A metamodel-based flexible insulin therapy for type 1 diabetes patients subjected to aerobic physical activity.

Patients with type 1 diabetes are subject to exogenous insulin injections, whether manually or through (semi)automated insulin pumps. Basic knowledge of the patient's characteristics and flexible insulin therapy (FIT) parameters are then needed. Specifically, artificial pancreas-like closed-loop insulin delivery systems are some of the most promising devices for substituting for endogenous insulin secretion in type 1 diabetes patients. However, these devices require self-reported information such as carbohydrates or physical activity from the patient, introducing potential miscalculations and delays that can have life-threatening consequences. Here, we display a metamodel for glucose-insulin dynamics that is subject to carbohydrate ingestion and aerobic physical activity. This metamodel incorporates major existing knowledge-based models. We derive comprehensive and universal definitions of the underlying FIT parameters to form an insulin sensitivity factor (ISF). In addition, the relevance of physical activity modelling is assessed, and the FIT is updated to take physical exercise into account. Specifically, we cope with physical activity by using heart rate sensors (watches) with a fully automated closed insulin loop, aiming to maximize the time spent in the glycaemic range (75.5% in the range and 1.3% below the range for hypoglycaemia on a virtual patient simulator).These mathematical parameter definitions are interesting on their own, may be new tools for assessing mathematical models and can ultimately be used in closed-loop artificial pancreas algorithms or to extend distinguished FIT.

Highly Accurate Real-Time Decomposition of Single Channel Intramuscular EMG.

OBJECTIVE: Real-time intramuscular electromyography (iEMG) decomposition, as an identification procedure of individual motor neuron (MN) discharge timings from a streaming iEMG recording, has the potential to be used in human-machine interfacing. However, for these applications, the decomposition accuracy and speed of current approaches need to be improved. METHODS: In our previous work, a real-time decomposition algorithm based on a Hidden Markov Model of EMG, using GPU-implemented Bayesian filter to estimate the spike trains of motor units (MU) and their action potentials (MUAPs), was proposed. In this paper, a substantially extended version of this algorithm that boosts the accuracy while maintaining real-time implementation, is introduced. Specifically, multiple heuristics that aim at resolving the problems leading to performance degradation, are applied to the original model. In addition, the recursive maximum likelihood (RML) estimator previously used to estimate the statistical parameters of the spike trains, is replaced by a linear regression (LR) estimator, which is computationally more efficient, in order to ensure real-time decomposition with the new heuristics. RESULTS: The algorithm was validated using twenty-one experimental iEMG signals acquired from the tibialis anterior muscle of five subjects by fine wire electrodes. All signals were decomposed in real time. The decomposition accuracy depended on the level of muscle activation and was when less than 10 MUs were identified, substantially exceeding previous real-time results. CONCLUSION: Single channel iEMG signals can be very accurately decomposed in real time with the proposed algorithm. SIGNIFICANCE: The proposed highly accurate algorithm for single-channel iEMG decomposition has the potential of providing neural information on motor tasks for human interfacing.

Recursive Decomposition of Electromyographic Signals With a Varying Number of Active Sources: Bayesian Modeling and Filtering.

OBJECTIVE: This paper describes a sequential decomposition algorithm for single-channel intramuscular electromyography (iEMG) generated by a varying number of active motor neurons. METHODS: As in previous work, we establish a hidden Markov model of iEMG, in which each motor neuron spike train is modeled as a renewal process with inter-spike intervals following a discrete Weibull law and motor unit action potentials are modeled as impulse responses of linear time-invariant systems with known prior. We then expand this model by introducing an activation vector associated with the state vector of the hidden Markov model. This activation vector represents recruitment/derecruitment of motor units and is estimated together with the state vector using Bayesian filtering. Non-stationarity of the model parameters is addressed by means of a sliding window approach, thus making the algorithm adaptive to variations in contraction force and motor unit action potential waveforms. RESULTS: The algorithm was validated using simulated and experimental iEMG signals with varying number of active motor units. The experimental signals were acquired from the tibialis anterior and abductor digiti minimi muscles by fine wire and needle electrodes. The decomposition accuracy in both simulated and experimental signals exceeded 90%. CONCLUSION: The recruitment/derecruitment was successfully tracked by the algorithm. Because of its parallel structure, this algorithm can be efficiently accelerated, which lays the basis for its real-time applications in human-machine interfaces. SIGNIFICANCE: The proposed method substantially broadens the domains of applicability of the algorithm.

