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Results for “parameter optimization”

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PubMed2006

Serum-free suspension large-scale transient transfection of CHO cells in WAVE bioreactors.

Here, we report the development of a large-scale transient expression platform utilizing Chinese hamster ovary (CHO) cells. The majority of recombinant proteins and antibodies that are produced for preclinical models and clinical trials are expressed in stably transfected CHO cells. A protocol for transient transfection of CHO cells that is rapid, reproducible, and cost-effective would therefore streamline the process from research to development and help avoid any potential host species induced variation in the molecule of interest. CHO cells were adapted to grow in serum-free suspension conditions in spinner flask cultures in a proprietary in-house developed growth medium. In developing this transient transfection protocol, the parameters optimized included the transfection reagent of choice, the cell density at the time of transfection, the plasmid DNA concentration, and the transfection reagent concentration. Using this optimized protocol, we have expressed recombinant proteins, including antibodies, at an expression level of up to 9.4 mg/L. We also report transient transfections from 500 mL working volume (w.v.) up to 20 L w.v. in a WAVE bioreactor. Using this optimized protocol, it is possible to rapidly (within 10 d) produce up to 100 mg of recombinant protein for further study.

Animals↗

PubMedDate not supplied

Determination of the electron density profile of the nerve myelin membrane in different states taking paracrystalline lattice distortions into account.

Recent investigations point to the presence of paracrystalline lattice disorder in the nerve myelin in the swollen, fixed and to some extent also in the native state. This leads to the distortion of the Q-function of the membrane stack and the Q0-function of the unit cell, which can be experimentally isolated through swelling. Representing the distance statistics between neighbouring membranes and the electron density distribution of the single membrane with Gaussian functions, the Q-function can be expressed analytically as a convolution polynomial of the electron density, its mirror image and the distance statistics functions. Fitting this model Q-function to the experimental Q-function obtained by the inverse Fourier transformation of the scattered intensity, one can determine the optimal parameters of the electron density distribution and the distance statistics functions. The elimination of the distance fluctuations between neighbouring membranes permits the calculation of the undistorted Q-function or the Q0-function. The Fourier Analytical Deconvolution of the undistorted Q0-function enables a unique determination of the double membrane profile, thus a unique phase determination.

Absorptiometry, Photon↗

PubMed1994

Luteinizing hormone secretory pattern before and after removal of Leydig cell tumor of the testis.

We studied the luteinizing hormone (LH) secretory pattern in three patients, aged 30, 23 and 43 years, with gynecomastia due to Leydig cell tumor of the testis, before and 6 months after unilateral orchidectomy. The results were compared to those of 11 normal fertile controls aged 20-35 years. Blood sampling was done at 20-min intervals from 22.00 h to 10.00 h. The LH data were analyzed with the Cluster analysis algorithm with "optimal parameters for LH male data" to determine the pulse interval and pulse amplitude. The Expfit program was applied to LH pulses to calculate the apparent half-life of immunoreactive LH. Before surgery, when compared to controls, the patients had a low to normal testosterone/estradiol ratio (0.053, 0.110, 0.046 vs 0.148 +/- 0.038) and mean LH levels (1.96, 3.7, 2.55 vs 4.0 +/- 1.9 IU/l), decreased pulse amplitude (2.65, 3.01, 2.21 vs 3.31 +/- 1.41 IU/l) and reduced apparent half-life of LH (74, 69, 78 vs 97 +/- 16 min). After removal of the Leydig cell tumor, the testosterone/estradiol ratio returned to the normal range (0.141, 0.177, 0.093) while an increase in mean LH levels (5.75, 7.90, 4.88 IU/l), LH pulse amplitude (3.07, 6.05, 2.86 IU/l) and apparent half-life of LH (138, 106, 104 min) was observed in all three patients. Our data indicate that endogenous hyperestrogenism in patients with Leydig cell tumor of the testis results in an inhibition of LH secretion, and suggests that such inhibition could result from a reduction in pulse amplitude and apparent half-life.

