Four-parameter Logistic Equation Research Articles

Response surface optimisation of technological parameters for producing Ganoderma lucidum by solid-state fermentation from Panax notoginseng residues and kinetics

The present work aimed to investigate the optimal control strategy for Ganoderma lucidum (Chuanzhi No. 6) fermentation from Panax notoginseng residue. Optimisation of the solid-state fermentation (SSF) technical parameters, including inoculum dosage, fermentation temperature, and fermentation time was carried out using the single-factor and Box-Behnken design methods. Results showed that the optimal technical parameters were inoculum dosage of 15.28%, fermentation temperature of 28.42°C, and fermentation time of 14 d. The predicted maximum biomass of G. lucidum was 0.4327 g, which was also verified by validation experiments. The biomass of G. lucidum increased significantly with optimised technological parameters. The logistic equation, the Boltzmann function, and the four-parameter logistic equation were respectively suitable for modelling G. lucidum growth kinetics (R2 = 0.9754), Ganoderma triterpene production kinetics (R2 = 0.9897), and the matrix consumption kinetics (R2 = 0.9826). These models can be used to predict the SSF process of G. lucidum, thus providing a theoretical basis for the development of new G. lucidum drugs, while at the same time recycle Panax notoginseng residue.

Anthelmintic-Like Activity and Ultrastructure Changes Produced by Two Polyphenolic Combinations against Cooperia punctata Adult Worms and Infective Larvae.

Cooperia punctata is one of the most prevalent gastrointestinal nematodes affecting cattle under grazing conditions, and the increasing reports of anthelmintic resistance forces researchers to look for novel control measures. Previous reports have proposed the use of polyphenolic compound (PC) combinations (Coumarin:Quercetin (CuQ) and Caffeic-acid:Rutin (CaR)) against free-living stages (L3) of C. punctata. The objective of this study was to assess the in vitro motility inhibition of C. punctata adult worms and infective larvae using the Larval Motility Inhibition Assay (LMIA) and Adult Motility Inhibition Assay (AMIA), and to assess the structural and ultrastructural changes induced by both treatments using Scanning and Transmission Electron Microscopy. For the LMIA, infective larvae were incubated for 3 h in 0.8 mg mL-1 and 0.84 mg mL-1 of CuQ and CaR, respectively. For AMIA, six concentrations and five incubation periods (2, 4, 6, 12 and 24 h) were assessed using each PC combination. Cooperia punctata motility was calculated as a percentage and corrected using control motility percentages. A multiple comparisons Brown-Forsythe and Welch ANOVA test was used to compare larval motility; and to fit the dose-response in AMIA, data were analyzed with a non-linear regression four-parameter logistic equation with a variable slope, using the computer program GraphPad Prism® V.9.2.0. Although larval motility was barely affected by both treatments (p > 0.05), adult worm motility was inhibited 100% and 86.9% after 24 h incubation with CuQ and CaR, respectively (p < 0.05). The best fit EC50 for adult worm motility inhibition were 0.073 ± 0.071 mg mL-1 and 0.051 ± 0.164 mg mL-1 for CuQ and CaR, respectively. Main structural and ultrastructural lesions observed in both biological stages were: (i) L3 sheath-cuticle complex disruption, (ii) collagen fibers degradation; (iii) hypodermic detachment, (iv) seam cell apoptosis and (v) mitochondrial swelling. The alterations observed suggest that the PC combinations interfere with the anatomy and physiology of the locomotive apparatus of the nematodes.

Kinetic Exclusion Assay of Biomolecules by Aptamer Capture.

