An MEKC assay for the therapeutic drug monitoring of cefepime

The development of a robust assay based on MEKC for cefepime in human serum and plasma with internal quality assurance is reported. Sample preparation comprises protein precipitation in the presence of SDS at pH 4.5. This is a gentle approach for which decomposition of cefepime during sample handling is negligible. After hydrodynamic sample injection of the supernatant, analysis occurs in a phosphate/borate buffer at pH 9.1 with 75 mM SDS using normal polarity and analyte detection at 257 nm. The MEKC run time interval and throughput are about 5 min and seven samples per hour, respectively. The calibration range for cefepime is 1-60 μg/mL, with 1 μg/mL being the LOQ. The performance of the assay with multilevel internal calibration was assessed with calibration and control samples. The assay is shown to be simple, inexpensive, reproducible, and robust. It was applied to determine cefepime levels in the sera of critically ill patients and to assess the instability of cefepime in patient and control samples. Our data revealed that serum containing cefepime can be stored at -20°C for a short time, whereas for long-term storage, samples have to be kept at -70°C.

A nonlinear QCT-based finite element model validation study for the human femur tested in two configurations in vitro

Purpose Femoral fracture is a common medical problem in osteoporotic individuals. Bone mineral density (BMD) is the gold standard measure to evaluate fracture risk in vivo. Quantitative computed tomography (QCT)-based homogenized voxel finite element (hvFE) models have been proved to be more accurate predictors of femoral strength than BMD by adding geometrical and material properties. The aim of this study was to evaluate the ability of hvFE models in predicting femoral stiffness, strength and failure location for a large number of pairs of human femora tested in two different loading scenarios. Methods Thirty-six pairs of femora were scanned with QCT and total proximal BMD and BMC were evaluated. For each pair, one femur was positioned in one-legged stance configuration (STANCE) and the other in a sideways configuration (SIDE). Nonlinear hvFE models were generated from QCT images by reproducing the same loading configurations imposed in the experiments. For experiments and models, the structural properties (stiffness and ultimate load), the failure location and the motion of the femoral head were computed and compared. Results In both configurations, hvFE models predicted both stiffness (R2=0.82 for STANCE and R2=0.74 for SIDE) and femoral ultimate load (R2=0.80 for STANCE and R2=0.85 for SIDE) better than BMD and BMC. Moreover, the models predicted qualitatively well the failure location (66% of cases) and the motion of the femoral head. Conclusions The subject specific QCT-based nonlinear hvFE model cannot only predict femoral apparent mechanical properties better than densitometric measures, but can additionally provide useful qualitative information about failure location.

Monitoring cerebral oxygenation during balloon occlusion with multichannel NIRS

We report on oxygenation changes noninvasively recorded by multichannel continuous-wave near infrared spectroscopy (CW-NIRS) during endovascular neuroradiologic interventions requiring temporary balloon occlusion of arteries supplying the cerebral circulation. Digital subtraction angiography (DSA) provides reference data on the site, timing, and effectiveness of the flow stagnation as well as on the amount and direction of collateral circulation. This setting allows us to relate CW-NIRS findings to brain specific perfusion changes. We focused our analysis on the transition from normal perfusion to vessel occlusion, i.e., before hypoxia becomes clinically apparent. The localization of the maximal response correlated either with the core (occlusion of the middle cerebral artery) or with the watershed areas (occlusion of the internal carotid artery) of the respective vascular territories. In one patient with clinically and angiographically confirmed insufficient collateral flow during carotid artery occlusion, the total hemoglobin concentration became significantly asymmetric, with decreased values in the ipsilateral watershed area and contralaterally increased values. Multichannel CW-NIRS monitoring might serve as an objective and early predictive marker of critical perfusion changes during interventions-to prevent hypoxic damage of the brain. It also might provide valuable human reference data on oxygenation changes as they typically occur during acute stroke.