Adult↗

PubMed1991

Visualization of the heart and determination of left ventricular mass in rats by echocardiography.

A high-frequency transducer was used to determine the optimal parameters for visualizing the heart in 40 normal Wistar, 15 SHR, and 10 aorta-banded rats. The rats were 5 to 30 weeks old and weighed between 105 and 705 grams. Two-dimensional and M-mode views of the ventricles, atria, valves, and great arteries were obtained by placing the transducer beneath the rats through the left or right parasternal window in either the prone or the right decubitus positions, respectively. Left ventricular (LV) mass was determined on the basis of a spheroid model; these values correlated well with the LV weight for both the Wistar rats (r = 0.94, p less than 0.001) and the rats with cardiac hypertrophy due to pressure load (r = 0.87, p less than 0.001). These results were highly reproducible. This indicates that echocardiography is useful for obtaining quantitative measurements in rats.

Animals↗

PubMed2001

Investigation and development of ultrasonic vibrodrives for traumatology.

An investigation was made of the dynamics of ultrasonic vibrodrills with a common source of rotational and ultrasonic reciprocal motions. The source was constructed on the basis of an electroacoustic transducer of longitudinal vibrations. The dependences of power, rotation speed, and flutter rates were plotted for various pushers as a function of biological tissue drag torque. The results obtained allowed us to determine the optimal parameters of basic ultrasonic modes used in treating biological tissues. Apart from that, ultrasonic vibrodrill pilot models, which are new in principle, are presented. They were designed to be applied in traumatology. A distinguishing feature of these vibrodrills is that an electroacoustic transducer of longitudinal vibrations is a source of two motion types: rotational and ultrasonic reciprocal.

Equipment Design↗

PubMed1987

An animal model of excessive eating: schedule-induced hyperphagia in food-satiated rats.

Nineteen rats were maintained throughout the experiment on ad libitum wet mash and water and were trained to press a lever on fixed-interval or fixed-ratio schedules of reinforcement with electrical brain stimulation. Fourteen rats ate at least 150% more mash during intermittent reinforcement sessions than during baseline, massed reinforcement control, and/or extinction sessions. In a 3-hr session, 11 of those 14 consumed more than 22 g of wet mash (13 g dry weight), the equivalent of nearly half an animal's daily food intake. In subsequent control sessions, the electrodes did not support stimulus-bound eating despite attempts to make stimulation parameters optimal. These results indicate that the eating was schedule induced or adjunctive, and suggest that the procedure may provide an animal model of excessive nonregulatory eating that contributes to obesity in humans.

Animals↗

PubMed2002

Bacteria-modified amperometric immunosensor for a Brucella melitensis antibody assay.

A novel amperometric immunosensor setup is described which uses horseradish peroxidase (HRP) as a label in conjunction with a current-based Brucella sensor. The Bacteria modified immunosensor was constructed by using a biocomposite formed by dispersing graphite powder into a mixture of Brucella melitensis and silicate polymer gel. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen (Brucella melitensis), which retains its binding properties, and the association reaction is easily detected at the surface exposed to the solution. The use of an oaminophenol (o-AP) substrate and amperometric detection at -150 mV (vs. SCE) results in a relatively low detection limit of 3.5 ng/ml and a linear detection range of 3.5 ng/ml to 200 ng/ml. Based on an optimized parameter, the prepared sensor was used to detect the Brucella melitensis antibody in serum samples by using a competitive binding assay. The results demonstrate the feasibility of employing the proposed immunosensor for the detection for Brucella melitensis antibody in a clinical analysis.

Animals↗

PubMed2002

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

Calcium Phosphates↗

PubMed1999

Use of enhanced green fluorescent protein to optimize and quantitate infection of target cells with recombinant retroviruses.

