Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Effect of metals on the regulation of acidogenic metabolism enhancing biohydrogen and carboxylic acids production from brewery spent grains: Microbial dynamics and biochemical analysis

The present study reports the mixed culture acidogenic production of biohydrogen and carboxylic acids (CA) from brewery spent grains (BSG) in the presence of high concentrations of cobalt, iron, nickel, and zinc. The metals enhanced biohydrogen output by 2.39 times along with CA biosynthesis by 1.73 times. Cobalt and iron promoted the acetate and butyrate pathways, leading to the accumulation of 5.14 gCOD/L of acetic and 11.36 gCOD/L of butyric acid. The production of solvents (ethanol + butanol) was higher with zinc (4.68 gCOD/L) and cobalt (4.45 gCOD/L). A combination of all four metals further enhanced CA accumulation to 42.98 gCOD/L, thus surpassing the benefits accrued from supplementation with individual metals. Additionally, 0.36 and 0.31 mol green ammonium were obtained from protein‐rich brewery spent grain upon supplementation with iron and cobalt, respectively. Metagenomic analysis revealed the high relative abundance of Firmicutes (>90%), of which 85.02% were Clostridium, in mixed metal‐containing reactors. Finally, a significant correlation of dehydrogenase activity with CA and biohydrogen evolution was observed upon metal addition.     

Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.     

Genomics in a changing arctic: critical questions await the molecular ecologist

Molecular ecology is poised to tackle a host of interesting questions in the coming years. The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. These questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high‐latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate.     

Low C-Reactive Protein Levels in a Traditional West-African Population Living in a Malaria Endemic Area

Background

C-reactive protein (CRP) levels are reported to be elevated in populations of African descent living in affluent environments compared to populations of European ancestry. However, the natural history of CRP levels in populations of African descent living under adverse environments remains largely unknown.

Methods

CRP levels were measured with a high sensitivity assay in 624 apparently healthy individuals who contributed blood as part of a study on innate immune responsiveness in a traditional Ghanaian population living under adverse environmental conditions in a malaria endemic area. As a comparison, we included CRP measurements from 2931 apparently healthy individuals from the Dutch population that were included in the same batch of CRP analyses. Associations between CRP and body mass index (BMI), immune responsiveness, and P. falciparum parasitaemia were investigated.

Results

In an age- and sex-adjusted model, CRP levels were 0.54 mg/L lower in the Ghanaian compared to the Dutch cohort (1.52 vs. 0.98 mg/L, p<0.001). When accounting for the substantially higher average BMI in the Dutch compared to the Ghanaians (25.6 vs. 18.4 kg/m²) the difference in CRP levels disappeared. BMI associated positively with CRP in the Dutch but not in the Ghanaians. In individuals with an acute phase response, CRP levels were higher in the Ghanaian compared to the Dutch cohort (24.6 vs. 17.3 mg/L, p = 0.04). Levels of CRP were positively related to immune responsiveness and P. falciparum parasitaemia (all p<0.001) among Ghanaians.

Conclusions

Our study demonstrates that West-Africans do not exhibit an inherently high inflammatory state. The role of genes, environment and gene-environment interaction in explaining reports of elevated CRP levels in populations of African ancestry when compared to other ethnicities living in affluent environments thus merits further investigation.     

Type 2 Endoleaks: The Diagnostic Performance of Non-Specialized Readers on Arterial and Venous Phase Multi-Slice CT Angiography

PurposeTo define the diagnostic precision of non-specialized readers in the detection of type 2 endoleaks (T2EL) in arterial versus venous phase acquisitions, and to evaluate an approach for radiation dose reduction.MethodsThe pre-discharge and final follow-up multi-slice CT angiographies of 167 patients were retrospectively analyzed. Image data were separated into an arterial and a venous phase reading set. Two radiology residents assessed the reading sets for the presence of a T2EL, feeding vessels, and aneurysm sac size. Findings were compared with a standard of reference established by two experts in interventional radiology. The effective dose was calculated.ResultsOverall, experts detected 131 T2ELs, and 331 feeding vessels in 334 examinations. Persistent T2ELs causing aneurysm sac growth > 5 mm were detected in 20 patients. Radiation in arterial and venous phases contributed to a mean of 58.6% and 39.0% of the total effective dose. Findings of reader 1 and 2 showed comparable sensitivities in arterial sets of 80.9 versus 85.5 (p = 0.09), and in venous sets of 73.3 versus 79.4 (p = 0.15), respectively. Reader 1 and 2 achieved a significant higher detection rate of feeding vessels with arterial compared to venous set (p = 0.04, p < 0.01). Both readers correctly identified T2ELs with growing aneurysm sac in all cases, independent of the acquisition phase.ConclusionArterial acquisitions enable non-specialized readers an accurate detection of T2ELs, and a significant better identification of feeding vessels. Based on our results, it seems reasonable to eliminate venous phase acquisitions.     

Simvastatin Efficiently Lowers Small LDL-IgG Immune Complex Levels: A Therapeutic Quality beyond the Lipid-Lowering Effect

We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions (salt gradient separation) were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis.