Production of entomopathogenic nematodes in submerged monoxenic culture: A review

Monoxenic liquid culture is the most suitable technology for scaling up to industrial production of entomopathogenic nematodes (EPNs); however, the variability of the yield production remains a current problem in the process. The aim of this study was to analyze the parameters and criteria for EPN production in liquid culture based on scientific and technological knowledge from the last two decades. While experimental research has permitted the yield production of Heterorhabditis bacteriophora (362 × 10³ infective juveniles [IJs]/ml) and Steinernema carpocapsae (252 × 10³ IJs/ml), simultaneously, theoretical approaches have contributed to the understanding of the culture process, based on biological parameters of the bacterium–nematode complex and hydrodynamic and rheological parameters of the complex gas–liquid–solid system. Under this interdisciplinary research approach, bioprocess and biosystem engineering can contribute to design the various control strategies of the process variables, increase the productivity, and reduce the variability that until now distinguishes the in vitro production of EPNs by the liquid culture.     

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.     

Pay attention to membrane tension: Mechanobiology of the cell surface

The cell surface is a mechanobiological unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could determine whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes.     

Plant-derived smoke solutions stimulate the growth of Lycopersicon esculentum roots in vitro

Aqueous extracts of smoke, derived from Themeda triandra, a fire-climax grass, and Passerina vulgaris, a fynbos plant, stimulated the growth of primary root sections of tomato roots in suspension culture. The optimal dilution for both extracts was 1:2000. Several of the fractions obtained from TLC separation of the Themeda and the Passerina extracts significantly promoted primary root growth. The auxins naphthaleneacetic acid (NAA), indolebutyric acid (IBA) and indoleacetic acid (IAA) were found to stimulate the growth of the primary root axis, with IAA and NAA significantly promoting lateral root number. Similarly, the naturally occurring cytokinins, zeatin and its derivatives (zeatin-O-glucoside; dihydrozeatin and zeatin riboside) stimulated primary root length. Zeatin and dihydrozeatin promoted secondary root growth, but only at very low concentrations.     

A method for the harvest,culture, and characterization of human adult atrial myocardial cells: Correlation with age of donor

Summary Myocardial cell culture methods are now well established for animal and fetal human tissue. We present here a method for harvesting and culturing adult human atrial myocardiocytes. Cells are obtained from fresh atrial tissue normally discarded after being removed to cannulate the right atrium during open heart surgery. The atrial tissue is minced and then digested using collagenase. The single cell suspension is initially cultured in serum-containing growth medium, then transferred to defined medium, selective for myocardial cell growth. The cells are characterized by immunoperoxidase stains and transmission electron microscopy. The cultured cells stain positive for myoglobin, whereas control cultured fibroblasts and endothelial cells do not. Electron microscopy shows the presence of numerous myofibrils, Z-bodies, pleomorphic mitochondria, and secretory granules. The chronological age of the donor was an important factor in culturing the adult tissue, the younger tissue correlated with a higher success rate. This method provides a means for in vitro study of human adult myocardial cells and provides guidelines for appropriate atrial tissue to use.     

Continuous bioprocessing: The real thing this time?: 10th Annual bioProcessUK Conference,December 3–4, 2013, London,UK

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10^th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.     

Adaptation to salinity at the plant cell level

Summary Various mechanisms of adaptation of plant cells to salinity are reviewed: (1) protection of enzymes and maintenance of turgor by organic solutes; (2) prevention of ion toxicity by compartmentation; and (3) energization of solute transport by the proton pump. All these mechanisms seem to play a role in adaptation. The particular advantages of using salt-adapted cells in suspension culture to identify mechanisms of adaptation are pointed out.     

Detection of cellular responses to toxicants by dielectrophoresis

The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C_mem) of the cells decreased while the specific membrane conductance (G_mem) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.     

Dictyosome vesicle production and plasma membrane turnover in auxin-stimulated outer epidermal cells of coleoptile segments fromAvena sativa (L.)

Summary Dark grown coleoptile segments were floated on solutions of IAA alone and of IAA and the secretion inhibitors cytochalasin and monensin. The secretion inhibitors prevented normal elongation of the tissue segments, the monensin inhibition being virtually complete while cytochalasin gave a 40% reduction over the first six hours with little further further elongation in the following 18 hours. Vesicle production was assessed in outer epidermal cells after 6 hours of IAA-stimulated elongation using the vesicle accumulation method following a cytochalasin-block of vesicle transport. The results were compared with the area of plasma membrane required to enable cell elongation to proceed at the observed rate. The area of vesicle membrane delivered to the cell surface exceeded this requirement to such an extent that at least 65% of the delivered membrane must be recycled back into the cytoplasm. Expressed in terms of the whole cell, the plasma membrane turnover rate was found to be once every 200 minutes. It is concluded that limitation of elongation by secretion inhibitors is more likely to reflect a requirement for the vesicle contents than the vesicle membrane. These results are compared with those obtained from other secretory systems using a similar approach.Abbreviations IAA indole acetic acid - DMSO dimethyl sulphoxide - D dictyosome - ER endoplasmic reticulum - V vesicle     

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.]

Adventitious bud formation on Sitka spruce [ Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.