On-Line Recursive Decomposition of Intramuscular EMG Signals Using GPU-Implemented Bayesian Filtering.

OBJECTIVE: Real-time intramuscular electromyography (iEMG) decomposition, which is needed in biofeedback studies and interfacing applications, is a complex procedure that involves identifying the motor neuron spike trains from a streaming iEMG recording. METHODS: We have previously proposed a sequential decomposition algorithm based on a Hidden Markov Model of EMG, which used Bayesian filter to estimate unknown parameters of motor unit (MU) spike trains, as well as their action potentials (MUAPs). Here, we present a modification of this original model in order to achieve a real-time performance of the algorithm as well as a parallel computation implementation of the algorithm on Graphics Processing Unit (GPU). Specifically, the Kalman filter previously used to estimate the MUAPs, is replaced by a least-mean-square filter. Additionally, we introduce a number of heuristics that help to omit the most improbable decomposition scenarios while searching for the best solution. Then, a GPU-implementation of the proposed algorithm is presented. RESULTS: Simulated iEMG signals containing up to 10 active MUs, as well as five experimental fine-wire iEMG signals acquired from the tibialis anterior muscle, were decomposed in real time. The accuracy of decompositions depended on the level of muscle activation, but in all cases exceeded 85 %. CONCLUSION: The proposed method and implementation provide an accurate, real-time interface with spinal motor neurons. SIGNIFICANCE: The presented real time implementation of the decomposition algorithm substantially broadens the domain of its application.

Simulation of Motor Unit Action Potential Recordings From Intramuscular Multichannel Scanning Electrodes.

Multi-channel intramuscular EMG (iEMG) provides information on motor neuron behavior, muscle fiber (MF) innervation geometry and, recently, has been proposed as a means to establish a human-machine interface. OBJECTIVE: to provide a reliable benchmark for computational methods applied to such recordings, we propose a simulation model for iEMG signals acquired by intramuscular multi-channel electrodes. METHODS: we propose several modifications to the existing motor unit action potentials (MUAPs) simulation methods, such as farthest point sampling (FPS) for the distribution of motor unit territory centers in the muscle cross-section, accurate fiber-neuron assignment algorithm, modeling of motor neuron action potential propagation delay, and a model of multi-channel scanning electrode. RESULTS: we provide representative applications of this model to the estimation of motor unit territories and the iEMG decomposition evaluation. Also, we extend it to a full multi-channel iEMG simulator using classic linear EMG modeling. CONCLUSIONS: altogether, the proposed models provide accurate MUAPs across the entire motor unit territories and for various electrode configurations. SIGNIFICANCE: they can be used for the development and evaluation of mathematical methods for multi-channel iEMG processing and analysis.

Individuals have unique muscle activation signatures as revealed during gait and pedaling.