IMPROVING QUANTITATIVE TREATMENT RESPONSE MONITORING WITH DEFORMABLE IMAGE REGISTRATION

Quantitative imaging with 18F-FDG PET/CT has the potential to provide an in vivo assessment of response to radiotherapy (RT). However, comparing tissue tracer uptake in longitudinal studies is often confounded by variations in patient setup and potential treatment induced gross anatomic changes. These variations make true response monitoring for the same anatomic volume a challenge, not only for tumors, but also for normal organs-at-risk (OAR). The central hypothesis of this study is that more accurate image registration will lead to improved quantitation of tissue response to RT with 18F-FDG PET/CT. Employing an in-house developed “demons” based deformable image registration algorithm, pre-RT tumor and parotid gland volumes can be more accurately mapped to serial functional images. To test the hypothesis, specific aim 1 was designed to analyze whether deformably mapping tumor volumes rather than aligning to bony structures leads to superior tumor response assessment. We found that deformable mapping of the most metabolically avid regions improved response prediction (P<0.05). The positive predictive power for residual disease was 63% compared to 50% for contrast enhanced post-RT CT. Specific aim 2 was designed to use parotid gland standardized uptake value (SUV) as an objective imaging biomarker for salivary toxicity. We found that relative change in parotid gland SUV correlated strongly with salivary toxicity as defined by the RTOG/EORTC late effects analytic scale (Spearman’s ρ = -0.96, P<0.01). Finally, the goal of specific aim 3 was to create a phenomenological dose-SUV response model for the human parotid glands. Utilizing only baseline metabolic function and the planned dose distribution, predicting parotid SUV change or salivary toxicity, based upon specific aim 2, became possible. We found that the predicted and observed parotid SUV relative changes were significantly correlated (Spearman’s ρ = 0.94, P<0.01). The application of deformable image registration to quantitative treatment response monitoring with 18F-FDG PET/CT could have a profound impact on patient management. Accurate and early identification of residual disease may allow for more timely intervention, while the ability to quantify and predict toxicity of normal OAR might permit individualized refinement of radiation treatment plan designs.

THE ISOLATION AND IDENTIFICATION OF O-ACETYLATED SIALIC ACIDS ON HUMAN CELLS (NEURAMINIC ACIDS, PAPER CHROMATOGRAPHY, HUMAN MALIGNANCY, BIOSYNTHETIC LABELING)

Unlike most carbohydrates, sialic acids have a restricted distribution in nature, being present in higher animals and in certain bacteriae. Unfortunately, most studies have not taken into account the fact that the parent sialic acid molecules, N-acetyl(or N-glycolyl)-neuraminic acid can be O-substituted at the 4, 7, 8 and 9 positions, generating many compounds and isomers. The approach and results of this research study demonstrates that proportions of non-, mono-, di-, and tri-O-acetylated sialic acids can be identified and quantitated on normal and malignant human cells. This was accomplished using a paper chromatographic technique to isolate and resolve individual species of non and O-substituted sialic acids. The chemical nature of these O-substituents, as an acetyl ester, was determined on the basis of chemical degradation, enzymatic and fast atom bombardment-mass spectrometry analysis.^ The working hypothesis of this study, that O-acetylated sialic acids are expressed in a restricted manner on normal and malignant cells, was confirmed using the above experimental approach; which identified mono-, di-, and tri-O-acetylated sialic acids on a variety of normal and malignant human cells. These O-acetylated sialic acids were expressed in restricted manner on subpopulations and subcellular fractions of PHL melanoma cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.^ Thus, the ability to isolate and identify biosynthetically radiolabeled O-acetylated sialic acids offers an efficient method of monitoring the expression of O-acetylated sialic acids in biochemical and cellular interactions. Furthermore, the ability to identify abnormal ratios of O-acetylated sialic acids in the human colon, represents a possible diagnostic tool to evaluate and identify patients who may be genetically or culturally predisposed to the development of adenocarcinoma of the colon. ^