DNA aptamers are short nucleotide oligomers selected to bind a target ligand with affinity and specificity rivaling that of antibodies. These remarkable features recommend aptamers as candidates for analytical and therapeutic applications that traditionally use antibodies as biorecognition elements. Numerous traditional and emerging analytical techniques have been proposed and successfully implemented to utilize aptamers for sensing purposes. In this work, we exploited the analytical capabilities offered by the kinetic exclusion assay technology to measure the affinity of fluorescent aptamers for their thrombin target and quantify the concentration of analyte in solution. Standard binding curves constructed by using equilibrated mixtures of aptamers titrated with thrombin were fitted with a 1:1 binding model and provided an effective Kd of the binding in the sub-nanomolar range. However, our experimental results suggest that this simple model does not satisfactorily describe the binding process; therefore, the possibility that the aptamer is composed of a mixture of two or more distinct Kd populations is discussed. The same standard curves, together with a four-parameter logistic equation, were used to determine “unknown” concentrations of thrombin in mock samples. The ability to identify and characterize complex binding stoichiometry, together with the determination of target analyte concentrations in the pM–nM range, supports the adoption of this technology for kinetics, equilibrium, and analytical purposes by employing aptamers as biorecognition elements.

Quantifying the Spatio-Temporal Process of Township Urbanization: A Large-Scale Data-Driven Approach

The integrated recognition of spatio-temporal characteristics (e.g., speed, interaction with surrounding areas, and driving forces) of urbanization facilitates regional comprehensive development. In this study, a large-scale data-driven approach was formed for exploring the township urbanization process. The approach integrated logistic models to quantify urbanization speed, partial triadic analysis to reveal dynamic relationships between rural population migration and urbanization, and random forest analysis to identify the response of urbanization to spatial driving forces. A typical subtropical town was chosen to verify the approach by quantifying the spatio-temporal process of township urbanization from 1933 to 2012. The results showed that (i) urbanization speed was well reflected by the changes of time-course areas of urban cores fitted by a four-parameter logistic equation (R2 = 0.95–1.00, p &lt; 0.001), and the relatively fast and steady developing periods were also successfully predicted, respectively; (ii) the spatio-temporal sprawl of urban cores and their interactions with the surrounding rural residential areas were well revealed and implied that the town experienced different historically aggregating and splitting trajectories; and (iii) the key drivers (township merger, elevation and distance to roads, as well as population migration) were identified in the spatial sprawl of urban cores. Our findings proved that a comprehensive approach is powerful for quantifying the spatio-temporal characteristics of the urbanization process at the township level and emphasized the importance of applying long-term historical data when researching the urbanization process.

Rapid prediction of the re-watering time point of Orychophragmus violaceus L. based on the online monitoring of electrophysiological indexes

Karst arid environments are not conducive to the normal growth of plants. Mastering the irrigation time point in advance can not only guarantee yield but can also save water resources in these regions. The seedlings of Orychophragmus violaceus L. during winter drought periods were processed by simulating a karst osmotic stress environment (high pH, high bicarbonate concentration, and drought) in a growth chamber to study the responses of photosynthesis, carbonic anhydrase (CA), leaf tensity (Td) and growth traits. All the plants were exposed to osmotic stress induced by poly-ethylene glycol (PEG 6000) at 5 levels (0, 10, 20, 40, and 80 g L−1 PEG) in the presence of 10 mmol L-1 NaHCO3. The physiological osmotic stress tolerance threshold of O. violaceus was investigated. Leaf area and dry weight biomass (DW) estimation models were established, a non-destructive and rapid measurement of the DW was achieved. By analysing the temporal dynamic variations in the DW, combined with the relationship between Td and DW, a prediction of the re-watering time point of O. violaceus was achieved. The results indicated the following: (1) O. violaceus maintained good water status and high photosynthetic efficiency when the stress level was not higher than 20 g L-1 PEG, and its physiological osmotic stress tolerance threshold was 20 g L-1 PEG; (2) the growth rate of DW was calculated according to the derivative of the four-parameter logistic growth equation at each osmotic stress level. The appropriate percentage for the decline in growth rate under osmotic stress was 30% or 50% to achieve an improvement in product quality. The re-watering time points of O. violaceus that corresponded to the given decline in the growth rate at 10 g L-1 PEG was day 4.90 or day 2.66, and that at 20 g L-1 PEG was day 1.41. Subsequently, the Td corresponding to the above-mentioned re-watering time point was calculated according to the four-parameter logistic equation and mathematical model between Td and DW. O. violaceus at 10 g L-1 PEG should be re-watered when the Td is 0.99 or 0.48, and O. violaceus at 20 g L-1 PEG should be re-watered when the Td is 0.69. O. violaceus at 10 g L-1 PEG exhibited greater long-term osmotic stress resistance than that at 20 g L-1 PEG. Re-watering for O. violaceus at 10 g L-1 PEG could minimize the water demand with corresponding minimal impact on the DW, thereby leading to improved economic returns to water; and (3) as such, the rapid prediction of the re-watering time point can be achieved through online monitoring of electrophysiological parameters such as Td.

