Optimization of heterologous production of the polyketide 6-MSA in Saccharomyces cerevisiae

Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes.     

Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.     

Macrophage migration inhibitory factor (MIF) promotes fibroblast migration in scratch-wounded monolayers in vitro

MIF was recently redefined as an inflammatory cytokine, which functions as a critical mediator of diseases such as septic shock, rheumatoid arthritis, atherosclerosis, and cancer. MIF also regulates wound healing processes. Given that fibroblast migration is a central event in wound healing and that MIF was recently demonstrated to promote leukocyte migration through an interaction with G-protein-coupled receptors, we investigated the effect of MIF on fibroblast migration in wounded monolayers in vitro. Transient but not permanent exposure of primary mouse or human fibroblasts with MIF significantly promoted wound closure, a response that encompassed both a proliferative and a pro-migratory component. Importantly, MIF-induced fibroblast activation was accompanied by an induction of calcium signalling, whereas chronic exposure with MIF down-regulated the calcium transient, suggesting receptor desensitization as the underlying mechanism.     

Weight loss maintenance in severely obese adults after an intensive lifestyle intervention: 2- to 4-year follow-up

Objective: To evaluate the long‐term weight loss maintenance after 2 to 4 years in severely obese subjects after a lifestyle intervention at a weight loss camp in Denmark. Research Methods and Procedures: In a retrospective follow‐up study, we assessed weight loss after 21 weeks of treatment at a weight loss camp, weight loss maintenance after 2 to 4 years, and numbers of subjects with a weight loss maintenance of ≥10% of a total number of 435 severely obese adults participating in an intensive lifestyle intervention with a primary focus on physical activity. Results: We obtained follow‐up data of 249 subjects (180 women and 69 men) with an initial body weight of 142 ± 32 kg. After 21 weeks at the camp, the subjects had reduced their body weight with a mean of 21.9 ± 13 kg (corresponding to a 15% weight loss). The average weight loss maintenance was 5.3% at a follow‐up after 2 to 4 years, and 28.3% had maintained a weight loss above 10% after 4 years of follow‐up. Discussion: Weight loss camps are a relatively new commercial approach in treating severely obese subjects. However, the results demonstrate that even with a multidisciplinary intensive setting with focus on diet, exercise, and psychological counseling, only 28% had maintained a weight loss above 10% after 4 years. This emphasizes that obesity is a chronic condition that needs additional strategies after a weight loss intervention in the efforts to maintain a sufficient weight loss.     

The Sandfish＇s Skin： Morphology, Chemistry and Reconstruction

The sandfish is a lizard having the remarkable ability to move in desert sand in a swimming-like fashion. The most outstanding adaptations to this mode of life are the low friction behaviour and the extensive abrasion resistance of the sandfish skin against sand, outperforming even steel. We investigated the topography, the composition and the mechanical properties of sandfish scales. These consist of glycosylated keratins with high amount of sulphur but no hard inorganic material, such as silicates or lime. Remarkably, atomic force microscopy shows an almost complete absence of attractive forces between the scale surface and a silicon tip, suggesting that this is responsible for the unusual tribological properties. The unusual glycosylation of the keratins was found to be absolutely necessary for the described phenomenon. The scales were dissolved and reconstituted on a polymer surface resulting in properties similar to the original scale. Thus, we provide a pathway towards exploitation of the reconstituted scale material for future engineering applications.     

High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop March 7-9, 2007 Wellcome Trust Conference Centre, Hinxton, UK

The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled "High Performance Proteomics". It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics.     

Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii

The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress-related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harboring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photoheterotrophic to primarily heterotrophic metabolism, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.     

Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Autonomic cardiovascular and respiratory control during prolonged spaceflights aboard the International Space Station.

Impaired autonomic control represents a cardiovascular risk factor during long-term spaceflight. Little has been reported on blood pressure (BP), heart rate (HR), and heart rate variability (HRV) during and after prolonged spaceflight. We tested the hypothesis that cardiovascular control remains stable during prolonged spaceflight. Electrocardiography, photoplethysmography, and respiratory frequency (RF) were assessed in eight male cosmonauts (age 41-50 yr, body-mass index of 22-28 kg/m2) during long-term missions (flight lengths of 162-196 days). Recordings were made 60 and 30 days before the flight, every 4 wk during flight, and on days 3 and 6 postflight during spontaneous and controlled respiration. Orthostatic testing was performed pre- and postflight. RF and BP decreased during spaceflight (P < 0.05). Mean HR and HRV in the low- and high-frequency bands did not change during spaceflight. However, the individual responses were different and correlated with preflight values. Pulse-wave transit time decreased during spaceflight (P < 0.05). HRV reached during controlled respiration (6 breaths/min) decreased in six and increased in one cosmonaut during flight. The most pronounced changes in HR, BP, and HRV occurred after landing. The decreases in BP and RF combined with stable HR and HRV during flight suggest functional adaptation rather than pathological changes. Pulse-wave transit time shortening in our study is surprising and may reflect cardiac output redistribution in space. The decrease in HRV during controlled respiration (6 breaths/min) indicates reduced parasympathetic reserve, which may contribute to postflight disturbances.