Recombinant retroviral vectors are useful tools for gene transfer in both gene therapy and research applications. An enhanced form of green fluorescent protein has been incorporated into recombinant retroviruses as a marker to follow infected cells. In this paper, we extended the use of the fluorescent reporter to quantify protein expression using such analytical tools as fluorescent microscopy, flow cytometry and fluorescent plate reader analysis. These tools enabled us to rapidly assess the titer of recombinant retrovirus harvested from packaging cells and to optimize parameters for infection of different cell lines.

3T3 Cells↗

PubMed2006

Sequencing batch reactor as a post-treatment of anaerobically treated dairy effluent.

Wastewater from dairy industries, characterized by its high COD content and relative high COD/TKN ratio, requires post-treatment after anaerobic treatment to complete the removal of organic matter and nutrients. Due to its simplicity, robustness and low maintenance costs, sequencing batch reactors (SBR) result in an attractive system, especially in case of small dairy industries in order to comply with the emission standards. The goal of this work was to determine the operational parameters, optimize the performance, and study the stability of the microbial population of a SBR system for the post-treatment of an anaerobic pond effluent. High and stable removal of COD and TKN was achieved in the reactor, which can easily be set up in dairy industries. An active nitrifying population was selected during reactor operation and maintained relatively stable, while the heterotrophic (total and denitrifying) communities were more unstable and susceptible to changes in the operating conditions.

Anaerobiosis↗

PubMed1996

Helical (spiral) CT of the upper airway with three-dimensional imaging: technique and clinical assessment.

OBJECTIVE: The purpose of this study was to evaluate the application of helical CT-generated three-dimensional images of the upper airway. MATERIALS AND METHODS: Thirty patients, 10 healthy and 20 with upper-airway disease, were studied with helical CT (5-mm collimation). Overlapping images at 2-mm intervals were retrospectively generated. In the group of healthy patients, two radiologists in independently compared overlapping with nonoverlapping images, ranked confidence in identifying small airway structures on a scale of 1-5, and tabulated the number of images demonstrating these structures. In the 20 patients with disease, three-dimensional (3D) surface models were rendered on an independent workstation and were reviewed by two radiologists and one otolaryngologist for image quality, appreciation of lesion morphology, and ability to judge lesion extent, using a similar scale. A phantom was used to optimize parameters for the 3D reconstructions. RESULTS: Viewing of the retrospectively generated overlapping images increased by 122% the number of images in which laryngeal and hypopharyngeal structures could be identified (p < .01). Image confidence scores for the radiologists averaged 3.3 for nonoverlapping and 4.0 for overlapping (p < .05). Radiologists and otolaryngologist rated the quality of the 3D images equally. The otolaryngologist's assessment of the value of the models for understanding the lesion morphology was 3.5 compared with the radiologists assessment of 2.5; and for judging the lesion extent, the otolaryngologist's assessment was 3.8 compared with 2.7 for the radiologist, a statistical significance of p < .01. CONCLUSION: Helical CT with the application of overlapping images and 3D reconstructions significantly assists the understanding of upper-airway disease.

Case-Control Studies↗

PubMedDate not supplied

Body fat mass in normal weight subjects.

Obesity is characterized by excessive body fat accumulation which may lead to serious health problems and complications. Body mass index is the most optimal parameter to evaluate the level of nutritional status and diagnose obesity. However, modern techniques studying body composition can more accurately determine whether the gain of body weight was on the account of body fat, lean body mass or total body water. If one's body mass index is in the range of normal values but the amount of body fat is above normal range, we talk about sarcopenic obesity. In order to evaluate presence of sarcopenic obesity, a group of 140 normal weight students of the Faculty of Medicine in Novi Sad were measured. Apart from standard anthropometrical parameters the amount of body fat was also determined by using the method of bioelectrical impedance analysis. Sarcopenic obesity was diagnosed in 25.71% of examined students. By using body mass index values this type of obesity cannot be diagnosed, and knowing that a higher amount of body fat in normal weight persons can lead to complications, especially metabolic, it is of great importance to evaluate the amount of body fat accurately.