Multiple-Point Evaluation Algorithms for Enhanced Precision of Pathogen-Specific Cerebrospinal Fluid/Serum Antibody Index Calculation.

The determination of pathogen-specific antibody indices (AIs) of CSF and serum is an essential cornerstone in assessing neurological diseases and demands reliable high precision. Various companies provide ELISA kits for the detection of respective antibody concentrations and base AI calculation on a single CSF/serum pair of optical densities (ODs), combined with selection rules. The remainder of OD measurements is not used. There is no averaging of measurement errors and result stabilization. OD data from Siemens Enzygnost ELISA measurements of 2012-2016 proficiency survey samples for measles/rubella/varicella zoster/herpes simplex virus (MRZH) reaction (INSTAND e.V.) were reanalyzed. Several reference methods for calculating Q values from ODs using multiple-point evaluation are described. The methods are based on the α method and the four-parameter logistic (4PL) equation. Statistical analysis shows standard deviations of relative AI differences from AI target values to be significantly lower if derived from multiple-point evaluation instead of single-pair evaluation. Thus, the virus-averaged hit rate of a 10% target AI environment can be improved from 49% up to 69%. Waiving the usage of a standard curve in favor of parameter fitting significantly improves calculational precision for Siemens Enzygnost assays. Patient safety, diagnostic assay costs, and laboratory effectiveness might be improved for other test distributors as well.

Detection of Pyricularia oryzae Based on Indirect ELISA Using Germinating Conidia as the Antigen Preparation

One of the most destructive fungal diseases in rice crop in Malaysia is the Rice blast disease or Penyakit Karah, which is caused by Pyricularia oryzae Cav. (previously known as Magnoporthe grisea). The disease can cause a loss of yield of more than 50 percent. In this study a sensitive indirect ELISA method was developed for the detection of Pyricularia oryzae through an indirect ELISA method using rabbit polyclonal antibody against different concentrations of Pyricularia oryzae conidia to give seven calibration standard points ranging from 1×101 to 1×109 conidia/mL. The calibration curve shows a typical sigmoidal curve and the fitting of the curve was based on the four-parameter logistic equation. The calculated LOD value was 21.60 conidia/mL (95% confidence interval, 11.63 to 31.57 conidia/mL) with a good correlation coefficient value of 0.999 indicating the model fit the experimental data in an excellent manner. The sensitivity was comparable to another published work which claimed to be the most sensitive to date. This indicate that the current developed method can be utilized in the future as a sensitive method for the rapid and simple detection of this pathogen.

Remodelling of the calibration curve for the potentiometric sensing of lamotrigine based on molecularly imprinted polymers using the four-parameter logistic model

A standard or a calibration curve allows the determination of an analyte concentration from an experiment. The calibration curve is constructed using a known concentration of analyte. The unidentified concentration of an analyte will then be determined utilizing this standard curve. Similar to immunoassay, ligand-binding interaction in Molecularly Imprinted Polymers (MIPs) also mimics immunoassay in that the calibration curve is sigmoidal in many cases. In a previous study an ion selective electrode based on molecularly imprinted polymers calibration curve for the detection of lamotrigine (LTG) showed a sigmoidal calibration curve but was not modelled according to a sigmoidal dose response model. Using the four-parameter logistic (4PL) equation, the calibration curve was remodelled according to this equation resulting in the calculated values of 19.87, -87.43, -4.614 and 0.6354 for the parameters a and d (maximum and minimum responses), Log EC50 (value that produces a 50% signal response) and Hillslope (slope-like parameter or Hill coefficient). The use of the 4PL model gave a correlation coefficient value of 0.9985 indicating the model excellently fit the experimental data

