In Situ Detection of nDNA Fragmentation during the Differentiation of Tracheary Elements in Higher Plants

Programmed cell death (pcd) is thought to occur during the autolysis of xylem vessels. Although several ultrastructural aspects of this differentiation process have been characterized, certain key aspects of this process remain unsolved. Here we demonstrate in pea (Pisum sativum) that nuclei of vessel elements undergoing pcd contain fragmented nDNA. This finding may provide evidence for the activation of a DNA degradation mechanism prior to the final disruption of the nucleus that occurs during the autolysis stage of this differentiation process. In situ detection of DNA fragmentation in nuclei of vessel elements undergoing pcd may therefore suggest that this death process involves the activation of a mechanism for DNA degradation, similar to that activated during apoptosis in animal cells. In addition, this differentiation process may serve as a useful positive control for the in situ detection of pcd in other developmental pathways and during the hypersensitive response of plants to avirulent pathogens.     

The roles of a process development group in biopharmaceutical process startup

The transfer of processes for biotherapeutic products into finalmanufacturing facilities was frequently problematic during the 1980's and early 1990's, resulting in costly delays to licensure(Pisano 1997). While plant startups for this class of products can become chaotic affairs, this is not an inherent or intrinsic feature. Major classes of process startup problems have been identified andmechanisms have been developed to reduce their likelihood of occurrence. These classes of process startup problems and resolution mechanisms are the major topic of this article. With proper planning and sufficient staffing, the probably of a smooth process startup for a biopharmaceutical product can be very high – i.e., successful process performance will often beachieved within the first two full-scale process lots in the plant. The primary focus of this article is the role of the Process Development Group in helping to assure this high probability of success.     

A novel regeneration of iron citrate solution by biooxidation of iron-oxidizing bacteria

Liquid phase oxidation process using chelated iron solution is among the most promising techniques for the hydrogen sulfide removal due to its double advantage of waste minimization and resource recovery. Regeneration of chelated iron is a core reaction in this process. Regeneration of chelated iron in acidic solution is very difficult. In this paper, a novel regeneration of iron citrate in acidic solution by biooxidation of iron-oxidizing bacteria was reported firstly. By using such a process, the influence of iron-oxidizing bacteria on the regeneration rate was investigated. The results demonstrated the regeneration rate with the new technology was increased significantly. The process may contribute to the biooxidation of iron-oxidizing bacteria. Application of this novel process increased the regeneration rate under the optimum conditions, suggesting the iron citrate regeneration process may be a feasible and economical method in application.     

Thermodynamic analysis of lignocellulosic biofuel production via a biochemical process: guiding technology selection and research focus

The aim of this paper is to present an exergy analysis of bioethanol production process from lignocellulosic feedstock via a biochemical process to asses the overall thermodynamic efficiency and identify the main loss processes. The thermodynamic efficiency of the biochemical process was found to be 35% and the major inefficiencies of this process were identified as: the combustion of lignin for process heat and power production and the simultaneous scarification and co-fermentation process accounting for 67% and 27% of the lost exergy, respectively. These results were also compared with a previous analysis of a thermochemical process for producing biofuel. Despite fundamental differences, the biochemical and thermochemical processes considered here had similar levels of thermodynamic efficiency. Process heat and power production was the major contributor to exergy loss in both of the processes. Unlike the thermochemical process, the overall efficiency of the biochemical process largely depends on how the lignin is utilized.     

Lag time in microbe growth as an age-dependent branching process with two phase types

We study a two-type, age-dependent branching process in which the branching probabilities of one of the types may vary with time. Specifically this modification of the Bellman-Harris process starts with a Type I particle which may either die or change to a Type II particle depending upon a time varying probability. A Type II particle may either die or reproduce with fixed probabilities but may not return to a particle of Type I. In this way the process models the lag phenomenon observed in microbe growth subsequent to transfer to a new culture medium while the organism is adapting to its new environment. We show that if the mean reproduction rate of Type II particles exceeds 1, then the population size grows exponentially. Further the extinction probability for this process is related to that of the Bellman-Harris process. Finally the governing equations are solved for several choices of the growth parameters and the solutions are graphically displayed showing that a wide variety of behavior can be modeled by this process.     

Trypanosomatids: mitochondrial RNA editing

RNA editing is a genetic regulatory process that was recently discovered in the mitochondrion of trypanosomatid parasites. It alters mRNA by the addition and deletion of uridines. Much remains to be learned about this process, including identification and characterization of the macromolecules that catalyze and regulate this process and the mechanism of editing.     