Adipose Tissue↗

PubMedDate not supplied

[Thoracic electrical bioimpedance in the follow-up of patients with cardiovascular disorders].

Thoracic electrical bioimpedance is a new non-invasive technique for obtaining haemodynamic parameters. The method involves the passing of low amperage, high frequency current through the thorax. The current induces a change of resistance within the thorax, which is registered by electrodes. The overall impedance of the thorax is a measure of the electrical resistance of the thorax to this high frequency, low amplitude current. In clinical practice, thoracic electrical bioimpedance has been applied in diagnostic, therapeutic, and prognostic use in patients with heart failure and hypertension, patients with pacemakers, within the scope of early diagnosis of implant rejection following heart transplantation, as well as in patients with kidney disorders, before and after dialysis. However, one of its most important applications is in the determination of antihypertensive therapy, because it provides the parameters that reflect the genesis of arterial blood pressure. In assessing these parameters, optimal therapy adjusted to the haemodynamic status of each patient can be prescribed.

Cardiography, Impedance↗

PubMed2006

Acute and long-term outcome of transvenous cryothermal catheter ablation of supraventricular arrhythmias involving the perinodal region.

OBJECTIVE: Cryoenergy is a new valuable treatment option to perform ablation close to the atrioventricular (AV) node in the cure of supraventricular tachycardias because of its favourable properties, such as the possibility of creating reversible lesions. The aim of this study was to report our experience on the effectiveness and safety of catheter cryoablation performed in "critical areas" to treat a large cohort of patients with supraventricular arrhythmias. METHODS: One hundred and thirty-one patients suffering from supraventricular tachycardias underwent catheter cryoablation using a 7F catheter. Eighty-seven patients presented with AV nodal re-entrant tachycardia (AVNRT), 39 had accessory pathways (APs) either manifest or concealed (15 midseptal, 24 parahissian), three had ectopic right atrial tachycardia (AT), and two patients had a permanent junctional reciprocating tachycardia (PJRT). When the optimal parameters were recorded, ice mapping at -30 degrees C was performed for 80 s to validate the ablation site. If the expected result occurred, cryoablation was carried out by lowering the temperature to -75 degrees C for 4 min. RESULTS: In two patients cryoablation was not performed because of technical reasons. Cryoablation was acutely successful in 84 out of 85 patients with AVNRT, in 37 of 39 with APs and in all patients with AT and PJRT. No complications occurred in any patient. Transient AV conduction impairment occurred in seven patients with midseptal APs and in two patients with AVNRT. In particular, in these patients no late permanent AV block was observed at follow-up. At a mean follow-up of 27 +/- 12.9 months, clinical success rate was 87%. CONCLUSIONS: Cryoablation is a safe and effective technique with a high success rate in the long term. It may be particularly useful when performing ablation close to the AV node or His bundle owing to the possibility of validating the ablation site with ice mapping, which creates only a reversible lesion, mainly in the midseptal APs.

Adolescent↗

PubMed1995

The effects of different stretch velocities on average force of the shortening phase in the stretch-shorten cycle.

The function of muscle is thought to be enhanced through the use of a stretch-shorten cycle. Although this pattern of muscle function is commonly used, optimal parameters of the cycle are not well understood. The purpose of this paper is to review the stretch-shorten cycle pattern of muscle function, as it applies to human movement, and to present a research study that investigated the effects of different stretching speeds on the average concentric forces during stretch-shorten cycles. Twenty-two women performed repeated plantar flexor stretch-shorten cycles on a Kin-Com dynamometer at approximately 60% of their isometric plantar flexor maximum. Sequential trials were recorded for 15 seconds at three different speed combinations (eccentric speed/concentric speed): 50/180 degrees/sec, 100/180 degrees/sec, and 150/180 degrees/sec. Eighteen cycles from each speed combination were chosen for analysis based on contributing to an equal plantar flexor activity among the speed combinations. Analysis of variance indicated that the speed combination that most closely approximated normal walking (50/180 degrees/sec) resulted in the highest average concentric forces. Analysis of covariance indicated that the highest forces were due, at least in part, to a higher averaged interphase force.