Although it is known that the muscle activation patterns used to produce even simple movements can vary between individuals, these differences have not been considered to prove the existence of individual muscle activation strategies (or signatures). We used a machine learning approach (support vector machine) to test the hypothesis that each individual has unique muscle activation signatures. Eighty participants performed a series of pedaling and gait tasks, and 53 of these participants performed a second experimental session on a subsequent day. Myoelectrical activity was measured from eight muscles: vastus lateralis and medialis, rectus femoris, gastrocnemius lateralis and medialis, soleus, tibialis anterior, and biceps femoris-long head. The classification task involved separating data into training and testing sets. For the within-day classification, each pedaling/gait cycle was tested using the classifier, which had been trained on the remaining cycles. For the between-day classification, each cycle from day 2 was tested using the classifier, which had been trained on the cycles from day 1. When considering all eight muscles, the activation profiles were assigned to the corresponding individuals with a classification rate of up to 99.28% (2,353/2,370 cycles) and 91.22% (1,341/1,470 cycles) for the within-day and between-day classification, respectively. When considering the within-day classification, a combination of two muscles was sufficient to obtain a classification rate >80% for both pedaling and gait. When considering between-day classification, a combination of four to five muscles was sufficient to obtain a classification rate >80% for pedaling and gait. These results demonstrate that strategies not only vary between individuals, as is often assumed, but are unique to each individual.NEW & NOTEWORTHY We used a machine learning approach to test the uniqueness and robustness of muscle activation patterns. We considered that, if an algorithm can accurately identify participants, one can conclude that these participants exhibit discernible differences and thus have unique muscle activation signatures. Our results show that activation patterns not only vary between individuals, but are unique to each individual. Individual differences should, therefore, be considered relevant information for addressing fundamental questions about the control of movement.

Walking Gait Step Length Asymmetry Induced by Handheld Device.

The modeling and feature extraction of human gait motion are crucial in biomechanics studies, human localization, and robotics applications. Recent studies in pedestrian navigation aim at extracting gait features based on the data of low-cost sensors embedded in handheld devices, such as smartphones. The general assumption in pedestrian dead reckoning (PDR) strategy for navigation application is that the presence of a device in hand does not impact the gait symmetry and that all steps are identical. This hypothesis, which is used to estimate the traveled distance, is investigated in this paper with an experimental study. Ten healthy volunteers participated in motion lab tests with a 0.190 kg device in hand. Several walking trials with different device carrying modes and several gait speeds were performed. For a fixed walking speed, it is shown that the steps differ in their duration when holding a mass equivalent to a smartphone mass, which invalidates classical symmetry hypothesis in the PDR step length modeling. It is also shown that this hypothesis can lead to a 2.5% to 6.3% error on the PDR estimated traveled distance for the different walking trials.

ß-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1.

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic ß cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via ß-arrestins. Further, G protein- and ß-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses ("biased agonism"). Whether GPR40 engages ß-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-ß-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting ß-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of ß-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a ß-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.

A Long-Term Model of the Glucose-Insulin Dynamics of Type 1 Diabetes.

A new glucose-insulin model is introduced which fits with the clinical data from in- and outpatients for two days. Its stability property is consistent with the glycemia behavior for type 1 diabetes. This is in contrast to traditional glucose-insulin models. Prior models fit with clinical data for a few hours only or display some nonnatural equilibria. The parameters of this new model are identifiable from standard clinical data as continuous glucose monitoring, insulin injection, and carbohydrate estimate. Moreover, it is shown that the parameters from the model allow the computation of the standard tools used in functional insulin therapy as the basal rate of insulin and the insulin sensitivity factor. This is a major outcome as they are required in therapeutic education of type 1 diabetic patients.

Biased signaling favoring gi over ß-arrestin promoted by an apelin fragment lacking the C-terminal phenylalanine.

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but ß-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes ß-arrestin recruitment to the receptor, K16P had a much reduced ability to promote ß-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both ß-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the ß-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease.

Sequential decoding of intramuscular EMG signals via estimation of a Markov model.

This paper addresses the sequential decoding of intramuscular single-channel electromyographic (EMG) signals to extract the activity of individual motor neurons. A hidden Markov model is derived from the physiological generation of the EMG signal. The EMG signal is described as a sum of several action potentials (wavelet) trains, embedded in noise. For each train, the time interval between wavelets is modeled by a process that parameters are linked to the muscular activity. The parameters of this process are estimated sequentially by a Bayes filter, along with the firing instants. The method was tested on some simulated signals and an experimental one, from which the rates of detection and classification of action potentials were above 95% with respect to the reference decomposition. The method works sequentially in time, and is the first to address the problem of intramuscular EMG decomposition online. It has potential applications for man-machine interfacing based on motor neuron activities.

