. In a group of 16 workers performing dynamic tasks a significant correlation (r = 0.57) was found between the two methods. A similar result was observed among 14 sedentary workers (r = 0.62). Although significant correlations between direct observation and continuous recording techniques were present, large differences were found between data obtained from individual subjects. The results cast doubt on the validity of assessments of percentage of worktime with bent trunk at an absolute level using only one measurement method. It is suggested that greater consideration has to be given in future studies to the reliability of measurement of postural load due to trunk bending.

The effect on sitting posture of a desk with a ten-degree inclination using an adjustable chair and table.

By means of continuous recording of the positions of the trunk and head, an investigation was carried out to examine the effect of the Erasmus office-desk - a desk with a 10 degrees inclination - on sitting posture whilst reading and writing, sitting in an adjustable chair at an adjustable table. The posture assumed by a person who is doing mixed reading and writing work at a table with the Erasmus office-desk is more upright than the posture of the same person doing the same work without the desk. This applies for both the head and the trunk. The average position of the trunk was changed from 26.0 degrees to 18.2 degrees when working with the desk placed on the table. The moment of force caused by gravity is proportional to the sine of the angle. Therefore the change of angle results in a decrease of the average moment of force on the back at L5-S1 of 29%. The largest individual decrease found was 86%. The average change of the position of the head was changed from 38.5 degrees to 29.6 degrees , resulting in an average decrease of the moment of force, on C7-T1, of 21%. The largest individual decrease was 51%. Lateroflexion of the back was less often assumed when the subjects were using the desk. The posture assumed by the subjects when they were doing writing work only was generally more upright when working at a desk with a 10 degrees inclination than when working at a flat desk. Marked differences were found between the postures of the subjects. The change of posture while using the desk also showed marked differences for each subject individually.

Rigidity of unilateral external fixators--a biomechanical study.

INTRODUCTION: External fixation is the primary choice of temporary fracture stabilisation for specific polytrauma patients. Adequate initial fracture healing requires sufficient stability at the fracture site. The purpose of this study was to compare the rigidity of the Dynafix DFS(®) Standard Fixator (4 joints) with the Orthofix ProCallus Fixator(®) (2 joints), which differ in possibilities for adapting the configuration for clinical needs. MATERIALS AND METHODS: Both devices were tested 10 times in a standardised model. In steps of 10N, loading was increased to a maximum of 160N in parallel, transversal and axial direction (distraction and compression). Translation resultant and rotation resultant were calculated. RESULTS: With a force of 100N in parallel direction the mean translation resultant (Tr(mean)) of the Dynafix DFS(®) Standard Fixator (6.65±1.43mm) was significantly higher than the ProCallus Fixator(®) (3.29±0.83mm, p<0.001; Student's t-test). With a maximum load of 60N in transverse direction the Tr(mean) of the Dynafix DFS(®) Standard Fixator was significantly lower (8.14±1.20mm versus 9.83±0.63mm, p<0.005). Translation was significantly higher with the Dynafix DFS(®) Standard Fixator, for both distraction (2.13±0.32mm versus 1.69±0.44mm, p<0.05) and compression (1.55±1.08mm versus 0.15±0.33mm, p<0.005). The mean rotation resultant (Rr(mean)) at 160N distraction was lower for the Dynafix DFS(®) Standard Fixator (0.70±0.17° versus 0.97±0.21°, p<0.005). CONCLUSIONS: Both fixators were most sensitive to transverse forces. The Dynafix DFS(®) Standard Fixator was less rigid with parallel and axial forces, whereas transverse forces and rotation at distraction forces favoured the Dynafix DFS(®) Standard Fixator. Repeated heavy loading did not influence the rigidity of both devices.

Comparison of three different pelvic circumferential compression devices: a biomechanical cadaver study.