Adult↗

PubMed1991

Focal dorsal raphe stimulation and pinnal electrical stimulation modulate spontaneous and noxious evoked responses in thalamic neurons.

This study investigated the nocieceptive responses of single neurons within the nucleus parafascicularis (PF) thalami of the rat following two modes of electrical stimulation known to induce analgesia. It was found that both focal electrical dorsal raphe stimulation (DRS) and bilateral pinnal (ear) electrical stimulation (PES) converge on the same PF neurons, affecting both the spontaneous discharges and the noxious evoked responses toward these neurons. The effects of different stimulus current intensity, frequency and pulse duration were also examined. It was found that for both DRS and PES at pulse frequency of 10 Hz and current amplitude of 10 microA are the optimal parameters to modulate both the spontaneous and the noxious evoked responses. These stimuli produced prolonged effects related to the duration of stimulation. The external (PES) low current stimulation which was delivered below the sensory threshold was as effective in modulating noxious responses as the invasive DRS in intact animals and in animals with bilateral dorsolateral-funiculus ablation. It was observed that dorsal lateral funiculus ablation (DLFx) did not modify the DRS and the PES effects. These observations further support the existence of an ascending pain modulation pathway.

Animals↗

PubMed1992

Respiration in vitro: II. Electrical stimulation.

The present report describes electrical-stimulation-induced activity recorded from intercostal muscles in the neonatal rat rib-attached, in vitro brain stem-spinal cord preparation. The muscle bursts induced by electrical stimulation included a short-latency twitch contraction and a long-latency modulated contraction similar to that observed during spontaneous respiratory-like activity. Multilevel recordings showed a cephalocaudal order of recruitment of intercostal muscles similar to that observed during spontaneous activity. The optimal parameters of stimulation were 2-msec pulses delivered at 0.1-0.2 Hz. Trains of pulses also were effective. These movements could be induced following stimulation of various sites within each segment of the spinal cord, with the lowest threshold sites located in the ventrolateral funiculus and intermediate gray. Stimulation of every cervical segment was effective in inducing respiratory-like activity, with the lowest-threshold segments being C1, C2, and C5. These results suggest that low-frequency, long-duration pulses applied directly to the spinal cord can induce respiratory-like activity similar to that observed during spontaneous activity in the neonatal rat, rib-attached in vitro brain stem-spinal cord preparation. The ability to elicit a coordinated respiratory pattern even after a high spinal transection suggests that such stimulation may be effective in inducing respiratory-like activity in the absence of descending brain stem connections.

Animals↗

PubMed1988

A study of caudate inhibition on an epileptic focus in the cat hippocampus.

The mechanisms whereby the caudate nucleus modifies hippocampal spiking activity have been studied. Epileptiform activity was induced in the cat hippocampus by topical application of sodium penicillin in different concentrations. The frequency of induced spikes appeared to be directly correlated to the two doses of epileptogenic agent. The inhibitory effect of 10 Hz caudate stimulation on spike frequency was present even when stimulation lasted for 180 s. Likewise 25 Hz caudate stimulation brought about an inhibition which was maintained by stimulus trains lasting up to 90 s, while the degree of inhibition was reduced by trains of longer duration (120, 150 and 180 s); similar results were also noted in some atropine-treated cats. The time course of spikes in cats with electrolytic lesions of the caudate exhibited an increase in both frequency and duration. The results indicate that there is an optimal parameter for caudate stimulation causing inhibition of penicillin-induced hippocampal spiking activity, and suggest the possibility of tonic control of hippocampal excitability exerted by the caudate nucleus.

Animals↗