Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis

The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities. Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis. By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved. Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part of ComGA of B. subtilis overcomes the slow translation initiation that is provoked by the eukaryotic codon bias present in the original cfp and yfp genes. Using these new vectors, we demonstrate that, within an isogenic population of sporulating B. subtilis cells, expression of the abrB and spoIIA genes is distinct in individual cells.     

Determining sites of interaction between prenisin and its modification enzymes NisB and NisC

Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Δ(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.     

Genetic Footprints of Iberian Cattle in America 500 Years after the Arrival of Columbus

Background

American Creole cattle presumably descend from animals imported from the Iberian Peninsula during the period of colonization and settlement, through different migration routes, and may have also suffered the influence of cattle directly imported from Africa. The introduction of European cattle, which began in the 18th century, and later of Zebu from India, has threatened the survival of Creole populations, some of which have nearly disappeared or were admixed with exotic breeds. Assessment of the genetic status of Creole cattle is essential for the establishment of conservation programs of these historical resources.

Methodology/Principal Findings

We sampled 27 Creole populations, 39 Iberian, 9 European and 6 Zebu breeds. We used microsatellite markers to assess the origins of Creole cattle, and to investigate the influence of different breeds on their genetic make-up. The major ancestral contributions are from breeds of southern Spain and Portugal, in agreement with the historical ports of departure of ships sailing towards the Western Hemisphere. This Iberian contribution to Creoles may also include some African influence, given the influential role that African cattle have had in the development of Iberian breeds, but the possibility of a direct influence on Creoles of African cattle imported to America can not be discarded. In addition to the Iberian influence, the admixture with other European breeds was minor. The Creoles from tropical areas, especially those from the Caribbean, show clear signs of admixture with Zebu.

Conclusions/Significance

Nearly five centuries since cattle were first brought to the Americas, Creoles still show a strong and predominant signature of their Iberian ancestors. Creole breeds differ widely from each other, both in genetic structure and influences from other breeds. Efforts are needed to avoid their extinction or further genetic erosion, which would compromise centuries of selective adaptation to a wide range of environmental conditions.     

Human Herpesvirus 6A Partially Suppresses Functional Properties of DC without Viral Replication

Human herpesvirus 6A (HHV-6A) is a common virus with a worldwide distribution that has been associated with multiple sclerosis. Whether HHV-6A can replicate in dendritic cells (DC) and how the infection might modulate the functional properties of the cell are currently not well known and need further investigations. Here, we show that a non-productive infection of HHV-6A in DC leads to the up-regulation of HLA-ABC, via autocrine IFN-α signaling, as well as the up-regulation of HLA-DR and CD86. However, HHV-6A exposure reduces IL-8 secretion by DC and their capacity to stimulate allogenic T cell proliferation. The ability to suppress DC functions important for activation of innate and adaptive immune responses might be one successful strategy by which HHV-6A avoids the induction of appropriate host defense mechanisms, and thus facilitating persistent infection.     

Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: Benefits of FAIMS separation and gas-phase fractionation DIA

Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co-purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and efficacy and their potential immunogenicity. Enzyme-linked immunosorbent assays (ELISA) commonly used for global HCP monitoring present limitations in terms of identification and quantification of individual HCPs. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as a promising alternative. Challenging DP samples show an extreme dynamic range requiring high performing methods to detect and reliably quantify trace-level HCPs. Here, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation and gas phase fractionation (GPF) prior to data independent acquisition (DIA). FAIMS LC-MS/MS analysis allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been successfully applied to two FDA/EMA approved DPs and allowed digging deeper into the HCP landscape with the identification and quantification of a few tens of HCPs with sensitivity down to the sub-ng/mg of mAb level.     

Identification of the natural breeding sites of sandflies (Diptera: Psychodidae: Phlebotominae), potential vectors of leishmaniasis, in the province of Chaco, Argentina

The aim of this work was to identify the natural breeding sites of sandflies in the province of Chaco, Argentina, for the first time. Preliminary studies were conducted in two different phytogeographic regions: dry Chaco (Parque Provincial Pampa del Indio), in January 2010, and humid Chaco (Resistencia, Margarita Belén and Colonia Benítez), from May-September 2010. A total of 127 samples were collected (Pampa del Indio: 15, Resistencia: 37, Margarita Belén: 36, Colonia Benítez: 39). A female of Migonemyia migonei was found in Pampa del Indio at the base of a bromeliad in the summer (January) and a pupal exuvium of a phlebotomine fly was found in Resistencia, in a place where dogs rested, in the winter (July). These findings highlighted these two sites as potential breeding sites. Because the existence of potential natural breeding sites for sandflies has been demonstrated in both forest and periurban areas, expanding the search efforts and characterising these sites will enable the development of specific study designs to gain insight into the spatial distribution of the risks posed by these vectors. The resulting information will serve as a basis for proposing and evaluating vector control measures.     

Anticipative management for coral reef ecosystem services in the 21st century

Under projections of global climate change and other stressors, significant changes in the ecology, structure and function of coral reefs are predicted. Current management strategies tend to look to the past to set goals, focusing on halting declines and restoring baseline conditions. Here, we explore a complementary approach to decision making that is based on the anticipation of future changes in ecosystem state, function and services. Reviewing the existing literature and utilizing a scenario planning approach, we explore how the structure of coral reef communities might change in the future in response to global climate change and overfishing. We incorporate uncertainties in our predictions by considering heterogeneity in reef types in relation to structural complexity and primary productivity. We examine 14 ecosystem services provided by reefs, and rate their sensitivity to a range of future scenarios and management options. Our predictions suggest that the efficacy of management is highly dependent on biophysical characteristics and reef state. Reserves are currently widely used and are predicted to remain effective for reefs with high structural complexity. However, when complexity is lost, maximizing service provision requires a broader portfolio of management approaches, including the provision of artificial complexity, coral restoration, fish aggregation devices and herbivore management. Increased use of such management tools will require capacity building and technique refinement and we therefore conclude that diversification of our management toolbox should be considered urgently to prepare for the challenges of managing reefs into the 21st century.