Simultaneous registration of ECG and cardiac motion by a single esophageal probe

Long-term surface ECG is routinely used to diagnose paroxysmal arrhythmias. However, this method only provides information about the heart's electrical activity. To this end, we investigated a novel esophageal catheter that features synchronous esophageal ECG and acceleration measurements, the latter being a record of the heart's mechanical activity. The acceleration data were quantified in a small study and successfully linked to the activity sequences of the heart in all subjects. The acceleration signals were additionally transformed into motion. The extracted cardiac motion was proved to be a valid reference input for an adaptive filter capable of removing relevant baseline wandering in the recorded esophageal ECGs. Taking both capabilities into account, the proposed recorder might be a promising tool for future long-term heart monitoring.

Image registration for triggered and non-triggered DTI of the human kidney: Reduced variability of diffusion parameter estimation

BACKGROUND: To investigate if non-rigid image-registration reduces motion artifacts in triggered and non-triggered diffusion tensor imaging (DTI) of native kidneys. A secondary aim was to determine, if improvements through registration allow for omitting respiratory-triggering. METHODS: Twenty volunteers underwent coronal DTI of the kidneys with nine b-values (10-700 s/mm2 ) at 3 Tesla. Image-registration was performed using a multimodal nonrigid registration algorithm. Data processing yielded the apparent diffusion coefficient (ADC), the contribution of perfusion (FP ), and the fractional anisotropy (FA). For comparison of the data stability, the root mean square error (RMSE) of the fitting and the standard deviations within the regions of interest (SDROI ) were evaluated. RESULTS: RMSEs decreased significantly after registration for triggered and also for non-triggered scans (P < 0.05). SDROI for ADC, FA, and FP were significantly lower after registration in both medulla and cortex of triggered scans (P < 0.01). Similarly the SDROI of FA and FP decreased significantly in non-triggered scans after registration (P < 0.05). RMSEs were significantly lower in triggered than in non-triggered scans, both with and without registration (P < 0.05). CONCLUSION: Respiratory motion correction by registration of individual echo-planar images leads to clearly reduced signal variations in renal DTI for both triggered and particularly non-triggered scans. Secondarily, the results suggest that respiratory-triggering still seems advantageous.J. Magn. Reson. Imaging 2014. (c) 2014 Wiley Periodicals, Inc.

Heterologous expression from the human D-Loop in organello

We report the expression of a linear reporter construct in isolated human mitochondria. The reporter construct contained the entire human D-Loop with adjacent tRNA (MTT) genes (mt.15956-647), the human ND1 gene with an in frame GFP gene and adjacent endogenous MTT genes and heterologous rat MTT genes. Natural competence of isolated human mitochondria of HepG2 cells was used to import reporter constructs. The import efficiency of various fluorescently labelled PCR-generated import substrates in the range of 250bp up to 3.5kb was assessed by quantitative PCR and evaluated by confocal microscopy. Heterologous expression of the imported construct was confirmed at RNA level by a circular RNA (cRNA)-RT-PCR assay for the expression of tRNAs and by in organello [α-(32)P]-UTP labelling and subsequent hybridisation to reporter-specific sequences for monitoring mRNA expression. Heterologous expression of rat mitochondrial tRNA(Leu(UUR)) (rMT-TL1) was confirmed by co-/post-transcriptional trinucleotide (CCA) addition. Interestingly, the rat-specific MT-TL1 was correctly processed in isolated human mitochondria at the 3' end, but showed an aberrant 5' end processing. Correct 3' end processing of the heterologous expressed mitochondrial rat tRNA(Ser2) (MT-TS2) was detected. These findings demonstrate the feasibility of genetic manipulation of human mitochondria, providing a tool for characterisation of cis-acting elements of the human mitochondrial genome and for the study of human mitochondrial tRNA processing in organello.