Protein tyrosine nitration: A new challenge in plants

Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO₂) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress.     

A systematic approach for scale-down model development and characterization of commercial cell culture processes

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.     

Efficient optimization of time-varying inputs in a fed-batch cell culture process using design of dynamic experiments

In cell culture process development, we rely largely on an iterative, one-factor-at-a-time procedure based on experiments that explore a limited process space. Design of experiments (DoE) addresses this issue by allowing us to analyze the effects of process inputs on process responses systematically and efficiently. However, DoE cannot be applied directly to study time-varying process inputs unless an impractically large number of bioreactors is used. Here, we adopt the methodology of design of dynamic experiments (DoDE) and incorporate dynamic feeding profiles efficiently in late-stage process development of the manufacture of therapeutic monoclonal antibodies. We found that, for the specific cell line used in this article, (1) not only can we estimate the effect of nutrient feed amount on various product attributes, but we can also estimate the effect, develop a statistical model, and use the model to optimize the slope of time-trended feed rates; (2) in addition to the slope, higher-order dynamic characteristics of time-trended feed rates can be incorporated in the design but do not have any significant effect on the responses we measured. Based on the DoDE data, we developed a statistical model and used the model to optimize several process conditions. Our effort resulted in a tangible improvement in productivity—compared with the baseline process without dynamic feeding, this optimized process in a 200-L batch achieved a 27% increase in titer and > 92% viability. We anticipate our application of DoDE to be a starting point for more efficient workflows to optimize dynamic process conditions in process development.     

An overview of mechanistic modeling of liquid chromatography

Abstract

Liquid chromatography is considered to be the bottleneck for purification of therapeutic proteins. Development and optimization of chromatography process is a cumbersome activity due to the increasing complexities in the types and content of impurities present in the high product titer cell culture harvest obtained from the upstream processing. Further, regulatory expectations are continuously rising with the recent initiatives of quality by design and process analytical technology expecting the manufacturer to have a deeper understanding of the process and the product. Mechanistic modeling is one approach to gain this deeper understanding of a process step. It involves modeling of the underlying physicochemical processes. A well calibrated model with acceptable predictability can be very effective in both process optimization and process characterization activities. In this paper we provide an overview of mechanistic modeling of liquid chromatography. We discuss the various components that such a model entails and also presents the status quo of this area.     

Equilibrium protein folding-unfolding process involving multiple intermediates

Mathematical models for the protein folding-unfolding process involving multiple intermediates have been derived. Computer fitting of the experimental data to this model generates various thermodynamic parameters for the folding-unfolding process. In this way, the complex folding-unfolding process of the multi-domain proteins can be analysed in a quantitative way. The application of the folding-unfolding model involving seven stages in human placental alkaline phosphatase is described. These authors contributed equally to this paper.     

On some Markov models of certain interacting populations

We give a stochastic foundation to the Volterra prey-predator population in the following case. We take Volterra's predator equations and let a free host birth and death process support the evolution of the predator population. The purpose of this article is to present a rigorous population sample path construction of this interacted predator process and study the properties of this interacted process. The constructions yields a strong Markov process. The existence of steady-state distribution for the interacted predator process means the existence of equilibrium population level. We find a necessary and sufficient condition for the existence of a steady-state distribution. Next we see that if the host process possesses a steady-state distribution, so does the interacted predator process and this distribution satisfies a difference equation. For special choices of the auto death and interaction parametersa andb of the predator, whenever the host process visits the particular statea ^*=a/b the predator takes rest (saturates) from its evolution. We find the probability of asymptotic saturating of the predator.     

Dynamical analysis of yeast protein interaction network during the sake brewing process

Proteins interact with each other for performing essential functions of an organism. They change partners to get involved in various processes at different times or locations. Studying variations of protein interactions within a specific process would help better understand the dynamic features of the protein interactions and their functions. We studied the protein interaction network of Saccharomyces cerevisiae (yeast) during the brewing of Japanese sake. In this process, yeast cells are exposed to several stresses. Analysis of protein interaction networks of yeast during this process helps to understand how protein interactions of yeast change during the sake brewing process. We used gene expression profiles of yeast cells for this purpose. Results of our experiments revealed some characteristics and behaviors of yeast hubs and non-hubs and their dynamical changes during the brewing process. We found that just a small portion of the proteins (12.8 to 21.6%) is responsible for the functional changes of the proteins in the sake brewing process. The changes in the number of edges and hubs of the yeast protein interaction networks increase in the first stages of the process and it then decreases at the final stages.     

