Single Dose Novel Salmonella Vaccine Enhances Resistance against Visceralizing L. major and L. donovani Infection in Susceptible BALB/c Mice

Visceral leishmaniasis is a major neglected tropical disease, with an estimated 500,000 new cases and more than 50,000 deaths attributable to this disease every year. Drug therapy is available but costly and resistance against several drug classes has evolved. Despite all efforts, no commercial, let alone affordable, vaccine is available to date. Thus, the development of cost effective, needle-independent vaccines is a high priority. Here, we have continued efforts to develop live vaccine carriers based on recombinant Salmonella. We used an in silico approach to select novel Leishmania parasite antigens from proteomic data sets, with selection criteria based on protein abundance, conservation across Leishmania species and low homology to host species. Five chosen antigens were differentially expressed on the surface or in the cytosol of Salmonella typhimurium SL3261. A two-step procedure was developed to select optimal Salmonella vaccine strains for each antigen, based on bacterial fitness and antigen expression levels. We show that vaccine strains of Salmonella expressing the novel Leishmania antigens LinJ08.1190 and LinJ23.0410 significantly reduced visceralisation of L. major and enhanced systemic resistance against L. donovani in susceptible BALB/c mice. The results show that Salmonella are valid vaccine carriers for inducing resistance against visceral leishmaniasis but that their use may not be suitable for all antigens

Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation

Gene and genon concept: coding versus regulation: A conceptual and information-theoretic analysis of genetic storage and expression in the light of modern molecular biology

We analyse here the definition of the gene in order to distinguish, on the basis of modern insight in molecular biology, what the gene is coding for, namely a specific polypeptide, and how its expression is realized and controlled. Before the coding role of the DNA was discovered, a gene was identified with a specific phenotypic trait, from Mendel through Morgan up to Benzer. Subsequently, however, molecular biologists ventured to define a gene at the level of the DNA sequence in terms of coding. As is becoming ever more evident, the relations between information stored at DNA level and functional products are very intricate, and the regulatory aspects are as important and essential as the information coding for products. This approach led, thus, to a conceptual hybrid that confused coding, regulation and functional aspects. In this essay, we develop a definition of the gene that once again starts from the functional aspect. A cellular function can be represented by a polypeptide or an RNA. In the case of the polypeptide, its biochemical identity is determined by the mRNA prior to translation, and that is where we locate the gene. The steps from specific, but possibly separated sequence fragments at DNA level to that final mRNA then can be analysed in terms of regulation. For that purpose, we coin the new term “genon”. In that manner, we can clearly separate product and regulative information while keeping the fundamental relation between coding and function without the need to introduce a conceptual hybrid. In mRNA, the program regulating the expression of a gene is superimposed onto and added to the coding sequence in cis - we call it the genon. The complementary external control of a given mRNA by trans-acting factors is incorporated in its transgenon. A consequence of this definition is that, in eukaryotes, the gene is, in most cases, not yet present at DNA level. Rather, it is assembled by RNA processing, including differential splicing, from various pieces, as steered by the genon. It emerges finally as an uninterrupted nucleic acid sequence at mRNA level just prior to translation, in faithful correspondence with the amino acid sequence to be produced as a polypeptide. After translation, the genon has fulfilled its role and expires. The distinction between the protein coding information as materialised in the final polypeptide and the processing information represented by the genon allows us to set up a new information theoretic scheme. The standard sequence information determined by the genetic code expresses the relation between coding sequence and product. Backward analysis asks from which coding region in the DNA a given polypeptide originates. The (more interesting) forward analysis asks in how many polypeptides of how many different types a given DNA segment is expressed. This concerns the control of the expression process for which we have introduced the genon concept. Thus, the information theoretic analysis can capture the complementary aspects of coding and regulation, of gene and genon

C70 ordering on nanostructured SrTiO3(001)

The nanostructured (7 x 4) surface of SrTiO3(001) is used as a template to order C70 into single-molecule-wide chains and linear islands. © The Royal Society of Chemistry

Template ordered open-grid arrays of paired endohedral fullerenes

Developing useful molecular systems, such as planar networks for novel molecular electronics, requires the ability to control the way molecules assemble at surfaces. Here we report how an oxide crystal surface can be used as a template to controllably order endohedral fullerenes, ErN@C, into two-dimensional (2D) open-grid arrays. The crystal surface is made of highly ordered oxide nanostructures which self-assemble on the surface of SrTiO(001). This method of molecular ordering can be applied to other fullerenes and has the potential to provide a basis for developing a wide range of molecular architectures. Copyright © 2006 American Chemical Society

Ordering of TiO2-based nanostructures on SrTiO3(001) surfaces.

A class of nanostructured surface phases on SrTiO3(001) is reported and characterized through atomic-resolution scanning tunneling microscopy and Auger electron spectroscopy. These surface phases are created via argon ion sputtering and UHV annealing and form close-packed domains of highly ordered nanostructures. Depending on the type of nanostructures present, the domain ordering exhibit either (6 x 2), (9 x 2), (12 x 2), (6 x 8), or (7 x 4) surface patterning. The nanostructures are composed of TiO2-derived complexes surrounded by a TiO2 surface termination. Such surface ordering phenomena introduce another level of complexity in the chemistry of perovskite oxide surfaces and provide a basis from which potential photocatalytic and molecular-ordering applications may be developed

Structure of vapour deposited adenine on a nanostructured perovskite surface studied by STM.

The structures of vapour deposited layers of adenine on a nanostructured SrTiO3(001) surface have been investigated by scanning tunneling microscopy (STM). The STM images reveal details of self-organization of adenine monolayers in which adsorption is controlled both by substrate nanostructure and by intermolecular H-bonding of adenine molecules. Detailed examination of STM images suggests that at least three different ordering structures are possible and two distinct orientations may exist with opposite chirality

Controlled surface ordering of endohedral fullerenes with a SrTiO(3) template.

The ability to select the way in which atoms and molecules self-organize on a surface is important for synthesizing nanometre scale devices. Here we show how endohedral fullerenes (Er(3)N@C(80)) can be assembled into four distinctive arrangements on a strontium titanate surface template. Each template pattern correlates to a particular reconstruction on n-doped SrTiO(3)(001), made in whole or in part by self-assembled arrays of non-stoichiometric oxide nanostructures. Close-packed assemblies of Er(3)N@C(80) molecules are formed, as well as one-dimensional chains and two-dimensional grids. This method of template-assisted molecular ordering provides a new platform for the development of experimental schemes of classical and quantum information processing at the molecular level

Hot STM of nanostructure dynamics on SrTiO(3)(001).

The dynamics of nanostructured surface phases on SrTiO(3)(001) have been analysed using in situ scanning tunnelling microscopy (STM) above 800 degrees C. During high-temperature annealing, the formation, growth and ordering of the nanostructures has been observed. Dilines, with a width of approximately 1 nm, are formed from a TiO(2)-rich intermediary at 800 degrees C. STM during annealing at 825 degrees C has enabled us to follow both the growth and dissolution of dilines. Following extended annealing, trilines with a width of approximately 2 nm and ordered two-dimensional (2D) nano-arrays form from the diline domains. Our observations of diline dissolution implies random nucleation and growth, followed by rearrangement at elevated temperature to form domains