Fusion of fundus images and MRI data of the human eye

Purpose Ophthalmologists are confronted with a set of different image modalities to diagnose eye tumors e.g., fundus photography, CT and MRI. However, these images are often complementary and represent pathologies differently. Some aspects of tumors can only be seen in a particular modality. A fusion of modalities would improve the contextual information for diagnosis. The presented work attempts to register color fundus photography with MRI volumes. This would complement the low resolution 3D information in the MRI with high resolution 2D fundus images. Methods MRI volumes were acquired from 12 infants under the age of 5 with unilateral retinoblastoma. The contrast-enhanced T1-FLAIR sequence was performed with an isotropic resolution of less than 0.5mm. Fundus images were acquired with a RetCam camera. For healthy eyes, two landmarks were used: the optic disk and the fovea. The eyes were detected and extracted from the MRI volume using a 3D adaption of the Fast Radial Symmetry Transform (FRST). The cropped volume was automatically segmented using the Split Bregman algorithm. The optic nerve was enhanced by a Frangi vessel filter. By intersection the nerve with the retina the optic disk was found. The fovea position was estimated by constraining the position with the angle between the optic and the visual axis as well as the distance from the optic disk. The optical axis was detected automatically by fitting a parable on to the lens surface. On the fundus, the optic disk and the fovea were detected by using the method of Budai et al. Finally, the image was projected on to the segmented surface using the lens position as the camera center. In tumor affected eyes, the manually segmented tumors were used instead of the optic disk and macula for the registration. Results In all of the 12 MRI volumes that were tested the 24 eyes were found correctly, including healthy and pathological cases. In healthy eyes the optic nerve head was found in all of the tested eyes with an error of 1.08 +/- 0.37mm. A successful registration can be seen in figure 1. Conclusions The presented method is a step toward automatic fusion of modalities in ophthalmology. The combination enhances the MRI volume with higher resolution from the color fundus on the retina. Tumor treatment planning is improved by avoiding critical structures and disease progression monitoring is made easier.

Modelling and Validation of a Mass Imbalance Oscillation Generator to Harvest Heart Motion Energy

An autonomous energy source within a human body is of key importance in the development of medical implants. This work deals with the modelling and the validation of an energy harvesting device which converts the myocardial contractions into electrical energy. The mechanism consists of a clockwork from a commercially available wrist watch. We developed a physical model which is able to predict the total amount of energy generated when applying an external excitation. For the validation of the model, a custom-made hexapod robot was used to accelerate the harvesting device along a given trajectory. We applied forward kinematics to determine the actual motion experienced by the harvesting device. The motion provides translational as well as rotational motion information for accurate simulations in three-dimensional space. The physical model could be successfully validated.

Detecting single-target changes in multiple object tracking: The case of peripheral vision.

In sports games, it is often necessary to perceive a large number of moving objects (e.g., the ball and players). In this context, the role of peripheral vision for processing motion information in the periphery is often discussed especially when motor responses are required. In an attempt to test the basal functionality of peripheral vision in those sports-games situations, a Multiple Object Tracking (MOT) task that requires to track a certain number of targets amidst distractors, was chosen. Participants’ primary task was to recall four targets (out of 10 rectangular stimuli) after six seconds of quasi-random motion. As a second task, a button had to be pressed if a target change occurred (Exp 1: stop vs. form change to a diamond for 0.5 s; Exp 2: stop vs. slowdown for 0.5 s). While eccentricities of changes (5-10° vs. 15-20°) were manipulated, decision accuracy (recall and button press correct), motor response time as well as saccadic reaction time were calculated as dependent variables. Results show that participants indeed used peripheral vision to detect changes, because either no or very late saccades to the changed target were executed in correct trials. Moreover, a saccade was more often executed when eccentricities were small. Response accuracies were higher and response times were lower in the stop conditions of both experiments while larger eccentricities led to higher response times in all conditions. Summing up, it could be shown that monitoring targets and detecting changes can be processed by peripheral vision only and that a monitoring strategy on the basis of peripheral vision may be the optimal one as saccades may be afflicted with certain costs. Further research is planned to address the question whether this functionality is also evident in sports tasks.