Determination of the detection limit using the four-parameter logistic model for the double-antibody sandwich ELISA for the rapid detection of Bacillus cereus in food

Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In a previous publication, a sandwich ELISA is developed for the detection of B. cereus whole cells. The curve showed a sigmoidal calibration curve, but the model used was linear. This study remodels the sigmoidal data obtained using a four-parameter logistic (4PL) equation that can cover the whole range of data obtained. The sigmoidal ELISA calibration curve was efficiently fit by the 4-PL equation. A good correlation coefficient value of 0.996 was obtained indicating good fitting. The LOD value was 5.36 x104 CFU/mL with the 95% confidence interval from 1.07x104 to 9.64x104 CFU/mL, which is about one order of magnitude less sensitive that the previously claimed LOD. The modelling exercise shows that the 4PL equation can fit the ELISA curve very well.

A model for in vivo analysis of sudomotor sympathetic C-fiber activation and human sweat gland output.

Quantitative assessment of small-fiber peripheral neuropathy often involves an evaluation of the interaction between the C-fiber sudomotor nerve and local sweat rate (SR). Typically, some sort of quantitative sudomotor axon reflex test (QSART) is performed to aid in diagnosing small-fiber dysfunction. The currently used QSART demonstrates only moderate test-retest reliability and therefore limits its usefulness in tracking small-fiber dysfunction. A new experimental model to examine small C-fiber function in the skin using intradermal electrical stimulation and simultaneous monitoring of SR is proposed. Using intradermal electrical stimulation (1.5 and 2.5 mA) and varying stimulus frequency from 0.2 to 64 Hz, a quantitative relationship between the area under the SR-time curve and log10 stimulus frequency is modeled using a four-parameter logistic equation, providing the following parameters: baseline, plateau, EC50, and Hill slope. The model has good to excellent repeatability within the same day (ICC = 0.98), on different days at the same skin site (ICC = 0.80), and when comparing two different skin sites (ICC = 0.78) with a small bias estimate and the line of identity always lying within the 95% limits of agreement. Atropine sulfate (0.1 mg/ml) blocked 90 ± 5% of the electrically induced sweating. Overall, the model provides control over sudomotor nerve activity and a quantitative assessment of SR. Finally, the ability to reproduce the quantitative stimulus-response curve on different days allows for a robust assessment of the relationship between the activation of a sympathetic C-fiber and local SR.NEW & NOTEWORTHY A model for quantitative assessment of C-fiber function in human skin using intradermal electrical stimulation and local sweat rate measurements has been developed. This new electrically induced sweating model is nonpainful and allows for a complete stimulus-response curve plotting the area under the local sweat rate-time curve vs. the log10 stimulus frequency. The model has good reproducibility and should provide a means of assessing the progression of small C-fiber peripheral neuropathy in humans.

Temperature Thresholds and Growing-Degree-Day Models for Red Sorrel (Rumex acetosella) Ramet Sprouting, Emergence, and Flowering in Wild Blueberry

