Bioconversion of hydrophobic compounds in a continuous closed-gas-loop bioreactor: feasibility assessment and epoxide production

Microorganisms can be used as catalysts to produce organic compounds in a highly chemo-, regio- and enantioselective manner, and whole cells do not require the costly addition of cofactors for redox reactions. However, bioconversions are slow compared to alternative chemical reactions, and the biocatalyst works at its best in an aqueous medium, while the transformations of interest frequently involve compounds with a low-aqueous solubility and that are toxic to microorganisms. This results in low-volumetric productivity in classical bioreactors. The Continuous Closed-Gas-Loop Bioreactor is described here-a reactor system with high productivity, but without the problems associated with two-phase systems, such as an emulsified product stream and phase toxicity. Its working principle is to recirculate a gas phase through a bioreaction compartment and a saturator/absorber module where the product accumulates as a clear organic solution. A wide range of bioconversions should be possible in this set-up, and proof of concept was established for the epoxidation of 1,7-octadiene to (R)-1,2-epoxyoct-7-ene by a native strain of Pseudomonas oleovorans. This reaction represents a group of terminal alkene epoxidations where the bioconversion substrate does not support growth of the microorganism. Practical results at a 5l-scale are presented for this bioconversion for both batch and continuous operation with respect to the aqueous phase, showing continuous stable epoxidation at productivities >14 micromol min(-1) L(-1) (U L(-1)). The results confirm that the metabolism does not allow a simple optimization strategy, because growth and biotransformation substrates compete for the same enzyme sites, and conversely growth on a substrate using this very enzyme system is necessary for longterm bioconversion. Integrated removal of the CO(2) formed via the liquid overflow was estimated from theory and verified in experimental work.     

Differential impact of simultaneous migration on coevolving hosts and parasites

Background  

The dynamics of antagonistic host-parasite coevolution are believed to be crucially dependent on the rate of migration between populations. We addressed how the rate of simultaneous migration of host and parasite affected resistance and infectivity evolution of coevolving meta-populations of the bacterium Pseudomonas fluorescens and a viral parasite (bacteriophage). The increase in genetic variation resulting from small amounts of migration is expected to increase rates of adaptation of both host and parasite. However, previous studies suggest phages should benefit more from migration than bacteria; because in the absence of migration, phages are more genetically limited and have a lower evolutionary potential compared to the bacteria.     

A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

Background  

Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Extreme cutaneous histamine sensitivity with hay fever and increased IgE concentrations in an unselected population.

Cutaneous sensitivity to histamine, responses to prick tests with allergens, and serum IgE concentrations were measured and hay fever assessed by questionnaire in an unselected population to determine whether increased sensitivity to histamine is an independent phenomenon contributing to allergic disorders or may be caused by allergic reactions. Increased cutaneous sensitivity to histamine was strongly associated with an increased number of positive responses to prick tests, high serum concentrations of IgE, and hay fever. This new test is simple, cheap, applicable to schoolchildren, and provides useful information.     

Molecular mechanism of the E99K mutation in cardiac actin (ACTC Gene) that causes apical hypertrophy in man and mouse

We generated a transgenic mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K at 50% of total heart actin and compared it with actin from patients carrying the same mutation. The actin mutation caused a higher Ca(2+) sensitivity in reconstituted thin filaments measured by in vitro motility assay (2.3-fold for mice and 1.3-fold for humans) and in skinned papillary muscle. The mutation also abolished the change in Ca(2+) sensitivity normally linked to troponin I phosphorylation. MyBP-C and troponin I phosphorylation levels were the same as controls in transgenic mice and human carrier heart samples. ACTC E99K mice exhibited a high death rate between 28 and 45 days (48% females and 22% males). At 21 weeks, the hearts of the male survivors had enlarged atria, increased interstitial fibrosis, and sarcomere disarray. MRI showed hypertrophy, predominantly at the apex of the heart. End-diastolic volume and end-diastolic pressure were increased, and relaxation rates were reduced compared with nontransgenic littermates. End-systolic pressures and volumes were unaltered. ECG abnormalities were present, and the contractile response to β-adrenergic stimulation was much reduced. Older mice (29-week-old females and 38-week-old males) developed dilated cardiomyopathy with increased end-systolic volume and continuing increased end-diastolic pressure and slower contraction and relaxation rates. ECG showed atrial flutter and frequent atrial ectopic beats at rest in some ACTC E99K mice. We propose that the ACTC E99K mutation causes higher myofibrillar Ca(2+) sensitivity that is responsible for the sudden cardiac death, apical hypertrophy, and subsequent development of heart failure in humans and mice.     