Are evolutionary rates really variable?

Summary Langley and Fitch (1974, 1976) have shown that the pattern of nucleotide substitutions in proteins is inconsistent with a Poisson process with constant rate. From this they conclude that the rate is temporally heterogeneous. It is pointed out in this note that a process which is temporally homogeneous but not a Poisson process is compatible with the data if the coefficient of variation of the time between substitutions is around 1.63. Furthermore, theoretical analysis of samples from neutral phylogenies shows that these samples should not appear to be samples from a Poisson process, but should deviate from a Poisson process in the same direction, though perhaps not to the same extent, as do the data.     

Autophagy pathway: Cellular and molecular mechanisms

Macroautophagy/autophagy is an essential, conserved self-eating process that cells perform to allow degradation of intracellular components, including soluble proteins, aggregated proteins, organelles, macromolecular complexes, and foreign bodies. The process requires formation of a double-membrane structure containing the sequestered cytoplasmic material, the autophagosome, that ultimately fuses with the lysosome. This review will define this process and the cellular pathways required, from the formation of the double membrane to the fusion with lysosomes in molecular terms, and in particular highlight the recent progress in our understanding of this complex process.     

Red blood cell aggredation: mechanisms, adrenergic regulation

Red blood cell aggregation is a complex multiple-factor process and exerts a substantial effect on realization of basic blood function: oxygen transport. Insufficient knowledge about mechanisms of erythrocyte aggregation in normal conditions and, especially, in pathological conditions, complicates the control and correction of possible negative consequences of this process. On addition, red blood cell aggregation process is a suitable model for the elucidation of basic patterns of intercellular interactions. The paper presents contemporary data on mechanisms of erythrocyte aggregation and contribution of plasma and membrane erythrocyte properties to this process. Currently available data on intracellular signal pathways are discussed.     

Recent developments in chromatographic purification of biopharmaceuticals

Over the last several decades, researchers have time and again proposed use of non-chromatographic methods for processing of biotherapeutic products. However, chromatography continues to be the backbone of downstream processing, particularly at process scale. There are many reasons for this, critical ones being the unparalleled scalability, robustness, and selectivity that process chromatography offers over its peers. It is no surprise then that process chromatography has been a topic of major developments in resin matrix, ligand chemistry, modalities, high throughput process development, process modelling, and approaches for control. In this review, we attempt to summarize major developments in the above-mentioned areas. Greater significance has been given to advancements in the last 5 years (2013–2017).     

Glucosamine oligomers: 4. Solid state-crystallization and sustained dissolution.

When glucosamine oligomers are stored in the solid state they undergo a process of crystallization. The extent to which this occurs depends on whether the samples are isolated in the -NH3+ or -NH2 form, on the storage time, and on the degree of polymerization of the isolated oligomer. The allomorph obtained by this process seems to correspond to the so-called 'tendon-chitosan'. Dissolution of such aged oligomer samples gives rise to a process of dissociation of the associated chains in the crystal, leading to the establishment of a pseudo-equilibrium between single and associated oligomer chains and hence the simultaneous presence of the 'monomeric', 'dimeric', 'trimeric', etc., forms of the oligomer. The phenomenon cannot be attributed to a process of aggregation in solution. The effects of various parameters on this behaviour have been investigated.     

Rhodopsin-detergent micelles aggregate upon activation of cyclic guanosine monophosphate phosphodiesterase

In the presence of G protein and phosphodiesterase, GTP induces aggregation of phospholipid-free rhodopsin-detergent micelles or rhodopsin reconstituted in phospholipid vesicles. The net electrical charge of the vesicle is not critical to the aggregation process since this phenomenon is not altered by reconstitution with phospholipids with different charge. The aggregation process is observed by monitoring changes in the light-scattering properties of the detergent micelles or vesicle suspension and by phase-contrast microscopy. The lowest light intensity which triggers the aggregation process and concomitant light-scattering changes in a rhodopsin-detergent micellar suspension bleaches 6% rhodopsin. Under these conditions, the signal saturates at 30% rhodopsin bleaching. The aggregation process appears likely to depend on the protein-protein interaction, and the presence of a disk membrane is not necessary for this process.