Model Predictive Feeding Rate Control in Conventional and Single-use Lab-scale Bioreactors: A Study on Practical Application

A developed solution for fed-batch process modeling and model predictive control (MPC), facilitating good manufacturing practice (GMP) based on process elaboration, control, and validation, is presented in the paper. The step-by-step evolution of the so-called “golden batch” optimal biomass growth profile and its control during the process is demonstrated. The case study of an advanced fed-batch control was performed on the recombinant E. coli BL21 lab-scale (5.4 L) biomass production process using the conventional stirred tank glass reactor. Additionally, a test experiment for control reproducibility and applicability assessment of the proposed approach was carried out in a single-use stirred tank reactor (5.7 L). Four sequentially performed experiments are demonstrated as an example for desirable feeding profile evolution for E. coli BL21 biomass production in a glucose-limited fed-batch process. Under different initial biomass and glucose conditions, as well as for different reference feeding profiles selected in the explorative experiments, good tracking quality of preset reference trajectories by the MPC system has been demonstrated. Estimated and experimentally measured biomass mean deviations from the preset reference value at the end of the processes were 4.6 and 3.8 %, respectively. Biomass concentration of 93.6 g L–1 (at 24 h) was reached in the most productive run. Better process controllability and safer process run, in terms of avoiding culture overfeeding but still maintaining a sufficiently high growth rate, was suggested for the process with biomass yield of 79.8 g L–1 (at 24 h). Practical recommendations on the approach application and adaptation for fed-batch cultures of interest are provided

Sudesna mjerenja γγ-raspada stanja 166Ho nastalih reakcijom (n,γ)

Levels of 166Ho were studied using thermal and average resonance neutron capture and with the (d,p) and (d,3He) reactions. We have devoted a large effort to the measurements of the γγ-coincidence spectra in the broad energy region 50 - 6243 keV. Based on these data and those of earlier studies, the levels are grouped into 23 rotational bands. Among them are 6 new ones. The results are in good agreement with the semiempirical and quasiparticle-phonon model, where Coriolis and residual interactions are taken into account. Details of model interpretation have been presented in a previously published paper.Proučavali smo stanja 166Ho nastala termičkim i prosječnim rezonantnim uhvatom neutrona, te (d,p) i (d,3He) reakcijama. Uložili smo velik trud u mjerenjima sudesnih γγ-spektara u širokom energijskom području od 50 do 6243 keV. Na osnovi tih i ranijih podataka, stanja su grupirana u 23 rotacijske vrpce. Među njima je i 6 novih. Ishodi su u suglasju s poluempiričkim i kvazičestično-fononskim modelom, uzimajući u obzir Coriolisovo međudjelovanje i rezidualne interakcije. Podrobnosti modelskog tumačenja objavljene u u ranijem članku

Proučavanje 194Ir uhvatom termičkih neutrona I (d, p) reakcijom

Levels of 194Ir were studied using thermal neutron capture reaction. A pair spectrometer was used to measure the high-energy γ-ray spectrum from thermal-neutron capture in enriched 193Ir target over the energy range 4640 - 6100 keV. The low-energy γ-radiation from the reaction was studied with crystal diffraction spectrometers, and conversion electrons were observed with magnetic spectrometers. The high-sensitivity measurements at the Grenoble reactor, evaluated for transition energies up to 500 keV, are compared with lower-sensitivity measurements at the Wuerenlingen and Salaspils reactors. The comparison helped to obtain reliable isotopic identification for a number of 194Ir lines. The multipolarity admixtures for 29 γ-transitions were determined on the basis of conversion lines from different electron subshells. Prompt and delayed γ-γ coincidences were measured using semiconductor and scintillation detectors. The 193Ir(d,p) high-resolution spectra, observed with a magnetic spectrometer, are given. All these data contributed to establishing a detailed level scheme of 194Ir. Additional data and the interpretation of the results in terms of current models will be presented in a forthcoming paper.Proučavala su se stanja u 194Ir reakcijama 193Ir(n, γ) i 193Ir(d, p). Mjerenja uhvata termičkih neutrona načinjena su uz reaktore u Grenoblu, Wuerenlingenu i Salapsisu. Za mjerenja γ-zračenja visoke energije upotrebljavao se spektrometar parova, a za niske energije difraktometar. Konverzijske elektrone se mjerilo magnetskim spektrometrom. Mjerenja reakcije (d, p) visokog razlučivanja izvedena su magnetskim spektrometrom. Usporedbe tih mjerenja omogućile su pouzdano izotopno prepoznavanje prijelaza u 194 Ir, a spektri konverzijskih elektrona i određivanje multipolnosti prijelaza. Dobiveni su podaci osnova sheme raspada 194Ir