Identification and characterization of an activating F229V substitution in the V2 vasopressin receptor in an infant with NSIAD.

Gain-of-function mutations in the gene encoding the V2 vasopressin receptor (V2R) cause nephrogenic syndrome of inappropriate antidiuresis. To date, reported mutations lead to the substitution of arginine 137 by either a cysteine or leucine (R137C/L). Here, we describe a 3-month-old hyponatremic infant found to have a phenylalanine 229 to valine (F229V) substitution in V2R. Characterization of this substitution in vitro revealed that it leads to high constitutive activity of the receptor, compatible with spontaneous antidiuresis. In contrast to R137C/L mutant receptors, F229V receptors do not undergo spontaneous desensitization, which results in sustained, high basal activity. Notably, the V2R-selective inverse agonists tolvaptan and satavaptan completely silenced the constitutive signaling activity of the F229V mutant receptor, indicating that this substitution does not lock the receptor in an irreversible active state. Thus, inverse agonists might prove to be effective therapies for treating patients with this or other spontaneously activating mutations that do not lock the V2R in its active state. These results emphasize the importance of genetic testing and the functional characterization of mutant receptors for patients with nephrogenic syndrome of inappropriate antidiuresis because the results might inform treatment decisions.

The surgical treatment of peroneal tendinopathy (excluding subluxations): a series of 17 patients.

Peroneal tendon pathology is rare, but is probably underestimated because it is frequently undiagnosed. It should always be in the differential diagnosis of lateral ankle pain. Surgical treatment of peroneal tendinopathy is indicated after failure of conservative measures. The aim of this retrospective study is to evaluate the medium-term clinical results of 17 patients operated for peroneal tendinopathy without tendon subluxation. A series of 17 patients composed of 7 women and 10 men with a mean age of 53.6 ± 4.6 (range 45 to 60) years were reviewed. The mean preoperative Kitaoka score was 46.7 ± 17.1 (range 25 to 69) points. All patients had radiological evaluation, which demonstrated hindfoot varus in 6 of the 17. Surgical interventions comprised synovectomy, debridement, suture-tubularization, fibrous resection, or tenodesis depending on the preoperative findings and also a valgus osteotomy (Dwyer) in 6 cases and ankle ligament reconstruction (modified Blanchet) in 1 case. All patients were reviewed clinically with a mean follow-up of 4.3 ± 3.8 years (range 16 months to 14 years). Average time to return to sport was 8.5 ± 10.4 months (range 3 months to 3 years). The mean time to return to work was 2.5 ± 1.9 (range 0 to 6) months. The mean postoperative Kitaoka score was 90.1 ± 11 (range 64 to 100) points with a statistically significant improvement to the preoperative score (p < .0001). Sixteen patients were satisfied or very satisfied with their treatment (94.1%). Surgical treatment of peroneal tendinopathy after failed conservative treatment leads to significantly improved function. It is a simple treatment to undertake, which gives a good outcome for both the patient and surgeon.

Spike sorting by stochastic simulation.

The decomposition of multiunit signals consists of the restoration of spike trains and action potentials in neural or muscular recordings. Because of the complexity of automatic decomposition, semiautomatic procedures are sometimes chosen. The main difficulty in automatic decomposition is the resolution of temporally overlapped potentials. In a previous study , we proposed a Bayesian model coupled with a maximum a posteriori (MAP) estimator for fully automatic decomposition of multiunit recordings and we showed applications to intramuscular EMG signals. In this study, we propose a more complex signal model that includes the variability in amplitude of each unit potential. Moreover, we propose the Markov Chain Monte Carlo (MCMC) simulation and a Bayesian minimum mean square error (MMSE) estimator by averaging on samples that converge in distribution to the joint posterior law. We prove the convergence property of this approach mathematically and we test the method representatively on intramuscular multiunit recordings. The results showed that its average accuracy in spike identification is greater than 90% for intramuscular signals with up to 8 concurrently active units. In addition to intramuscular signals, the method can be applied for spike sorting of other types of multiunit recordings.

Functional characterization of vasopressin type 2 receptor substitutions (R137H/C/L) leading to nephrogenic diabetes insipidus and nephrogenic syndrome of inappropriate antidiuresis: implications for treatments.

Substitution of arginine-137 of the vasopressin type 2 receptor (V2R) for histidine (R137H-V2R) leads to nephrogenic diabetes insipidus (NDI), whereas substitution of the same residue to cysteine or leucine (R137C/L-V2R) causes the nephrogenic syndrome of inappropriate antidiuresis (NSIAD). These two diseases have opposite clinical outcomes. Still, the three mutant receptors were shown to share constitutive beta-arrestin recruitment and endocytosis, resistance to vasopressin-stimulated cAMP production and mitogen-activated protein kinase activation, and compromised cell surface targeting, raising questions about the contribution of these phenomenons to the diseases and their potential treatments. Blocking endocytosis exacerbated the elevated basal cAMP levels promoted by R137C/L-V2R but not the cAMP production elicited by R137H-V2R, demonstrating that substitution of Arg137 to Cys/Leu, but not His, leads to constitutive V2R-stimulated cAMP accumulation that most likely underlies NSIAD. The constitutively elevated endocytosis of R137C/L-V2R attenuates the signaling and most likely reduces the severity of NSIAD, whereas the elevated endocytosis of R137H-V2R probably contributes to NDI. The constitutive signaling of R137C/L-V2R was not inhibited by treatment with the V2R inverse agonist satavaptan (SR121463). In contrast, owing to its pharmacological chaperone property, SR121463 increased the R137C/L-V2R maturation and cell surface targeting, leading to a further increase in basal cAMP production, thus disqualifying it as a potential treatment for patients with R137C/L-V2R-induced NSIAD. However, vasopressin was found to promote beta-arrestin/AP-2-dependent internalization of R137H/C/L-V2R beyond their already elevated endocytosis levels, raising the possibility that vasopressin could have a therapeutic value for patients with R137C/L-V2R-induced NSIAD by reducing steady-state surface receptor levels, thus lowering basal cAMP production.

Unsupervised Bayesian decomposition of multiunit EMG recordings using Tabu search.

Intramuscular electromyography (EMG) signals are usually decomposed with semiautomatic procedures that involve the interaction with an expert operator. In this paper, a Bayesian statistical model and a maximum a posteriori (MAP) estimator are used to solve the problem of multiunit EMG decomposition in a fully automatic way. The MAP estimation exploits both the likelihood of the reconstructed EMG signal and some physiological constraints, such as the discharge pattern regularity and the refractory period of muscle fibers, as prior information integrated in a Bayesian framework. A Tabu search is proposed to efficiently tackle the nondeterministic polynomial-time-hard problem of optimization w.r.t the motor unit discharge patterns. The method is fully automatic and was tested on simulated and experimental EMG signals. Compared with the semiautomatic decomposition performed by an expert operator, the proposed method resulted in an accuracy of 90.0% +/- 3.8% when decomposing single-channel intramuscular EMG signals recorded from the abductor digiti minimi muscle at contraction forces of 5% and 10% of the maximal force. The method can also be applied to the automatic identification and classification of spikes from other neural recordings.

Vasopressin type 2 receptor V88M mutation: molecular basis of partial and complete nephrogenic diabetes insipidus.

