Quorum sensing involvement in response surface methodology for optimisation of sclerotiorin production by Penicillium sclerotiorum in shaken flasks and bioreactors

Sclerotiorin, an azaphilone produced by some filamentous fungi including Penicillium sclerotiorum, is a pigment with variety of biological activities including lipoxygenase inhibition, reduction of cholesterol levels, and anti-cancer properties. Sclerotiorin has potential use in pharmaceutical as well as food industries. In this context, the purpose of this study was to provide a simple and robust procedure for optimised production of sclerotiorin by P. sclerotiorum using a central composite design developed through response surface methodology (RSM) and to identify the molecule(s) involved in the signalling mechanism in P. sclerotiorum. The optimisation of sclerotiorin production was carried out using RSM in shaken flasks and the obtained results were then replicated using a 2-L stirred tank bioreactor. Penicillium sclerotiorum ethyl acetate culture extract was analysed using thin layer chromatography (TLC) and potential signalling molecules were identified using Gas chromatography-mass spectrometry (GC-MS). The experimental studies suggested an increase in the sclerotiorin production by 2.1-fold and 2.2-fold in shaken flasks and stirred tank bioreactors respectively. Further analysis of P. sclerotiorum ethyl acetate culture extract reported the presence of ricinoleic acid, an oxylipin, belonging to a family of signalling molecules tentatively involved in the enhancement of sclerotiorin production. This paper has highlighted the positive effect of the optimal supplementation of P. sclerotiorum culture extracts for enhanced production of sclerotiorin. It has also examined potential molecules involved in the signalling mechanism in P. sclerotiorum culture extract for the overproduction of sclerotiorin.     

Current status of the management of fall webworm,Hyphantria cunea: Towards the integrated pest management development

The fall‐webworm (FWW), Hyphantria cunea, is a highly polyphagous insect pest that is native to North America and distributed in different countries around the world. To manage this insect pest, various control methods have been independently evaluated in the invaded areas. Some of the control methods have been limited to the laboratory and need further study to verify their effectiveness in the field. On the other hand, currently, integrated pest management (IPM) has become a promising ecofriendly insect pest management option to reduce the adverse effect of insecticides on the environment. The development of an IPM for an insect pest must combine different management options in a compatible and applicable manner. In the native areas of the insect pests, there are some recommended management options. However, to date, there is no IPM for the management of the FWW in the newly invaded areas. Therefore, to develop an IPM for this insect pest, compilation of effective management option information is the first step. Thus, believing in the contribution of an IPM to the established management strategies, the chemical, biological, natural enemy, sex pheromone, and molecular studies regarding this insect were reviewed and potential future research areas were delineated in this review study. Therefore, using the currently existing management options, IPM development for this insect pest should be the subject of future research in the newly invaded areas.     

Cannabis sativa L. photoautotrophic micropropagation: a powerful tool for industrial scale in vitro propagation

In Vitro Cellular & Developmental Biology - Plant - Global demands for an in vitro culture of cannabis have never been more sought after as countries shift their paradigm towards legalization....     

Phenotyping of circulating CD8⁺ T cell subsets in human cutaneous leishmaniasis

Recovery from CL is usually accompanied with long-lasting protection and induction of strong immune response. The phenotypes, generation and maintenance of central (=T_CM) and effector (=T_EM) memory T cell subsets in human leishmaniasis are not well known. Profile of T cell subsets were analyzed on peripheral CD8⁺ T cells from volunteers with history of cutaneous leishmaniasis (HCL).In HCL and control groups, mean frequencies of CCR7⁺CD45RA⁺CD8⁺ naïve and CCR7^?CD45RA^?CD8⁺ T_EM cells were higher than other subsets before culture, but after stimulation with soluble Leishmania antigen, the frequency of naïve T cells was significantly decreased and the frequency of T_EM cells was significantly increased. T_EM phenotype composed the highest portion of proliferating Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE)-dim population which was significantly higher in HCL volunteers than in control group. Stimulation of isolated CD8⁺ memory T cells, but not naïve T cells, from HCL volunteers induced a significantly higher IFN-γ production compared with that of healthy controls. Intracellular IFN-γ staining provided the same result.Memory population is shown to be responsible for Leishmania-induced IFN-γ production. Leishmania-reactive proliferating T_EM cells were identified as the most frequent subset which may play a role in recall immune response and protection against Leishmania infection.     

Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran

The apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is a prime malaria asexual blood-stage vaccine candidate. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. The extracellular region of P. falciparum AMA-1 (PfAMA-1) consists of three domains (I-III), of which the domain I is the most diverse region of this antigen. The objective of our study was to investigate and analyze the extent of genetic diversity and the effectiveness of natural selection at the AMA-1 domain I of P. falciparum in isolates from Iran. A fragment of ama-1 gene spanning domain I was amplified by nested PCR from 48 P. falciparum isolates collected from two major malaria endemic areas of Iran during 2009 to August 2010 and sequenced. Genetic polymorphism and statistical analyses were performed using DnaSP and MEGA software packages. Analysis of intrapopulation diversity revealed relatively high nucleotide and haplotype diversity at the PfAMA-1 domain I of Iranian isolates. Neutrality tests provided strong evidence of positive natural selection acting on the sequenced gene region. The findings also demonstrated that, in addition to natural selection, intragenic recombination may contribute to the diversity observed at the domain I. The results obtained will have significant implications in the design and the development of an AMA-1-based vaccine against falciparum malaria.     

A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Step change in the efficiency of centrifugation through cell engineering: co‐expression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock

Cell engineering to enable step change improvements in bioprocessing can be directed at targets other than increasing product titer. The physical properties of the process suspension such as viscosity, for example, have a major impact on various downstream processing unit operations. The release of chromosomal DNA during homogenization of Escherichia coli and its influence on viscosity is well‐recognized. In this current article we demonstrate co‐expression of Staphylococcus aureus nuclease in E. coli to reduce viscosity through auto‐hydrolysis of nucleic acids. Viscosity reduction of up to 75% was achieved while the particle size distribution of cell debris was maintained approximately constant (d₅₀ = 0.5–0.6 µm). Critically, resultant step change improvements to the clarification performance under disc‐stack centrifugation conditions are shown. The cell‐engineered nuclease matched or exceeded the viscosity reduction performance seen with the addition of exogenous nuclease removing the expense and validation issues associated with such additions to a bioprocess. The resultant material dramatically altered performance in scale‐down mimics of continuous disc‐stack centrifugation. Laboratory scale data indicated that a fourfold reduction in the settling area of a disc‐stack centrifuge can be expected due to a less viscous process stream achieved through nuclease co‐expression with a recombinant protein. Biotechnol. Bioeng. 2009; 104: 134–142 © 2009 Wiley Periodicals, Inc.     

Spontaneous activity of dopaminergic retinal neurons

Dopaminergic local circuit neurons in the retina (DA cells) show robust, spontaneous, tetrodotoxin-sensitive pacemaking. To investigate the mechanism underlying this behavior, we characterized the sodium current and a subset of the potassium currents in the cells in voltage-clamp experiments. We found that there is a persistent component of the sodium current in DA cells which activates at more depolarized potentials than the transient component of the current. The transient component was completely inactivated at -50 mV, but DA cells remained able to fire spontaneous action potentials when potassium channels were partially blocked and the membrane potential remained above -40 mV. Based on these electrophysiological data, we developed a reduced computer model that reproduced the major features of DA cells. In simulations at the physiological resting potential, the persistent component of the sodium current was both necessary and sufficient to account for spontaneous activity, and the major contribution of the transient component of the sodium current was to initiate the depolarization of the model cell during the interspike interval. When tonic inhibition was simulated by lowering the input impedance of the model cell, the transient component played a larger role.     

The influence of small oligosaccharides on the immune system

In this study, oligosaccharides known to enhance the synthesis of penicillin by Penicillium chrysogenum have been presented to human immune cells and their effect measured. In addition a range of commercially available oligosaccharides have been tested. Results obtained indicate that oligosaccharides with a degree of polymerisation greater than 6 and with a tendency to form helical structures are most effective at influencing the immune system as measured by the production of reactive oxidising species. Laminariheptaose has been shown to increase reactive oxidising species production by up to 25%, whilst mannan-oligosaccharides with a DP of 6 to 7 decrease production by up to 44%. These and other results show that the immune system can recognise subtle differences in oligosaccharides and that these oligosaccharides could potentially be used to modulate the immune response.