A dynamic model linking cell growth to intracellular metabolism and extracellular by-product accumulation

Mathematical modeling of animal cell growth and metabolism is essential for the understanding and improvement of the production of biopharmaceuticals. Models can explain the dynamic behavior of cell growth and product formation, support the identification of the most relevant parameters for process design, and significantly reduce the number of experiments to be performed for process optimization. Few dynamic models have been established that describe both extracellular and intracellular dynamics of growth and metabolism of animal cells. In this study, a model was developed, which comprises a set of 33 ordinary differential equations to describe batch cultivations of suspension AGE1.HN.AAT cells considered for the production of α1-antitrypsin. This model combines a segregated cell growth model with a structured model of intracellular metabolism. Overall, it considers the viable cell concentration, mean cell diameter, viable cell volume, concentration of extracellular substrates, and intracellular concentrations of key metabolites from the central carbon metabolism. Furthermore, the release of metabolic by-products such as lactate and ammonium was estimated directly from the intracellular reactions. Based on the same set of parameters, this model simulates well the dynamics of four independent batch cultivations. Analysis of the simulated intracellular rates revealed at least two distinct cellular physiological states. The first physiological state was characterized by a high glycolytic rate and high lactate production. Whereas the second state was characterized by efficient adenosine triphosphate production, a low glycolytic rate, and reactions of the TCA cycle running in the reverse direction from α-ketoglutarate to citrate. Finally, we show possible applications of the model for cell line engineering and media optimization with two case studies.     

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.     

Effectiveness of Partial Sedation to Reduce Stress in Captured Mule Deer

Information garnered from the capture and handling of free-ranging animals helps advance understanding of wildlife ecology and can aid in decisions on wildlife management. Unfortunately, animals may experience increased levels of stress, injuries, and death resulting from captures (e.g., exertional myopathy, trauma). Partial sedation is a technique proposed to alleviate stress in animals during capture, yet efficacy of partial sedation for reducing stress and promoting survival post-capture remains unclear. We evaluated the effects of partial sedation on physiological, biochemical, and behavioral indicators of acute stress and probability of survival post-capture for mule deer (Odocoileus hemionus) that were captured via helicopter net-gunning in the eastern Greater Yellowstone Ecosystem, Wyoming, USA. We administered 10–30 mg of midazolam and 15 mg of azaperone intramuscularly (IM) to 32 mule deer in 2016 and 53 mule deer in 2017, and maintained a control group (captured but not sedated) of 38 mule deer in 2016 and 54 mule deer in 2017. To evaluate indicators of acute stress, we measured heart rate, blood-oxygen saturation, body temperature, respiration rate, and levels of serum cortisol. We recorded number of kicks and vocalizations of deer during handling and evaluated behavior during release. We also measured levels of fecal glucocorticoids as an indicator of baseline stress. Midazolam and azaperone did not reduce physiological, biochemical, or behavioral indicators of acute stress or influence probability of survival post-capture. Mule deer that were administered midazolam and azaperone, however, were more likely to hesitate, stumble or fall, and walk during release compared with individuals in the control group, which were more likely to trot, stot, or run without stumbling or falling. Our findings suggest that midazolam (10–30 mg IM) and azaperone (15 mg IM) may not yield physiological or demographic benefits for captured mule deer as previously assumed and may pose adverse effects that can complicate safety for captured animals, including drug-induced lethargy. Although we failed to find efficacy of midazolam and azaperone as a method for reducing stress in captured mule deer, the efficacy of midazolam and azaperone or other combinations of partial sedatives in reducing stress may depend on the dose of tranquilizer, study animal, capture setting, and how stress is defined. © 2020 The Wildlife Society.     