Red sorrel is a common herbaceous creeping perennial weed in wild blueberry fields in Nova Scotia that spreads by seeds and an extensive creeping root system. Experiments were established to determine temperature thresholds for ramet sprouting from creeping root fragments and to develop growing-degree-day (GDD) models for predicting ramet emergence and flowering under field conditions in wild blueberry fields in Nova Scotia. Ramets sprouted from root fragments at temperatures as low as 1 C, with an optimum temperature for ramet sprouting around 22 C. Ramet sprouting was completely inhibited at temperatures above 35 C. Cumulative ramet emergence and flowering under field conditions were adequately explained as functions of GDD by a three-parameter power equation (R2= 0.98) and a four-parameter logistic equation (R2= 0.87), respectively. Ramet emergence began between 110 and 265 GDD and continued throughout the season at each site. Model prediction for the initiation of emergence was 92 GDD, and 50 and 95% emergence were predicted to occur at 1,322 and 2,696 GDD, respectively. Red sorrel ramets began to flower in the field between 308 and 515 GDD. Model prediction for the initiation of flowering was 289 GDD, and 50 and 95% flowering were predicted to occur at 545 and 1,336 GDD, respectively. Model validation was conducted with the use of two additional independent data sets for emergence and flowering and indicated good performance of the proposed models (R2and root-mean-square error values ranging from 0.96 to 0.99 and 4.0 to 13.8, respectively). The models allow for direct comparison of red sorrel phenology to that of the wild blueberry and will aid in the development of new management strategies.

Micro-flow-injection analysis (μFIA) immunoassay of herbicide residue 2,6-dichlorobenzamide – towards automated at-line monitoring using modular microfluidics.

As a part of developing new systems for continuously monitoring the presence of pesticides in groundwater, a microfluidic amperometric immunosensor was developed for detecting the herbicide residue 2,6-dichlorobenzamide (BAM) in water. A competitive immunosorbent assay served as the sensing mechanism and amperometry was applied for detection. Both the immunoreaction chip (IRC) and detection (D) unit are integrated on a modular microfluidic platform with in-built micro-flow-injection analysis (μFIA) function. The immunosorbent, immobilized in the channel of the IRC, was found to have high long-term stability and withstand many regeneration cycles, both of which are key requirements for systems utilized in continuous monitoring. The IRC was regenerated during 51 cycles in a heterogeneous competitive assay out of which 27 were without the analyte (the highest possible signal level) in order to assess the regeneration capability of the immunosorbent. Detection of BAM standard solutions was performed in the concentration range from 62.5 μg L(-1) to 0.0008 μg L(-1). Non-linear regression of the data using the four-parameter logistic equation generated a sigmoidal standard curve showing an IC50 value (concentration that reduces the signal by 50%) of 0.25 μg L(-1). The strongest signal variation is observed in the concentration range between 0.02 and 2.5 μg L(-1), which includes the 0.1 μg L(-1) threshold limit set by the European Commission for BAM in drinking water. The presented results demonstrate the potential of the constructed μFIA immunosensor as an at-line monitoring system for controlling the quality of ground water supply.

Baroreflex and somato-reflex control of blood pressure, heart rate and renal sympathetic nerve activity in the obese Zucker rat

It has been reported that the baroreflex control of heart rate (HR) and sympathetic nerve activity (SNA) is attenuated in obese Zucker rats (OZRs) compared with age-matched lean animals (LZRs). What is not known, however, is the extent to which the baroreflex control of mean arterial blood pressure (MAP) is altered in the OZR. In addition, it is not known whether the interactions of other sensory nerve inputs on autonomic control are altered in the OZR compared with the LZR. The aim of this study was to determine the baroreflex control of MAP, HR and renal SNA (RSNA) in the OZR and LZR using an open-loop baroreflex approach. In addition, the effect of brachial nerve stimulation (BNS) on the baroreflex control was determined in these animals. Age-matched, male LZRs and OZRs were anaesthetized, and the carotid baroreceptors were vascularly isolated, bilaterally. The carotid sinus pressure was increased in 20 mmHg increments from 60 to 180 mmHg using an oscillating pressure stimulus. Baroreflex function curves were constructed using a four-parameter logistic equation, and gain was calculated from the first derivative, which gave a measure of baroreceptor sensitivity, before and during BNS. The range over which the baroreflex could change MAP (28 ± 6 versus 87 ± 5 mmHg; mean ± SEM), HR (17 ± 4 versus 62 ± 11 beats min(-1)) and normalized RSNA (NormNA; 22 ± 4 versus 76 ± 11%) was significantly decreased in the OZR compared with the LZR. Likewise, the maximal gain was lower in the OZR, as follows: MAP -0.88 ± 0.22 versus -2.26 ± 0.17; HR -0.42 ± 0.18 versus -1.44 ± 0.22 beats min(-1); and NormNA -0.54 ± 0.14 versus -1.65 ± 0.30% mmHg(-1). There was no difference in the mid-point of the baroreflex curve for each variable between the OZR and LZR. However, the minimal values obtained when the baroreceptors were maximally loaded were higher in the OZR (MAP 68 ± 5 versus 53 ± 4 mmHg; HR 455 ± 7 versus 390 ± 13 beats min(-1); and NormNA -19 ± 4 versus -48 ± 8%). Brachial nerve stimulation in the LZR resulted in an upward and rightward resetting of the baroreflex control of MAP and RSNA, and abolished baroreflex control of HR. The baroreflex control of RSNA in the OZR during BNS was further attenuated and reset upwards and to the right, while the HR response was abolished. With respect to MAP, the baroreflex curve reset upwards and to the right to a point comparable with the LZR during BNS. These data show that there is an attenuated baroreflex control in the OZR and that the ability to reset to higher arterial pressure during somatic afferent nerve stimulation is similar to that in the LZR.

