Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

Accuracy of cardiac ablation catheter guidance by means of a single equivalent moving dipole inverse algorithm to identify sites of origin of cardiac electrical activation.

We have developed a system that could potentially be used to identify the site of origin of ventricular tachycardia (VT) and to guide a catheter to that site to deliver radio-frequency ablation therapy. This system employs the Inverse Solution Guidance Algorithm based upon Single Equivalent Moving Dipole (SEMD) localization method. The system was evaluated in in vivo swine experiments. Arrays consisting of 9 or 16 bipolar epicardial electrodes and an additional mid-myocardial pacing lead were sutured to each ventricle. Focal tachycardia was simulated by applying pacing pulses to each epicardial electrode at multiple pacing rates during breath hold at the end-expiration phase. Surface potentials were recorded from 64 surface electrodes and then analyzed using the SEMD method to localize the position of the pacing electrodes. We found a close correlation between the locations of the pacing electrodes as measured in computational and real spaces. The reproducibility error of the SEMD estimation of electrode location was 0.21 ± 0.07 cm. The vectors between every pair of bipolar electrodes were computed in computational and real spaces. At 120 bpm, the lengths of the vectors in the computational and real space had a 95% correlation. Computational space vectors were used in catheter guidance simulations which showed that this method could reduce the distance between the real space locations of the emulated catheter tip and the emulated arrhythmia origin site by approximately 72% with each movement. We have demonstrated the feasibility of using our system to guide a catheter to the site of the emulated VT origin.

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of KPC-Producing Klebsiella pneumoniae.

We evaluated a real-time single-peak (11.109-Da) detection assay based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae Our results demonstrated that the 11.109-Da peak was detected in 88.2% of the KPC producers. Analysis of blaKPC-producing K. pneumoniae showed that the gene encoding the 11.109-Da protein was commonly (97.8%) associated with the Tn4401a isoform.

PATIENT: Physical Anatomical Trainer Instrumented for Education and Non-Subjective Testing.

The PATIENT manikin (Physical Anatomical Trainer Instrumented for Education and Non-subjective Testing) is designed with the conflicting needs of a highly modular system for ultimate scenario flexibility and cost containment, and a highly realistic system. PATIENT provides a unique combination of capital and disposable components, with each organ treated as a limited-reuse component. The organs contain unobtrusive instrumentation which informs the PATIENT control unit of the organ status. The control unit can adjust whole-body parameters to reflect the local physiology. PATIENT enables tailored simulations, from Point of Injury training of basic life support, through hospital training including surgical interventions.

A method to noninvasively identify cardiac bioelectrical sources.

BACKGROUND: We have introduced a method to guide radiofrequency catheter ablation (RCA) procedures that estimates the location of a catheter tip used to pace the ventricles and the target site for ablation using the single equivalent moving dipole (SEMD). OBJECTIVE: To investigate the accuracy of this method in resolving epicardial and endocardial electrical sources. METHODS: Two electrode arrays, each of nine pacing electrodes at known distances from each other, sutured on the left- and right-ventricular (LV and RV) epicardial surfaces of swine, were used to pace the heart at multiple rates, while body surface potentials from 64 sites were recorded and used to estimate the SEMD location. A similar approach was followed for pacing from catheters in the LV and RV. RESULTS: The overall (RV & LV) error in estimating the interelectrode distance of adjacent epicardial electrodes was 0.38 ± 0.45 cm. The overall endocardial (RV & LV) interelectrode distance error, was 0.44 ± 0.26 cm. Heart rate did not significantly affect the error of the estimated SEMD location (P > 0.05). The guiding process error became progressively smaller as the SEMD approached an epicardial target site and close to the target, the overall absolute error was ∼ 0.28 cm. The estimated epicardial SEMD locations preserved their topology in image space with respect to their corresponding physical location of the epicardial electrodes. CONCLUSION: The proposed algorithm suggests one can efficiently and accurately resolve epicardial electrical sources without the need of an imaging modality. In addition, the error in resolving these sources is sufficient to guide RCA procedures.

Electrogastrographic and autonomic responses during oculovestibular recoupling in flight simulation.