BACKGROUND/AIMS: Mutations in the type 2 vasopressin receptor gene (AVPR2) underlie X-linked recessive nephrogenic diabetes insipidus (NDI). Here, we report on a family with a mutation in AVPR2, c.262G>A (p.V88M). This recurrently identified mutation was previously shown to abolish AVPR2 function, yet in some affected members, urine osmolalities of up to 570 mosm/kg were observed. We detail the variable clinical phenotype and investigate its molecular basis. METHODS: Retrospective analysis of clinical data and in vitro assessment of wild-type and V88M-mutant receptors. RESULTS: Clinical data were available on 6 patients. Four of these demonstrated a substantial increase in urinary concentration after 1-desamino[8-D-arginine] vasopressin, consistent with partial NDI, while 2 did not respond. In vitro analysis revealed a reduced cell surface expression and decreased binding affinity for arginine-vasopressin of the mutant receptor, leading to blunted signaling activity. Treatment with the pharmacological chaperone SR121463 enhanced cell surface expression. CONCLUSION: The V88M mutation is associated with phenotypical diversity, which may be explained by the fact that both the expression level and the hormone-binding affinity are affected by the mutation. Our results provide a rational basis for treatment trials with vasopressin analogues in combination with pharmacologic chaperones in patients with this recurrently identified mutation.

Effect of the structure of bile salt aggregates on the binding of aromatic guests and the accessibility of anions.

The binding of naphthalene (Np), 1-ethylnaphthalene (EtNp), acenaphthene (AcN), and 1-naphthyl-1-ethanol (NpOH) as guests to the aggregates of sodium cholate (NaCh), taurocholate (NaTC), deoxycholate (NaDC), and deoxytaurocholate (NaTDC) was studied with the objective of determining how the structure of the bile salts affects the binding dynamics of guests and quenchers with the bile salt aggregates. Time-resolved and steady-state fluorescence experiments were used to determine the binding efficiency of the guests with the aggregates and were also employed to investigate the quenching of the singlet excited state of the guests by iodide anions. Quenching studies of the triplet excited states using laser flash photolysis were employed to determine the accessibility to the aggregate of nitrite anions, used as quenchers, and the dissociation rate constants of the guests from the bile salt aggregates. The binding efficiency of the guests to NaDC and NaTDC is higher than for NaCh and NaTC, and the protection efficiency is also higher for NaDC and NaTDC, in line with the larger aggregates formed for the latter bile salts. The formation of aggregates is in part driven by the structure of the guest, where an increased protection efficiency and residence time can be achieved by the introduction of short alkyl substituents (AcN or EtNp vs Np). NpOH was shown to be located in a very different environment in all four bile salts when compared to AcN, EtNp, and Np, suggesting that hydrogen bonding plays an important role in the formation of the aggregate around NpOH.

Limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells.

Transient gene expression in mammalian cells is intensively used for the rapid generation of recombinant proteins for biochemical, biophysical and pre-clinical studies. Still, the principles behind DNA transfer to the cells and the cellular cascade of events that ultimately dictate protein expression levels are not fully understood. Using polyethylenimine (PEI) mediated transfection of HEK293-EBNA1 cells, we sought to determine the most critical parameters that drive and limit recombinant protein production. Our results showed that a maximum of 65,000 plasmid copies/cell can be recovered in total extracts at 1 day post-transfection. Analyses performed after cell sorting revealed equal amounts of plasmid DNA in GFP-positive and -negative populations. However, nuclear plasmid content was three-fold higher in GFP-positive cells (1850 copies) than in GFP-negative cells (550 copies). The fact that significant amounts of plasmid DNA are found in the nucleus of GFP-negative cells suggests that its transcriptional competency is impaired. Interestingly, transfecting cells using a wide range of plasmid quantities at the optimal DNA:PEI ratio did not significantly affect the number of expressing cells. Thus, it appears that successful transgene expression is more likely to depend on a cellular "competent" state than to the quantity of plasmid DNA delivered per cell. Moreover, Northern blot analysis and SEAP/GFP measurement following plasmid titration experiments showed that transcriptional and translational processes are operating near to saturation under optimal transfection conditions. Overall, our results suggest that events that regulate nuclear translocation of plasmid DNA and its transcriptional competency as well as translational/post-translational limitations represent major bottlenecks in the success of a PEI-mediated protein production.

Improving glucose and glutamine metabolism of human HEK 293 and Trichoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme.

Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.