Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

BACKGROUND: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. RESULTS: Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase) promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter. CONCLUSION: The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism

Results of the Anaconda endovascular graft in abdominal aortic aneurysm with a severe angulated infrarenal neck

Objective: Proximal neck anatomy of an abdominal aortic aneurysm (AAA), especially a severe angulated neck of more than 60 degrees, predicts adverse outcome in endovascular aneurysm repair. In the present study, we evaluate the feasibility of the use of the Anaconda endovascular graft (Vascutec, Terumo, Inchinnan, Scotland) for treating infrarenal AAA with a severe angulated neck (>60 degrees) and report the midterm outcomes. Methods: In total, nine Dutch hospitals participated in this prospective cohort study. From December 2005 to January 2011, a total of 36 AAA patients, 30 men and six women, were included. Mean and median follow-up were both 40 months. Results: Mean infrarenal neck angulation was 82 degrees. Successful deployment was reached in 34 of 36 patients. Primary technical success was achieved in 30 of 36 patients (83%). There was no aneurysm-related death. Four-year primary clinical success was 69%. In the first year, eight clinical failures were reported including four leg occlusions which could be solved using standard procedures. After the first year, three patients with additional failures occurred; two of them were leg occlusions. Four patients needed conversion to open AAA exclusion. In six of 36 patients, one or more reinterventions were necessary. Three of them were performed for occlusion of one Anaconda leg and two were for occlusion of the body. Conclusions: The use of the Anaconda endovascular graft in AAA with a severe angulated infrarenal neck is feasible but has its side effects. Most clinical failures occur in the first year. Thereafter, few problems occur, and midterm results are acceptable. Summarizing the present experiences, we conclude that open AAA repair is still a preferable option in patients with challenging aortic neck anatomy and fit for open surgery

Assessment of Carbon Stock and Greenhouse Gas Emissions of Selected Coffee-Based Agroforestry Farms in Timor Leste

Agroforestry is an important carbon sequestration strategy because of carbon storage potential in its multiple plant species and soil as well as its applicability in agricultural lands and in reforestation.  The study was conducted in selected coffee-based agroforestry farms in Gleno, Ermera District, Timor Leste to determine the amount of carbon stocks and greenhouse gas emissions in the management of coffee-based agroforestry farms. Results reveal that agroforestry coffee cropping pattern had a carbon stock composed of 6.425 Mg ha-1 biomass, 2.01 Mg ha-1 necromass, and 157.17 Mg ha-1 soil organic carbon. For the carbon stock stored in the monoculture coffee cropping pattern, a biomass of 5.321 Mg ha-1, necromass of 1.32 Mg ha-1, and soil organic carbon of 128.74. Mg ha-1 were recorded while for the pruned coffee cropping pattern, biomass of 2.771 Mg ha-1, necromass of 1.82 Mg ha-1, and soil organic carbon of 69.29 Mg ha-1 were found.  Moreover, the greenhouse gas emission CO2 absorbed by coffee plants across cropping patterns were the following: agroforestry coffee, 24.156 t/ha-1, monoculture coffee, 19.520 t/ha-1 and pruned coffee, 10.177 t/ha-1.  Abovementioned results reveal that coffee-based agroforestry system a coffee-based agroforestry is considered as an effective solution in combatting heightened atmospheric GHGs concentration and climate change while at the same time addressing concerns on socio-economic stability

Exposure to androstenes influences processing of emotional words

There is evidence that human-produced androstenes affect attitudinal, emotional, and physiological states in a context-dependent manner, suggesting that they could be involved in modulating social interactions. For instance, androstadienone appears to increase attention specifically to emotional information. Most of the previous work focused on one or two androstenes. Here, we tested whether androstenes affect linguistic processing, using three different androstene compounds. Participants (90 women and 77 men) performed a lexical decision task after being exposed to an androstene or to a control treatment (all compounds were applied on the philtrum). We tested effects on three categories of target words, varying in emotional valence: positive, competitive, and neutral words (e.g., hope, war, and century, respectively). Results show that response times were modulated by androstene treatment and by emotional valence of words. Androstenone, but not androstadienone and androstenol, significantly slowed down the reaction time to words with competitive valence. Moreover, men exposed to androstenol showed a significantly reduced error rate, although men tended to make more errors than women in general. This suggests that these androstenes modulate the processing of emotional words, namely some particular lexical emotional content may become more salient under the effect of androstenes

Aggregated Mycobacterium tuberculosis Enhances the Inflammatory Response

Mycobacterium tuberculosis (Mtb) bacilli readily aggregate. We previously reported that Mtb aggregates lead to phagocyte death and subsequent efficient replication in the dead infected cells. Here, we examined the transcriptional response of human monocyte derived macrophages to phagocytosis of aggregated Mtb relative to phagocytosis of non-aggregated single or multiple bacilli. Infection with aggregated Mtb led to an early upregulation of pro-inflammatory associated genes and enhanced TNFα signaling via the NFκB pathway. These pathways were significantly more upregulated relative to infection with single or multiple non-aggregated bacilli per cell. Phagocytosis of aggregates led to a decreased phagosome acidification on a per bacillus basis and increased phagocyte cell death, which was not observed when Mtb aggregates were heat killed prior to phagocytosis. Mtb aggregates, observed in a granuloma from a patient, were found surrounding a lesion cavity. These observations suggest that TB aggregation may be a mechanism for pathogenesis. They raise the possibility that aggregated Mtb, if spread from individual to individual, could facilitate increased inflammation, Mtb growth, and macrophage cell death, potentially leading to active disease, cell necrosis, and additional cycles of transmission

An amphitropic cAMP-binding protein in yeast mitochondria

ABSTRACT: We describe the first example of a mitochondrial protein with a covalently attached phos-phatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cereuisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified CAMP-binding protein through a phospholipase activity. Only in recent years specific fatty acids have been recog-nized to play important roles in the association of proteins with membranes. Both noncovalent and covalent interactions be-tween fatty acids and proteins have been reported. Among the latter are GTP-binding proteins (Molenaar et al., 1988)

Towards a sustainable approach to clustering small-scale farmers to market their agricultural produce

The University of the Philippines Mindanao and Curtin University have been organising small-scale vegetable farmers in Mindanao into cluster marketing groups to improve their access to markets and returns from the sale of their vegetable products using the 8-Step Clustering Approach for Agroenterprise Development developed by the Catholic Relief Services (Philippines). This paper reports on an action research investigation with around 30 marketing clusters. Cluster marketing has led to: better and wider market access, improved prices, reduced costs, improved human capital and social capital, and higher incomes. However, the current process may not lead to sustainable cluster marketing without support and may lead to dependency. This paper discusses strategies to overcome key challenges identified and deficiencies in the current process and suggests changes to the 8-step process so that it may improve the chances of cluster success and sustainability