Process optimization for increased yield of surface-expressed protein in Escherichia coli

The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.     

Early growth delay in diabetic pregnancy: relation to psychomotor development at age 4

Ninety nine consecutive insulin dependent and 101 non-diabetic pregnant women were examined by ultrasonograph to assess early fetal growth. In 42 of the diabetic mothers and three of the non-diabetic mothers the scan showed early intrauterine growth delay. At 4-5 years of age all children available for study were evaluated by the Denver developmental screening test. Only 23 of the 34 children of diabetic mothers with early intrauterine growth delay had normal test scores compared with 46 of the 50 children of diabetic mothers with normal intrauterine growth. The children failed in personal-social development, gross motor development, and particularly in language and speech development. Children of diabetic mothers with normal early fetal growth had scores very similar to those of the children of non-diabetic mothers, of whom 76 of the 86 tested had normal scores.This study suggests that children with a history of growth delay in early diabetic pregnancy should be screened for possible developmental impairment.     

The transthyretin-related protein family.

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.     

Mosquitoes and West Nile virus along a river corridor from prairie to montane habitats in eastern Colorado

We conducted studies on mosquitoes and West Nile virus (WNV) along a riparian corridor following the South Platte River and Big Thompson River in northeastern Colorado and extending from an elevation of 1,215 m in the prairie landscape of the eastern Colorado plains to 1,840 m in low montane areas at the eastern edge of the Rocky Mountains in the central part of the state. Mosquito collection during June‐September 2007 in 20 sites along this riparian corridor yielded a total of 199,833 identifiable mosquitoes of 17 species. The most commonly collected mosquitoes were, in descending order: Aedes vexans, Culex tarsalis, Ae. dorsalis, Ae. trivittatus, Ae. melanimon, Cx. pipiens, and Culiseta inornata. Species richness was higher in the plains than in foothills‐montane areas, and abundances of several individual species, including the WNV vectors Cx. tarsalis and Cx. pipiens and the nuisance‐biter and potential secondary WNV vector Ae. vexans, decreased dramatically from the plains (1,215‐1,487 m) to foothills‐montane areas (1,524‐1,840 m). Ae. vexans and Cx. tarsalis had a striking pattern of uniformly high abundances between 1,200‐1,450 m followed by a gradual decrease in abundance above 1,450 m to reach very low numbers above 1,550 m. Culex species were commonly infected with WNV in the plains portion of the riparian corridor in 2007, with 14 of 16 sites yielding WNV‐infected Cx. tarsalis and infection rates for Cx. tarsalis females exceeding 2.0 per 1,000 individuals in ten of the sites. The Vector Index for abundance of WNV‐infected Cx. tarsalis females during June‐September exceeded 0.5 in six plains sites along the South Platte River but was uniformly low (0–0.1) in plains, foothills and montane sites above 1,500 m along the Big Thompson River. A population genetic analysis of Cx. tarsalis revealed that all collections from the ≈190 km riparian transect in northeastern Colorado were genetically uniform but that these collections were genetically distinct from collections from Delta County on the western slope of the Continental Divide. This suggests that major waterways in the Great Plains serve as important dispersal corridors for Cx. tarsalis but that the Continental Divide is a formidable barrier to this WNV vector.     

The feeding biology of Hydrobia ventrosa (Montagu). I. The assimilation of different components of the food

The assimilation efficiencies for 7 species of diatoms, two of blue-green algae, and one bacterium have been measured in the deposit-feeding prosobranch Hydrobia ventrosa (Montagu). Two methods have been used, a chemical and a radiotracer one.The results of the first method were difficult to evaluate due to the fact that the time taken to clear the gut is ill-defined. The second method gave more consistent results.All diatoms are assimilated efficiently (60–71 %) as is the bacterium (about 75 %). The blue-green algae are less efficiently utilized; an Oscillatoria sp. was assimilated with an efficiency of about 50 % whereas a Chroococcus sp. was only assimilated with an efficiency of 8 %. It is concluded that resource partitioning based on differential utilization of micro-organisms cannot play an important rôle in the co-existence of Hydrobia ventrosa with other deposit-feeders.     

Growing islands and sinking solutes: processes maintaining the endorheic Okavango Delta as a freshwater system

In the Okavango Delta 98–99% of the water from inflow and rainfall is lost to the atmosphere through evapotranspiration. As a consequence 94% of inflowing solutes are retained within the Delta landscape. This process might be expected to result in an entirely saline environment, but that is not the case: the surface waters have very low salinity, supporting a typical freshwater biota. It has been deduced that the numerous islands in the Delta (about 150,000 within an area of 13,500 km²) have been formed through evapotransporative concentration in the groundwater, of infiltrating solutes, followed by precipitation and volume increase. Evidence of this is the large amount of calcrete in island soils. These islands of 3–10 m thickness with clayey soils are underlain by fine Kalahari sand to a depth of 200–300 m, which also indicates that they are formed through surface processes. The infiltration rate of surface water from floodplains and streams into islands is very high, and is predominantly a lateral process that is unidirectional. Evapotranspiration in the riparian woodland zone cause the ground-waters in the central area of islands—with halophyte grasslands—to have very high salinities. By use of chloride as a conservative element the concentration factor between central island groundwater and surface water is calculated to be 500–1,000. This groundwater is depleted of calcium and magnesium supporting the early deductions that these elements have precipitated as calcrete. There is also a large depletion of silicate and potassium that probably have precipitated as well forming the clayey soils typical of the islands. The central island groundwater is dominated by sodium, bicarbonate and dissolved organic matter. The gradual increase of salinity here causes a periodic let off of this water through a density-driven process to deeper layers. This process together with island growth through precipitation of solutes are the two major sink processes of inflowing solutes and explains why the Okavango Delta is at present a freshwater system. The whole island complex is calculated to be 100,000–400,000 years old while some intensely studied islands may be younger: 80,000–240,000 years. The discrepancy is explained by a biassed selection of islands currently in flooded areas with better growth conditions. The uniqueness of the Okavango Delta and ideas for future research are discussed.     

Reconstruction of Cellular Signal Transduction Networks Using Perturbation Assays and Linear Programming

Perturbation experiments for example using RNA interference (RNAi) offer an attractive way to elucidate gene function in a high throughput fashion. The placement of hit genes in their functional context and the inference of underlying networks from such data, however, are challenging tasks. One of the problems in network inference is the exponential number of possible network topologies for a given number of genes. Here, we introduce a novel mathematical approach to address this question. We formulate network inference as a linear optimization problem, which can be solved efficiently even for large-scale systems. We use simulated data to evaluate our approach, and show improved performance in particular on larger networks over state-of-the art methods. We achieve increased sensitivity and specificity, as well as a significant reduction in computing time. Furthermore, we show superior performance on noisy data. We then apply our approach to study the intracellular signaling of human primary nave CD4⁺ T-cells, as well as ErbB signaling in trastuzumab resistant breast cancer cells. In both cases, our approach recovers known interactions and points to additional relevant processes. In ErbB signaling, our results predict an important role of negative and positive feedback in controlling the cell cycle progression.