Phosphorylation and Degradation of Tomosyn-2 De-represses Insulin Secretion

The abundance and functional activity of proteins involved in the formation of the SNARE complex are tightly regulated for efficient exocytosis. Tomosyn proteins are negative regulators of exocytosis. Tomosyn causes an attenuation of insulin secretion by limiting the formation of the SNARE complex. We hypothesized that glucose-dependent stimulation of insulin secretion from β-cells must involve reversing the inhibitory action of tomosyn. Here, we show that glucose increases tomosyn protein turnover. Within 1 h of exposure to 15 mm glucose, ∼50% of tomosyn was degraded. The degradation of tomosyn in response to high glucose was blocked by inhibitors of the proteasomal pathway. Using ³²P labeling and mass spectrometry, we showed that tomosyn-2 is phosphorylated in response to high glucose, phorbol esters, and analogs of cAMP, all key insulin secretagogues. We identified 11 phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (Ser → Asp) and loss-of-function (Ser → Ala) mutants. The Ser → Asp mutant had enhanced protein turnover compared with the Ser → Ala mutant and wild type tomosyn-2. Additionally, the Ser → Asp tomosyn-2 mutant was ineffective at inhibiting insulin secretion. Using a proteomic screen for tomosyn-2-binding proteins, we identified Hrd-1, an E3-ubiquitin ligase. We showed that tomosyn-2 ubiquitination is increased by Hrd-1, and knockdown of Hrd-1 by short hairpin RNA resulted in increased abundance in tomosyn-2 protein levels. Taken together, our results reveal a mechanism by which enhanced phosphorylation of a negative regulator of secretion, tomosyn-2, in response to insulin secretagogues targets it to degradation by the Hrd-1 E3-ubiquitin ligase.     

Assessment of Prosthesis Alignment after Revision Total Knee Arthroplasty Using EOS 2D and 3D Imaging: A Reliability Study

Introduction

A new low-dose X-ray device, called EOS, has been introduced for determining lower-limb alignment in 2D and 3D. Reliability has not yet been assessed when using EOS on lower limbs containing a knee prosthesis. Therefore purpose of this study was to determine intraobserver and interobserver reliability of EOS 2D and 3D knee prosthesis alignment measurements after revision total knee arthroplasty (rTKA).

Methods

Forty anteroposterior and lateral images of 37 rTKA patients were included. Two observers independently performed measurements on these images twice. Varus/valgus angles were measured in 2D (VV2D) and 3D (VV3D). Intraclass correlation coefficients and the Bland and Altman method were used to determine reliability. T-tests were used to test potential differences.

Results

Intraobserver and interobserver reliability were excellent for VV2D and VV3D. No significant difference or bias between the first and second measurements or the two observers was found. A significant mean and absolute difference of respectively 1.00° and 1.61° existed between 2D and 3D measurements.

Conclusions

EOS provides reliable varus/valgus measurements in 2D and 3D for the alignment of the knee joint with a knee prosthesis. However, significant differences exist between varus/valgus measurements in 2D and 3D.     

Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus.

An animal model for the heterosexual transmission of human immunodeficiency virus (HIV) was developed by the application of simian immunodeficiency virus (SIV) onto the genital mucosas of both mature and immature, male and female rhesus macaques. Virus preparations were infused into the vaginal vaults or the urethras (males) of the animals through a soft plastic pediatric nasogastric feeding tube. The macaques that were infected by this route (six males and nine females) developed SIV-specific antibodies, and SIV was isolated from peripheral mononuclear cells of all seropositive animals. One male and one female infected by this route developed severe acquired immunodeficiency syndrome-like disease with retroviral giant-cell pneumonia. As few as two inoculations of cell-free SIV containing 50 50% tissue culture infective doses induced persistent viremia. Cell-free virus preparations were capable of producing infection by the genital route. Much higher doses of virus were required to transmit SIV by this route than are required for transmission by intravenous inoculation. Thus, it appears that the mucous membranes of the genital tract act as a barrier to SIV infection. Spermatozoa and seminal plasma were not required for the genital transmission of SIV. Rarely, SIV was recovered from mononuclear cells in semen and vaginal secretions. The SIV-rhesus macaque model is suitable for assessing the role of cofactors in heterosexual transmission of HIV and will be useful for testing the effectiveness of spermicides, pharmacologic agents, and vaccines in preventing the heterosexual transmission of HIV.     

Circulating immune complexes and complement C3 and C4 levels in a selected group of patients with rhinitis in Lebanon

BACKGROUND: A number of reports indicate that circulating immune complexes (CIC) and activation of the complement system contribute to the pathogenesis of Type I allergy. The aim of this study was to investigate the status of CIC in 113 patients with rhinitis in Lebanon and determine complement components C3 and C4 serum levels in the CIC-positive patients. Serum specific IgE antibodies were previously detected and reported in 74 of the 113 patients. METHODS: CIC were detected by polyethylene glycol precipitation and serum C3 and C4 levels quantified by radial immunodiffusion. RESULTS: CIC was positive in 20 of the specific IgE-positive and 13 of the specific IgE-negative patients. C3 and C4 levels were within the normal range in all the 33 CIC-positive patients. CONCLUSIONS: The antibody class that constitutes the complexes does not seem to be IgG or IgM. Moreover, complement activation does not seem to be involved in the allergic reaction since both C3 and C4 levels were normal in all patients. The role of these complexes, if any, in the pathogenesis of rhinitis is yet to be determined.