Monoterpenoid biosynthesis in Saccharomyces cerevisiae

Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.     

Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin

The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.     

Development of a functional skin matrix requires deposition of collagen V heterotrimers

Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.     

Delivery of mengovirus-derived RNA replicons into tumoural liver enhances the anti-tumour efficacy of a peripheral peptide-based vaccine

Hepatocellular carcinoma is a deadly cancer with growing incidence for which immunotherapy is one of the most promising therapeutic approach. Peptide-based vaccines designed to induce strong, sustained CD8+ T cell responses are effective in animal models and cancer patients. We demonstrated the efficacy of curative peptide-based immunisation against a unique epitope of SV40 tumour antigen, through the induction of a strong CD8+ T cell-specific response, in our liver tumour model. However, as in human clinical trials, most tumour antigen epitopes did not induce a therapeutic effect, despite inducing strong CD8+ T cell responses. We therefore modified the tumour environment to enhance peptide-based vaccine efficacy by delivering mengovirus (MV)-derived RNA autoreplicating sequences (MV-RNA replicons) into the liver. The injection of replication-competent RNA replicons into the liver converted partial tumour regression into tumour eradication, whereas non-replicating RNA had no such effect. Replicating RNA replicon injection induced local recruitment of innate immunity effectors (NK and NKT) to the tumour and did not affect specific CD8+ T cell populations or other myelolymphoid subsets. The local delivery of such RNA replicons into tumour stroma is therefore a promising strategy complementary to the use of peripheral peptide-based vaccines for treating liver tumours.     

Phage display as a tool for the directed evolution of enzymes

Since its introduction in 1985, phage display has had a tremendous impact on the discovery of peptides that bind to a variety of receptors, the generation of binding sites within predefined scaffolds, and the creation of high-affinity antibodies without immunization. Its application to enzymology has required the development of techniques that couple enzymatic activity to selection protocols based on affinity chromatography. Here, we describe both indirect methods, using transition-state analogues and suicide substrates, and direct methods, using the ability of active phage-enzymes to transform substrate into product. The methods have been applied to large libraries for mechanistic-based studies and to generate variants with new or improved properties. In addition, such techniques have been successfully used to select catalytic antibodies and improve their catalytic efficiency.     

Polyhydric alcohol protective effect on Rhizomucor miehei lipase deactivation enhanced by pressure and temperature treatment

The influence of polyhydric alcohols (sorbitol, xylitol, erythritol, glycerol) on the thermal stability of Rhizomucor miehei lipase has been studied at high hydrostatic pressure (up to 500 MPa). In the absence of additives, a protective effect (PE) (the ratio between the residual activities determined at 480 MPa for the enzyme in the presence or absence of polyhydric alcohols) of low-applied pressures (from 50 MPa to 350 MPa) against thermal deactivations (at 50°C and 55°C) has been noticed. In the presence of additives, a strong correlation between PE and the total hydroxyl group concentration has been obtained, for the first time, under treatments of combining denaturing temperatures and high hydrostatic pressures. This relationship does not seem to be dependent on the nature polyhydric alcohols as the same effect could be observed with 1 M sorbitol and 2 M glycerol. This PE, against thermal and high pressure combined lipase deactivation, increases with polyhydric alcohol concentrations, and when temperature increases from 25°C to 55°C.     

Clonal tracking of hESCs reveals differential contribution to functional assays

Human embryonic stem cells (hESCs) have unique self-renewal and differentiation properties, which are experimentally measured using functional assays. hESC cultures are known to be heterogeneous, but whether subsets of cells contribute differently to functional assays has yet to be examined. Here, using clonal tracking by retroviral integration, we analyzed in situ the propensity of individual hESCs to contribute to different functional assays. We observed different clonal distributions in teratomas versus in vitro differentiation assays. Some hESC subsets apparently contributed substantially to lineage-specific embryoid body differentiation and lacked clonogenic capacity, although they had self-renewal ability. In contrast, other subsets of self-renewing hESCs with clonogenic ability contributed to teratoma formation but were less frequently observed after in vitro differentiation. Our study suggests that assays used to measure pluripotency may detect distinct subsets of hESCs. These findings have direct implications for hESC-based therapies that may be optimized based on such functional assays.     

Genomic structure,QTL mapping,and molecular markers of lipase genes responsible for palm oil acidity in the oil palm (<Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq.)

The degradation of triglycerides in oil palm fruit due to an endogenous lipase in the pulp is the main reason for acidification of palm oil, which causes major economic losses and is currently mainly associated with the FLL1 gene. We designed this study to identify all the major genes controlling differences in acidity and lipase activity in the oil palm fruit mesocarp and determine a molecular markers kit to allow marker-assisted selection of commercial varieties with low acidity. Not only one gene (FLL1) but three closely linked genes including FLL1 were found and characterized in LM2T_EgCIR184O12c, a bacterial artificial chromosome sequence of 231 kb. Intra-gene PCR-based markers were designed for these genes. A QTL gene co-localization analysis for oil acidity (percentage of fatty acids released) was performed on two mapping populations. It evidenced a single major QTL at our lipase gene loci, explaining 84 to 92% of phenotypic variation, and validating the main genetic control of palm oil acidification by FLL1 and/or by the two new lipase genes. The three lipase genes had high homology to demonstrated triacylglycerol lipases. While FLL1 shows the highest expression levels, the two other genes may also contribute to oil acidity. Our molecular markers of lipase genes and the associated major QTL is an important step towards marker-assisted selection of commercial varieties with low acidity.     

Circulating Endothelial Cells in Refractory Pulmonary Hypertension in Children: Markers of Treatment Efficacy and Clinical Worsening

Background

Pulmonary vasodilators in general and prostacyclin analogues in particular have improved the outcome of patients with pulmonary arterial hypertension (PAH). Endothelial dysfunction is a key feature of PAH and we previously described that circulating endothelial cell (CEC) level could be used as a biomarker of endothelial dysfunction in PAH. We now hypothesized that an efficient PAH-specific vasodilator therapy might decrease CEC level.

Methods/Results

CECs were prospectively quantified by immunomagnetic separation with mAb CD146-coated beads in peripheral blood from children with idiopathic PAH (iPAH, n = 30) or PAH secondary to congenital heart disease (PAH-CHD, n = 30): before, after treatment and during follow up. Controls were 23 children with reversible PAH. Oral treatment with endothelin receptor antagonists (ERA) and/or phosphodiesterase 5 inhibitors (PDE5) significantly reduced CEC counts in children. In 10 children with refractory PAH despite oral combination therapy, subcutaneous (SC) treprostinil was added and we observed a significant decrease in CEC counts during the first month of such treatment. CECs were quantified during a 6 to 36 month-follow-up after initiation of SC treprostinil and we found that CEC counts changed over time, with rising counts always preceding clinical deterioration.

Conclusion

CECs might be useful as a biomarker during follow-up of pediatric iPAH and PAH-CHD to assess response to treatment and to anticipate clinical worsening.     

A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species

Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV) as a biological model, we confirm that the noncirculative mode of transmission dominant in plant viruses (designated mechanical vector transmission in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the helper component (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.