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-Methyl-d-aspartate Receptors with Fast Off-rate by Novel Multifunctional Dimer Bis(propyl)-cognitin

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and gamma-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [(3)H]MK-801 with a K(i) value of 0.27 mum, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.

Complement dependent cytotoxicity (CDC) activity of a humanized anti Lewis-Y antibody: FACS-based assay versus the ‘classical’ radioactive method—Qualification, comparison and application of the FACS-based approach

The fully humanized Lewis-Y carbohydrate specific monoclonal antibody (mAb) IGN311 is currently tested in a passive immunotherapy approach in a clinical phase I trail and therefore regulatory requirements demand qualified assays for product analysis. To demonstrate the functionality of its Fc-region, the capacity of IGN311 to mediate complement dependent cytotoxicity (CDC) against human breast cancer cells was evaluated. The “classical” radioactive method using chromium-51 and a FACS-based assay were established and qualified according to ICH guidelines. Parameters evaluated were specificity, response function, bias, repeatability (intra-day precision), intermediate precision (operator–time different), and linearity (assay range). In the course of a fully nested design, a four-parameter logistic equation was identified as appropriate calibration model for both methods. For the radioactive assay, the bias ranged from −6.1% to −3.6%. The intermediate precision for future means of duplicate measurements revealed values from 12.5% to 15.9% and the total error (β-expectation tolerance interval) of the method was found to be <40%. For the FACS-based assay, the bias ranged from −8.3% to 0.6% and the intermediate precision for future means of duplicate measurements revealed values from 4.2% to 8.0%. The total error of the method was found to be <25%. The presented data demonstrate that the FACS-based CDC is more accurate than the radioactive assay. Also, the elimination of radioactivity and the ‘real-time’ counting of apoptotic cells further justifies the implementation of this method which was subsequently applied for testing the influence of storage at 4 °C and 25 °C (‘stability testing’) on the potency of IGN311 drug product. The obtained results demonstrate that the qualified functional assay represents a stability indicating test method.

Logistic Equations Effectively Model Mammalian Cell Batch and Fed-Batch Kinetics by Logically Constraining the Fit