INTRODUCTION: Simulator sickness causes vestibulo-autonomic responses that increase sympathetic activity and decrease parasympathetic activity. The purpose of the study was to quantify these responses through electrogastrography and cardiac interbeat intervals during flight simulation. METHODS: There were 29 subjects that were randomly assigned to 2 parallel arms: (1) oculovestibular recoupling, where galvanic vestibular stimulation was synchronous with the visual field; and (2) control. Electrogastrography and interbeat interval data were collected during baseline, simulation, and post-simulation periods. A simulator sickness questionnaire was administered. RESULTS: Statistically significant differences were observed in percentage of recording time with the dominant frequency of electrogastrography in normogastric and bradygastric domains between the oculovestibular recoupling and control groups. Normogastria was dominant during simulation in the oculovestibular recoupling group. In the control group, the percentage of recording time with the dominant frequency decreased by 22% in normogastria and increased by 20% in bradygastria. The percentage change of the dominant power instability coefficient from baseline to simulation was 26% in the oculovestibular recoupling group vs. 108% in the control group. The power of high-frequency components for interbeat intervals did not change significantly in the oculovestibular recoupling group and was decreased during simulation in the control group. DISCUSSION: Electrogastrography and interbeat intervals are sensitive indices of autonomic changes in subjects undergoing flight simulation. These data demonstrate the potential of oculovestibular recoupling to stabilize gastric activity and cardiac autonomic changes altered during simulator and motion sickness.

The single equivalent moving dipole model does not require spatial anatomical information to determine cardiac sources of activation.

Radio-frequency catheter ablation (RCA) is an established treatment for ventricular tachycardia (VT). A key feature of the RCA procedure is the need for a mapping approach that facilitates the identification of the target ablation site. In this study, we investigate the effect of the location of the reference potential and spatial anatomical constraints on the accuracy of an algorithm to identify the target site for ablation therapy of VT. This algorithm involves processing body surface potentials using the single equivalent moving dipole (SEMD) model embedded in an infinite homogeneous volume conductor to model cardiac electrical activity. We employed a swine animal model and an electrode array of nine electrodes that was sutured on the epicardial surface of the right ventricle. We identified two potential reference electrode locations: at an electrode most far away from the heart (R1) and at the average of all 64 body surface electrode potentials (R2). Also, we developed three spatial "constraining" schemes of the algorithm used to obtain the SEMD location: one that does not impose any constraint on the inverse solution (S1), one that constrains the solution into a volume that corresponds to the heart (S2), and one that constrains the solution into a volume that corresponds to the body surface (S3). We have found that R2S1 is the most accurate approach (p < 0.05 versus R1S1 at earliest activation time-EAT) for localizing epicardial electrical sources of known locations in vivo. Although the homogeneous volume conductor introduces systematic error in the estimated compared to the true dipole location, we have observed that the overall error of the estimated interelectrode distance compared to the true one was 0.4 ± 0.4 cm and 0.4 ± 0.1 cm for the R1S1 and R2S1 combinations, respectively, at the EAT (p = N.S.) and 1.0 ± 0.6 and 0.5 ± 0.4 cm, respectively, at the pacing spike time (PST, ). In conclusion, our algorithm to estimate the SEMD parameters from body surface potentials can potentially be a useful method to rapidly and accurately guide the catheter tip to the target site during a RCA procedure without the need for spatial anatomical information obtained by conventional imaging modalities.

Cardiac ablation catheter guidance by means of a single equivalent moving dipole inverse algorithm.

BACKGROUND: We developed and evaluated a novel system for guiding radiofrequency catheter ablation therapy of ventricular tachycardia. This guidance system employs an inverse solution guidance algorithm (ISGA) using a single equivalent moving dipole (SEMD) localization method. The method and system were evaluated in both a saline tank phantom model and in vivo animal (swine) experiments. METHODS: A catheter with two platinum electrodes spaced 3 mm apart was used as the dipole source in the phantom study. A 40-Hz sinusoidal signal was applied to the electrode pair. In the animal study, four to eight electrodes were sutured onto the right ventricle. These electrodes were connected to a stimulus generator delivering 1-ms duration pacing pulses. Signals were recorded from 64 electrodes, located either on the inner surface of the saline tank or on the body surface of the pig, and then processed by the ISGA to localize the physical or bioelectrical SEMD. RESULTS: In the phantom studies, the guidance algorithm was used to advance a catheter tip to the location of the source dipole. The distance from the final position of the catheter tip to the position of the target dipole was 2.22 ± 0.78 mm in real space and 1.38 ± 0.78 mm in image space (computational space). The ISGA successfully tracked the locations of electrodes sutured on the ventricular myocardium and the movement of an endocardial catheter placed in the animal's right ventricle. CONCLUSION: In conclusion, we successfully demonstrated the feasibility of using an SEMD inverse algorithm to guide a cardiac ablation catheter.

Oculo-vestibular recoupling using galvanic vestibular stimulation to mitigate simulator sickness.

