Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.     

Dynamic Changes in ANGUSTIFOLIA3 Complex Composition Reveal a Growth Regulatory Mechanism in the Maize Leaf

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.     

Effective production of the biodiesel additive STBE by a continuous flow process

A new fuel additive, namely solketal tert-butyl ether (STBE), was developed and optimized under continuous flow conditions using a Corning? Advanced-Flow? glass reactor. STBE was obtained in two steps from glycerol, a renewable building-block produced in large amount in the processing of biodiesel. The advantages of the highly engineered Corning glass reactor included high mixing and heat-exchange efficiency, chemical resistance under corrosive flow conditions and a small hold-up. A robust, continuous, green and safe industrial-scale process is described.     

High local substrate availability stabilizes a cooperative trait

Cooperative behavior is widely spread in microbial populations. An example is the expression of an extracellular protease by the lactic acid bacterium Lactococcus lactis, which degrades milk proteins into free utilizable peptides that are essential to allow growth to high cell densities in milk. Cheating, protease-negative strains can invade the population and drive the protease-positive strain to extinction. By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it. The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes.     

LINE-1 hypomethylation during primary colon cancer progression

Background

Methylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage.

Methodology/Principal Findings

DNA was collected by laser-capture microdissection (LCM) from normal, adenoma, and cancer tissue from 25 patients with TisN0M0 and from 92 primary CRC patients of various TNM-stages. The paraffin-embedded tissue sections were treated by in-situ DNA sodium bisulfite modification (SBM). LINE-1 hypomethylation index (LHI) was measured by absolute quantitative analysis of methylated alleles (AQAMA) realtime PCR; a greater index indicated enhanced hypomethylation. LHI in normal, cancer mesenchymal, adenoma, and CRC tissue was 0.38 (SD 0.07), 0.37 (SD 0.09), 0.49 (SD 0.10) and 0.53 (SD 0.08), respectively. LHI was significantly greater in adenoma tissue compared to its contiguous normal epithelium (P = 0.0003) and cancer mesenchymal tissue (P<0.0001). LHI did not differ significantly between adenoma and early cancer tissue of Tis stage (P = 0.20). LHI elevated with higher T-stage (P<0.04), was significantly greater in node-positive than node-negative CRC patients (P = 0.03), and was significantly greater in stage IV than all other disease stages (P<0.05).

Conclusion/Significance

By using in-situ SBM and LCM cell selection we demonstrated early onset of LINE-1 demethylation during adenomatous change of colorectal epithelial cells and demonstrated that LINE-1 demethylation progression is linear in relation to TNM-stage progression.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

High-level acetaldehyde production in Lactococcus lactis by metabolic engineering

Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.