BACKGROUND: Pelvic circumferential compression devices are designed to stabilize the pelvic ring and reduce the volume of the pelvis following trauma. It is uncertain whether pelvic circumferential compression devices can be safely applied for all types of pelvic fractures because the effects of the devices on the reduction of fracture fragments are unknown. The aim of this study was to compare the effects of circumferential compression devices on the dynamic realignment and final reduction of the pelvic fractures as a measure of the quality of reduction. METHODS: Three circumferential compression devices were evaluated: the Pelvic Binder, the SAM Sling, and the T-POD. In sixteen cadavers, four fracture types were generated according to the Tile classification system. Infrared retroreflective markers were fixed in the different fracture fragments of each pelvis. The circumferential compression device was applied sequentially in a randomized order with gradually increasing forces applied. Fracture fragment movement was studied with use of a three-dimensional infrared video system. Dynamic realignment and final reduction of the fracture fragments during closure of the circumferential compression devices were determined. A factorial repeated-measures analysis of variance with pairwise post hoc comparisons was performed to analyze the differences in pulling force between the circumferential compression devices. RESULTS: In the partially stable and unstable (Tile type-B and C) pelvic fractures, all circumferential compression devices accomplished closure of the pelvic ring and consequently reduced the pelvic volume. No adverse fracture displacement (>5 mm) was observed in these fracture types. The required pulling force to attain complete reduction at the symphysis pubis varied substantially among the three different circumferential compression devices, with a mean (and standard error of the mean) of 43 ± 7 N for the T-POD, 60 ± 9 N for the Pelvic Binder, and 112 ± 10 N for the SAM Sling. CONCLUSIONS: The Pelvic Binder, SAM Sling, and T-POD provided sufficient reduction in partially stable and unstable (Tile type-B1 and C) pelvic fractures. No undesirable overreduction was noted. The pulling force that was needed to attain complete reduction of the fracture parts varied significantly among the three devices, with the T-POD requiring the lowest pulling force for fracture reduction.

Physical load as risk factor for musculoskeletal complaints among tank terminal workers.

In a cross-sectional study, the relationship between physical load and musculoskeletal complaints was examined among operators (n = 77), office workers (n = 52), and miscellaneous workers (n = 15) in a tank terminal company. Information about history of musculoskeletal complaints, individual characteristics, and working conditions in past and present was obtained by a standardized interview. Assessment of physical load was performed by direct observation of awkward postures and forceful exertions during normal activities. Workers filled out a diary regarding the executed tasks during one shift. Physical load was also modeled by linking the average physical load of workers performing a particular task with the task distribution of each worker. The modeling approach was beneficial to estimate infrequent characteristics of physical load, whereas the observations were better for assessing frequent exposure to awkward trunk postures. After adjustment for age and other confounders the observed work time with trunk flexion and rotation was associated with the occurrence of back pain. The modeled work time with arms raised above shoulder showed a relationship with elbow pain, and the modeled work time with forceful exertions over 100 N showed relationships with elbow pain and wrist pain. The described relationships demonstrate that the assessment strategy chosen in a particular study strongly influences random measurement error of physical load, and that inappropriate strategies may mask true associations between physical load and musculoskeletal complaints. Modeling strategies offer the opportunity to assess physical load in dynamic work environments.

Artificial sphincter pressure versus detrusor leakage pressure.

Increasing urethral occlusion pressures were created with an artificial sphincter in fresh bladder-urethra specimens of adult pigs. Detrusor leakage pressures remained about 25 per cent below the associated sphincter cuff pressures. The same relation was found when, during abrupt variations of cuff pressure, the detrusor leakage pressures were measured with antegrade urethral perfusion pressure measurement through a new sleeve catheter. Side-hole perfusion pressure measurements showed 100 per cent transmission of abrupt variations of cuff pressures to the inner urethral wall. No rotational differences could be detected. From the results it has been concluded that a probable pressure loss of about 25 per cent must be taken into account when converting artificial sphincter pressures into bladder pressures at which leakage will occur.

Relative urethral leakage pressure versus maximum urethral closure pressure. The reliability of the measurement of urethral competence with the new tube-foil sleeve catheter in patients.

In 78 female patients the urethral leakage pressure, defined as the intravesical pressure at which leakage starts from the urethral orifice, was estimated with the tube-foil sleeve catheter. In accordance with the definition of maximum urethral closure pressure, the relative urethral leakage pressure was defined as urethral leakage pressure minus intravesical resting pressure. In the same patients urethral closure pressure was also measured by urethral pressure profilometry with a flexible micro pressure-sensor catheter. On the average the ratio of relative urethral leakage pressure/urethral closure pressure was about 0.5. Relative urethral leakage pressure and urethral closure pressure were higher than the maximum detrusor pressure increases during detrusor instabilities in 21 continent patients with motor urge. The relative urethral leakage pressure correlated better with the detrusor pressure elevations at which fluid loss from the urethral orifice started in 24 motor urge incontinent patients than urethral closure pressure did.

Molecular probing of the secretory pathway in peptide hormone-producing cells.

The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of black- and white-adapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC. Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H(+)-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.

The effect on sitting posture of a desk with a 10 degree inclination for reading and writing.