Molecular mechanisms of induction of antigen-specific allograft tolerance by intranasal peptide administration

We have previously shown that intranasal (i.n.) administration of a single MHC class II-restricted HY peptide to female mice induces tolerance to up to five additional epitopes expressed on test male grafts, a phenomenon known as linked suppression. In this study, we investigated the molecular mechanisms involved both in the induction phase following peptide administration and during linked suppression after grafting. We report that following initial i.n. administration, peptide is widely disseminated and is presented by functionally immature dendritic cells. These fail to cause optimal stimulation of the responding HY-specific CD4(+) T cells that express genes characteristic of regulatory T cells. Following i.n. peptide plus LPS administration, causing immunization, HY-specific CD4(+) T cells express genes characteristic of activated T cells. We further find that following male skin grafting, HY-specific CD8(+) T cells from peptide-treated tolerant mice display both quantitative and qualitative differences compared with similar cells from untreated mice that reject their grafts. In tolerant mice there are fewer HY-specific CD8(+) cells and they express several genes characteristic of exhausted T cells. Furthermore, associated with specific chemokine receptor and integrin expression, HY-specific CD8(+) T cells show more limited migration from the graft draining lymph node into other tissues.     

Crystal Structure of Patatin-17 in Complex with Aged and Non-Aged Organophosphorus Compounds

Patatin is a non-specific plant lipase and the eponymous member of a broad class of serine hydrolases termed the patatin-like phospholipase domain containing proteins (PNPLAs). Certain PNPLA family members can be inhibited by organophosphorus (OP) compounds. Currently, no structural data are available on the modes of interaction between the PNPLAs and OP compounds or their native substrates. To this end, we present the crystal structure of patatin-17 (pat17) in its native state as well as following inhibition with methyl arachidonyl fluorophosphonate (MAFP) and inhibition/aging with diisopropylphosphorofluoridate (DFP). The native pat17 structure revealed the existence of two portals (portal1 and portal2) that lead to its active-site chamber. The DFP-inhibited enzyme underwent the aging process with the negatively charged phosphoryl oxygen, resulting from the loss of an isopropyl group, being within hydrogen-binding distance to the oxyanion hole. The MAFP-inhibited pat17 structure showed that MAFP did not age following its interaction with the nucleophilic serine residue (Ser77) of pat17 since its O-methyl group was intact. The MAFP moiety is oriented with its phosphoryl oxygen in close proximity to the oxyanion hole of pat17 and its O-methyl group located farther away from the oxyanion hole of pat17 relative to the DFP-bound state. The orientation of the alkoxy oxygens within the two OP compounds suggests a role for the oxyanion hole in stabilizing the emerging negative charge on the oxygen during the aging reaction. The arachidonic acid side chain of MAFP could be contained within portals 1 or 2. Comparisons of pat17 in the native, inhibited, and aged states showed no significant global conformational changes with respect to their Cα backbones, consistent with observations from other α/β hydrolases such as group VIIA phospholipase A2.     

A novel approach to achieving modular retrovirus clearance for a parvovirus filter

Viral filtration is routinely incorporated into the downstream purification processes for the production of biologics produced in mammalian cell cultures (MCC) to remove potential viral contaminants. In recent years, the use of retentive filters designed for retaining parvovirus (～20 nm) has become an industry standard in a conscious effort to further improve product safety. Since retentive filters remove viruses primarily by the size exclusion mechanism, it is expected that filters designed for parvovirus removal can effectively clear larger viruses such as retroviruses (～100 nm). In an attempt to reduce the number of viral clearance studies, we have taken a novel approach to demonstrate the feasibility of claiming modular retrovirus clearance for Asahi Planova 20N filters. Porcine parvovirus (PPV) and xenotropic murine leukemia virus (XMuLV) were co‐spiked into six different feedstreams and then subjected to laboratory scale Planova 20N filtration. Our results indicate that Planova 20N filters consistently retain retroviruses and no retrovirus has ever been detected in the filtrates even when significant PPV breakthrough is observed. Based on the data from multiple in‐house viral validation studies and the results from the co‐spiking experiments, we have successfully claimed a modular retrovirus clearance of greater than 6 log₁₀ reduction factors (LRF) to support clinical trial applications in both USA and Europe. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:79–85, 2014