A four-parameter logistic equation was used to fit batch and fed-batch time profiles of viable cell density in order to estimate net growth rates from the inoculation through the cell death phase. Reduced three-parameter forms were used for nutrient uptake and metabolite/product formation rate calculations. These logistic equations constrained the fits to expected general concentration trends, either increasing followed by decreasing (four-parameter) or monotonic (three-parameter). The applicability of this approach was first verified for Chinese hamster ovary (CHO) cells cultivated in 15-L batch bioreactors. Cell density, metabolite, and nutrient concentrations were monitored over time and used to estimate the logistic parameters by nonlinear least squares. The logistic models fit the experimental data well, supporting the validity of this approach. Further evidence to this effect was obtained by applying the technique to three previously published batch studies for baby hamster kidney (BHK) and hybridoma cells in bioreactors ranging from 100 mL to 300 L. In 27 of the 30 batch data sets examined, the logistic models provided a statistically superior description of the experimental data than polynomial fitting. Two fed-batch experiments with hybridoma and CHO cells in benchtop bioreactors were also examined, and the logistic fits provided good representations of the experimental data in all 25 data sets. From a computational standpoint, this approach was simpler than classical approaches involving Monod-type kinetics. Since the logistic equations were analytically differentiable, specific rates could be readily estimated. Overall, the advantages of the logistic modeling approach should make it an attractive option for effectively estimating specific rates from batch and fed-batch cultures.

Validation of ELISA for the determination of anti-ricin immunoglobulin G concentration in mouse sera

An enzyme-linked immunosorbent assay (ELISA) for the determination of anti-ricin immunoglobulin G (IgG) concentration in mouse sera was systematically validated. The results obtained throughout the validation process strongly demonstrated that the ELISA was reliable, reproducible, and suitable for its intended use. The assay had a high level of precision within and between runs, was specific for the anti-ricin IgG, and showed no interference with a number of different serum matrices. The assay exhibited excellent accuracy, linearity, and stability. The mean recovery of four test samples with different known concentrations was 100.9 ± 11.3%, 102.7 ± 10.8%, 99.0 ± 7.2%, and 95.9 ± 11.3%, respectively ( n = 10). The mean recovery of the observed anti-ricin IgG concentration of three quality control samples run on 73 plates to their nominal concentrations was 100.1 ± 7.3%, 100.2 ± 5.8%, and 103.7 ± 8.1%; and the coefficient of variation (CV) was 7.3%, 5.8%, and 7.8%, respectively. The back-calculated anti-ricin IgG concentration, %CV, and relative error of seven standards from the calibration curves run in the entire validation study were analyzed ( n = 7 × 73). The results indicated that the four-parameter logistic (4PL) equation, y = ( a − d)/(1 + ( x/ c) b ) + d, provided an accurate representation of a sigmoidal relationship between the measured response and the logarithm of observed concentration of anti-ricin IgG in mouse sera for this ELISA. The lower limit of quantification and upper limit of quantification of the calibration curve were 3.3 ng/ml and 82.8 ng/ml, respectively. The measurable range of the assay would cover all possible anti-ricin IgG concentrations in mouse sera stimulated with a ricin vaccine candidate, when the test sera are measured at a 1:800 starting dilution followed by four additional fourfold serial dilutions.

Different Structural Requirements for the Constitutive and the Agonist-induced Activities of the β2-Adrenergic Receptor

We converted Ser-207, located in helix 5 of the beta2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to G(s) activation, we expressed the mutants in a G alpha(s)-tethered form. GTP exchange in such constructs is restricted to the fused alpha-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.

Metastasis-associated Protein S100A4 Induces Angiogenesis through Interaction with Annexin II and Accelerated Plasmin Formation

Many advanced tumors overexpress and secrete the S100A4 protein that is known to promote angiogenesis and metastasis development. The mechanisms of this effect and the endothelial receptor for S100A4 are both still unknown. Here we report that extracellular S100A4 interacts with annexin II, an endothelial plasminogen co-receptor. Co-localization and direct binding of S100A4 and annexin II were demonstrated, and the binding site was identified in the N-terminal region of annexin II. S100A4 alone or in a complex with annexin II accelerated tissue plasminogen activator-mediated plasminogen activation in solution and on the endothelial cell surface through interaction of the S100A4 C-terminal lysines with the lysine-binding domains of plasminogen. A synthetic peptide corresponding to the N terminus of annexin II prevented S100A4-induced plasmin formation in the endothelial cell culture. Local plasmin formation induced by circulating S100A4 could contribute to tumor-induced angiogenesis and metastasis formation that makes this protein an attractive target for new anti-cancer and anti-angiogenic therapies.