A pilot study was carried out on 10 subjects to examine the effect that a desk with a 10 degree inclination had on their sitting posture while reading and writing. A continuous recording of the subjects head and trunk positions was made. Each subject was studied during two periods of 1.5 h on different days. The subject worked at a flat desk on the first day and at a desk with a 10 degree inclination on the second. On average, the position of the head in the sagittal plane was found to be 6 degrees more erect and the position of the trunk 7 degrees more erect when working at a desk with a 10 degree inclination than when working at a flat desk. The maximal decrease in load observed on the cervical spine was 35% and on the thoracic spine 95%. The angle between the head and the trunk did not change significantly when using an inclined desk. Differences in posture in the frontal plane were not observed in this study.

A comparison of working postures of parcel sorters using three different working methods.

The postures of the trunk of eight parcel sorters were measured continuously under three different working conditions, during whole shifts. The working methods used were two different sorting tables and a hydraulic lifting platform on which the carts were to be placed. The question posed by the PTT-Post occupational health service was whether the latter method proved to be the better one with respect to the load on the musculoskeletal system. This study will help them to make a well-considered decision on which working method to choose for the new distribution centre in Rotterdam. The method used for measuring the working postures and movements of the trunk is the Portable Posture Registration Set (PPRS). This method was developed at the department of Biomedical Physics and Technology of the Erasmus University Rotterdam. While using the lifting platform, the subjects stood 8-11% more erect compared to the work at the sorting tables, thereby reducing the load on the lumbar spine. For this reason the lifting platform is preferred, although sagittal flexion of the back was somewhat more. It is stressed that the way in which sorters use the lifting platform is very important.

Translocon-associated protein TRAP delta and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary.

In the intermediate pituitary gland of Xenopus laevis, the expression levels of the prohormone pro-opiomelanocortin (POMC) can be readily manipulated. When the animal is placed on a black background, the gene for POMC is actively transcribed, whereas on a white background the gene is virtually inactive. In this study, we characterized two genes whose transcript levels in the intermediate pituitary are regulated in coordination with that for POMC. One of these codes for a protein homologous to translocon-associated protein TRAP delta, a subunit of a transmembrane protein complex located at the site where nascent secretory proteins enter the endoplasmic reticulum (ER). Both Xenopus and mice were found to express an alternatively spliced transcript that gives rise to a previously unknown version of the TRAP delta protein. The product of the second gene is a novel and highly conserved protein with structural similarity to glycoprotein gp25L, a constituent of another translocon-associated protein complex. A database search revealed the existence of a novel family of gp25L-related proteins whose members occur throughout the animal kingdom. Together, our data imply that (i) the group of ER proteins surrounding translocating polypeptide chains may be far more complex than previously expected, and (ii) a number of the accessory components of the translocon participate in early steps of prohormone biosynthesis.

Isodynamic evaluation of trunk muscles and low-back pain among workers in a steel factory.

A cross-sectional study was conducted to explore the relationship between dynamic parameters of trunk muscle strength and the occurrence of low-back pain (LBP). Isodynamic strength measurements were performed among 31 male maintenance workers with spells of low-back pain in the past 12 months and 28 maintenance workers without a history of low-back pain. In general, workers with low-back pain showed a lower trunk muscle performance than the workers without low-back pain. Only the measures of torque during lateral movement differed significantly between both groups. Regression analysis revealed the importance of the nature of low-back pain for trunk muscle strength. Most parameters of trunk muscle strength were significantly decreased among workers with chronic low-back pain of a rather severe nature, showing reductions from 18% to 50%. None of the work-related risk factors for low-back pain contributed significantly to the prediction of trunk strength measures. The study suggests that isodynamic evaluation of trunk muscle strength may assist in determining objective measures for the severity of low-back pain rather than in evaluating a subject's capability of successfully coping with a specific work load.

Cloning and sequence analysis of hypothalamic cDNA encoding Xenopus preproneuropeptide Y.

Neuropeptide Y (NPY) consists of 36 amino acids and it constitutes one of the most conserved neuropeptides. Here we report the complete sequence of the first amphibian NPY precursor by cloning of a hypothalamic cDNA encoding Xenopus laevis preproNPY. The overall amino acid sequence identity between Xenopus and other known NPY precursor proteins ranges from 59% (fish) to 82% (chicken); a low degree of identity was found for the signal peptide sequence (32-75%) and for the carboxy-terminal peptide of NPY (CPON; 43-73%), while the NPY peptide sequence itself constitutes the most highly-conserved region (89-100%) within the preproNPY structure.