INTRODUCTION: Despite improvement in the computational capabilities of visual displays in flight simulators, intersensory visual-vestibular conflict remains the leading cause of simulator sickness (SS). By using galvanic vestibular stimulation (GVS), the vestibular system can be synchronized with a moving visual field in order to lessen the mismatch of sensory inputs thought to result in SS. METHODS: A multisite electrode array was used to deliver combinations of GVS in 21 normal subjects. Optimal electrode combinations were identified and used to establish GVS dose-response predictions for the perception of roll, pitch, and yaw. Based on these data, an algorithm was then implemented in flight simulator hardware in order to synchronize visual and GVS-induced vestibular sensations (oculo-vestibular-recoupled or OVR simulation). Subjects were then randomly exposed to flight simulation either with or without OVR simulation. A self-report SS checklist was administered to all subjects after each session. An overall SS score was calculated for each category of symptoms for both groups. RESULTS: The analysis of GVS stimulation data yielded six unique combinations of electrode positions inducing motion perceptions in the three rotational axes. This provided the algorithm used for OVR simulation. The overall SS scores for gastrointestinal, central, and peripheral categories were 17%, 22.4%, and 20% for the Control group and 6.3%, 20%, and 8% for the OVR group, respectively. CONCLUSIONS: When virtual head signals produced by GVS are synchronized to the speed and direction of a moving visual field, manifestations of induced SS in a cockpit flight simulator are significantly reduced.

The effect of galvanic vestibular stimulation on distortion product otoacoustic emissions.

Galvanic stimulation has long been used as a nonmechanical means of activating the vestibular apparatus through direct action on the vestibular nerve endings. This stimulation has been reported to be safe, but no studies have examined the potential changes in the corresponding cochlear receptors. The aim of the present study was to evaluate the effect of galvanic vestibular stimulation (GVS) on distortion product otoacoustic emissions (DPOAEs). Fourteen subjects underwent DPOAEs during several conditions of GVS. The DPOAEs ranged from ∼ 1 kHz to ∼ 8 kHz at 65/55 dB for f1/f2 and with an f2/f1 ratio of 1.2. The subjects were evaluated at 10 stimulation conditions that ranged from -2.0 mA to +2.0 mA for each frequency. Statistical analysis showed no significant differences in DPOAE amplitudes for all conditions with and without GVS. Results also showed no significant differences between DPOAE amplitudes before and after GVS. Multivariate analysis found subject variability in DPOAE amplitude, which was not thought to be GVS related. Results indicated that GVS produced neither temporary nor permanent changes in DPOAEs.

Validation of a novel catheter guiding method for the ablative therapy of ventricular tachycardia in a phantom model.

Accurate guidance of an ablation catheter is critical in the RF ablation (RFA) of ventricular tachycardia (VT). With current technologies, it is challenging to rapidly and accurately localize the site of origin of an arrhythmia, often restricting treatment to patients with hemodynamically stable arrhythmias. We investigated the effectiveness of a new guidance method, the inverse solution guidance algorithm (ISGA), which is based on a single-equivalent dipole representation of cardiac electrical activity and is suitable for patients with hemodynamically unstable VT. Imaging was performed in homogeneous and inhomogeneous saline-filled torso phantoms in which a catheter tip was guided toward a stationary electrical dipole source over distances of more than 5 cm. Using ISGA, the moving catheter tip was guided to within 0.61 +/-0.43 and 0.55 +/-0.39 mm of the stationary source in the homogeneous and inhomogeneous phantoms, respectively. This accuracy was achieved with less than ten movements of the catheter. These results suggest that ISGA has potential to provide accurate and efficient guidance for RFA procedures in the patient population with hemodynamically unstable arrhythmias.

Truth cube: establishing physical standards for soft tissue simulation.

Accurate real-time models of soft tissue behavior are key elements in medical simulation systems. The need for fast computation in these simulations, however, often requires simplifications that limit deformation accuracy. Validation of these simplified models remains a challenge. Currently, real-time modeling is at best validated against finite element models that have their own intrinsic limitations. This study develops a physical standard to validate real-time soft tissue deformation models. We took CT images of a cube of silicone rubber with a pattern of embedded Teflon spheres that underwent uniaxial compression and spherical indentation tests. The known material properties, geometry and controlled boundary conditions resulted in a complete set of volumetric displacement data. The results were compared to a finite element model analysis of identical situations. This work has served as a proof of concept for a robust physical standard for use in validating soft tissue models. A web site has been created to provide access to our database: http://biorobotics.harvard.edu/truthcube/ (soon to be http://www.truthcube.org).