Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

Biomimetic reconstruction of the hematopoietic stem cell niche for in vitro amplification of human hematopoietic stem cells.

Hematopoietic stem cell transplantation is successfully applied since the late 1950s; however, its efficacy still needs to be increased. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an improved ex vivo culture system that supports proliferation and maintains HSC pluripotency would override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro, we optimized the HSC medium composition with a panel of cytokines and valproic acid and used an artificial 3D bone marrow-like scaffold made of polydimethylsiloxane (PDMS). This 3D scaffold offered a suitable platform to amplify human HSCs in vitro and, simultaneously, to support their viability, multipotency and ability for self-renewal. Silicon oxide-covering of PDMS structures further improved amplification of CD34+ cells, although the conservation of naïve HSCs was better on non-covered 3D PDMS. Finally, we found that HSC cultivated on non-covered 3D PDMS generated most pluripotent colonies within colony forming unit assays. In conclusion, by combining biological and biotechnological approaches, we optimized in vitro HSCs culture conditions, resulting in improved amplification, multipotency maintenance and vitality of HSCs.

Deletion of the gene for the membrane-bound acid phosphatase of Leishmania mexicana.

The membrane-bound acid phosphatase of Leishmania mexicana (LmxMBAP) has been shown to be a heterogeneously N-glycosylated type I transmembrane protein, which is localized predominantly in vesicular structures close to the flagellar pocket in promastigotes and amastigotes. Its expression in both life stages prompted us to analyse its function by performing deletion analysis. Both alleles of the single copy gene were sequentially replaced by resistance marker genes and the resulting deletion mutant was tested for its potential to infect Balb/c mice and peritoneal macrophages. There was no obvious difference detectable between the mutant and the wild-type. Therefore, we conclude that LmxMBAP is neither involved in the infection process nor required for amastigote survival in the infected host cell. LmxMBAP null mutant promastigotes were used to establish a system for homogeneous overexpression of LmxMBAP which will be useful to investigate protein sorting in L. mexicana.

The complete nucleotide sequence and functional organization of Bacillus subtilis bacteriophage SPP1.

The complete nucleotide sequence of the B. subtilis bacteriophage SPP1 is described. The genome is 44,007 bp in size and has a base composition of 43.7% dG + dC. Only 32.2 kb are essential for phage amplification under laboratory conditions. Transcription using only the 'heavy strand' is asymmetric. Eighty-one orfs organized in five early and four late operons were identified. Experiments have shown that 25 orfs are essential. Of the remaining orfs, functions could be predicted for the products of five of the orfs on the basis of comparison of the deduced amino acid sequence to known proteins. Intergenic regions include most of the 5 PE and the 4 PL promoters. Transcripts are polycistronic. Transcription from the PE promoters is mediated by host RP, whereas recognition of the PL promoters requires an additional unidentified phage-encoded product. Translation of mRNA transcribed from most of the orfs seems to be initiated independently, each from its own ribosomal binding and initiation site, although a few cases of coupled translation have been reported. The organization of SPP1 genes involved in the replication, DNA packaging and phage assembly proteins resembles the organization of genes of equivalent regions of different E. coli double-stranded DNA phages. Absence of aa sequence similarity between analogous proteins of different phages suggested that the conserved gene organization is representative of a primordial bacteriophage.

Nucleotide sequence and complementation studies of the gene 35 region of the Bacillus subtilis bacteriophage SPP1.

Genetic analysis of the Bacillus subtilis bacteriophage SPP1 defective in gene 35 shows that the gene 35 product (G35P) is essential for SPP1 growth. The defect in growth of SPP1tsl17 and SPP1tsl20F at nonpermissive temperature is overcome by wild-type gene 35 expressed from a plasmid. The region where gene 35 maps was cloned and sequenced. Analysis of the nucleotide sequence (5884-bp) around gene 35 revealed 13 open reading frames (orfs). We have assigned the term gene to three of these orfs; gene 35, gene 36, the product of which shares homology with SSB proteins, and gene 38, given the gene order orf 34-orf 34.1-orf 34.2-orf34.3-orf34.4-gene 35-gene 36-orf 36.1-orf 37-orf 37.1-orf 37.2-orf 37.3-gene 38. Gene 35 encodes a protein of 32.0 kDa. By using the T7 promoter-expression system for gene 35 a radioactive band of the expected molecular mass was detected.

Analysis of cis and trans acting elements required for the initiation of DNA replication in the Bacillus subtilis bacteriophage SPP1.

The development of SPP1 has been studied in several B. subtilis mutants conditionally defective in initiation of DNA replication. Initiation of SPP1 replication is independent of the host DnaA (replisome organizer), DnaB, DnaC and DnaI products, but requires the DnaG (DNA primase) and the DNA gyrase. Furthermore, SPP1 replication is independent of the DnaK (heat shock) protein. The phage-encoded products required for initiation of SPP1 replication have been genetically characterized. Analysis of the nucleotide sequence (3.292 kilobases) of the region where SPP1 initiation replication mutants map, revealed five open reading frames (orf). We have assigned genes 38, 39 and 40 to three of these orfs, which have the successive order gene 38-gene 39-orf39,1-gene 40-orf41. The direction of transcription of the reading frames, the lengths of the mRNAs as well as the transcription start point, upstream of gene 38 (PE2), were identified. Proteins of 29.9, 14.6 and 46.6 kDa were anticipated from translation of gene 38, gene 39 and gene 40, respectively. The purified G38P and G39P have estimated molecular masses of 31 and 15 kDa. G38P and G39P do not share significant identity with primary protein sequences currently available in protein databases, whereas G40P shares substantial homology with a family of DNA primase-associated DNA helicases. G38P binds specifically to two discrete SPP1 DNA restriction fragments (EcoRI-4 and EcoRI-3). The G38P binding site on EcoRI-4 was localized on a 393 bp DNA segment, which lies within the coding sequence of gene 38. The putative binding site on EcoRI-3 was inferred by DNA sequence homology, it maps in a non-coding segment. G39P, which does not bind to DNA, is able to form a complex with G38P. The organization of the SPP1 genes in the gene 38 to gene 40 interval resembles that one found in the replication origin regions of different Escherichia coli double-stranded DNA phages (lambda, phi 80 and P22). We propose that the conserved gene organization is representative of the replication origin region of a primordial phage.