Immunological Tolerance to Muscle Autoantigens Involves Peripheral Deletion of Autoreactive CD8+ T Cells

Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4+ T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8+ T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8+ tolerance in SM-Ova mice. We show herein that Ova-specific CD8+ T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8+ T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8+ T cells

Growing old with the immune system: a study of immunosenescence in the zebra finch (Taeniopygia guttata)

Immunosenescence has not received much attention in birds and the few existing studies indicate that the occurrence of immunosenescence and/or its extent may differ between species. In addition, not much information is available on the immunosenescence patterns of different immune parameters assessed simultaneously in both sexes within a single species. The present study reports the results on immunosenescence in innate immunity and both cellular and humoral acquired immunity of both sexes in a captive population of zebra finch (Taeniopygia guttata) using three age groups (approximately 0.2, 2.5 and 5.1 years). Both male and female finches showed an inverse U-shaped pattern in cellular immune function with age, quantified by a PHA response. Males showed stronger responses than females at all ages. In contrast, an increase with age in humoral immunity, quantified through total plasma immunoglobulin Y levels, was found in both sexes. However, our measurements of innate immunity measured through the bacteria-killing ability against Escherichia coli gave inconclusive results. Still, we conclude that both cellular and humoral acquired immunity are susceptible to immunosenescence, and that the sexes differ in cellular immunity

Computational Models of HIV-1 Resistance to Gene Therapy Elucidate Therapy Design Principles

Gene therapy is an emerging alternative to conventional anti-HIV-1 drugs, and can potentially control the virus while alleviating major limitations of current approaches. Yet, HIV-1's ability to rapidly acquire mutations and escape therapy presents a critical challenge to any novel treatment paradigm. Viral escape is thus a key consideration in the design of any gene-based technique. We develop a computational model of HIV's evolutionary dynamics in vivo in the presence of a genetic therapy to explore the impact of therapy parameters and strategies on the development of resistance. Our model is generic and captures the properties of a broad class of gene-based agents that inhibit early stages of the viral life cycle. We highlight the differences in viral resistance dynamics between gene and standard antiretroviral therapies, and identify key factors that impact long-term viral suppression. In particular, we underscore the importance of mutationally-induced viral fitness losses in cells that are not genetically modified, as these can severely constrain the replication of resistant virus. We also propose and investigate a novel treatment strategy that leverages upon gene therapy's unique capacity to deliver different genes to distinct cell populations, and we find that such a strategy can dramatically improve efficacy when used judiciously within a certain parametric regime. Finally, we revisit a previously-suggested idea of improving clinical outcomes by boosting the proliferation of the genetically-modified cells, but we find that such an approach has mixed effects on resistance dynamics. Our results provide insights into the short- and long-term effects of gene therapy and the role of its key properties in the evolution of resistance, which can serve as guidelines for the choice and optimization of effective therapeutic agents

Population mechanics: A mathematical framework to study T cell homeostasis

Unlike other cell types, T cells do not form spatially arranged tissues, but move independently throughout the body. Accordingly, the number of T cells in the organism does not depend on physical constraints imposed by the shape or size of specific organs. Instead, it is determined by competition for interleukins. From the perspective of classical population dynamics, competition for resources seems to be at odds with the observed high clone diversity, leading to the so-called diversity paradox. In this work we make use of population mechanics, a non-standard theoretical approach to T cell homeostasis that accounts for clone diversity as arising from competition for interleukins. The proposed models show that carrying capacities of T cell populations naturally emerge from the balance between interleukins production and consumption. These models also suggest remarkable functional differences in the maintenance of diversity in naïve and memory pools. In particular, the distribution of memory clones would be biased towards clones activated more recently, or responding to more aggressive pathogenic threats. In contrast, permanence of naïve T cell clones would be determined by their affinity for cognate antigens. From this viewpoint, positive and negative selection can be understood as mechanisms to maximize naïve T cell diversity