Development and characterization of a scaffold-free 3D spheroid model of iPSC-derived human cardiomyocytes

Purpose: Cardiomyocytes are terminally differentiated cells in the adult heart and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSC) from human origin was developed and characterized. Methods: Human cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. 2D cultures were examined using immunofluorescence microscopy and Western blotting while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. Results: iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to pro-hypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived cardiomyocytes formed spheroidal MTs within 4 days showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature, and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium-transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca2+-release, extracellular calcium levels. Conclusions: 3D culture using iPSC-derived human cardiomyocytes provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Virtopsy: postmortem imaging of the human heart in situ using MSCT and MRI.

The rapid further development of computed tomography (CT) and magnetic resonance imaging (MRI) induced the idea to use these techniques for postmortem documentation of forensic findings. Until now, only a few institutes of forensic medicine have acquired experience in postmortem cross-sectional imaging. Protocols, image interpretation and visualization have to be adapted to the postmortem conditions. Especially, postmortem alterations, such as putrefaction and livores, different temperature of the corpse and the loss of the circulation are a challenge for the imaging process and interpretation. Advantages of postmortem imaging are the higher exposure and resolution available in CT when there is no concern for biologic effects of ionizing radiation, and the lack of cardiac motion artifacts during scanning. CT and MRI may become useful tools for postmortem documentation in forensic medicine. In Bern, 80 human corpses underwent postmortem imaging by CT and MRI prior to traditional autopsy until the month of August 2003. Here, we describe the imaging appearance of postmortem alterations--internal livores, putrefaction, postmortem clotting--and distinguish them from the forensic findings of the heart, such as calcification, endocarditis, myocardial infarction, myocardial scarring, injury and other morphological alterations.

Photometric monitoring of non-resolved space debris and databases of optical light curves.

The population of space debris increased drastically during the last years. Collisions involving massive objects may produce large number of fragments leading to significantly growth of the space debris population. An effective remediation measure in order to stabilize the population in LEO, is therefore the removal of large, massive space debris. To remove these objects, not only precise orbits, but also more detailed information about their attitude states will be required. One important property of an object targeted for removal is its spin period and spin axis orientation. If we observe a rotating object, the observer sees different surface areas of the object which leads to changes in the measured intensity. Rotating objects will produce periodic brightness vari ations with frequencies which are related to the spin periods. Photometric monitoring is the real tool for remote diagnostics of the satellite rotation around its center of mass. This information is also useful, for example, in case of contingency. Moreover, it is also important to take into account the orientation of non-spherical body (e.g. space debris) in the numerical integration of its motion when a close approach with the another spacecr aft is predicted. We introduce the two databases of light curves: the AIUB data base, which contains about a thousand light curves of LEO, MEO and high-altitude debris objects (including a few functional objects) obtained over more than seven years, and the data base of the Astronomical Observatory of Odessa University (Ukraine), which contains the results of more than 10 years of photometric monitoring of functioning satellites and large space debris objects in low Earth orbit. AIUB used its 1m ZIMLAT telescope for all light curves. For tracking low-orbit satellites, the Astronomical Observatory of Odessa used the KT-50 telescope, which has an alt-azimuth mount and allows tracking objects moving at a high angular velocity. The diameter of the KT-50 main mirror is 0.5 m, and the focal length is 3 m. The Odessa's Atlas of light curves includes almost 5,5 thousand light curves for ~500 correlated objects from a time period of 2005-2014. The processing of light curves and the determination of the rotation period in the inertial frame is challenging. Extracted frequencies and reconstructed phases for some interesting targets, e.g. GLONASS satellites, for which also SLR data were available for confirmation, will be presented. The rotation of the Envisat satellite after its sudden failure will be analyzed. The deceleration of its rotation rate within 3 years is studied together with the attempt to determine the orientation of the rotation axis.