Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.Planetary protection efforts work toward protecting (i) solar system bodies from contamination by terrestrial biological material (forward contamination), thus preserving opportunities for future scientific investigation, and (ii) the Earth from harmful contamination by materials returned from outer space (back contamination) (5). These approaches apply directly to the control and eradication of microorganisms present on the surfaces of spacecraft intended to land, orbit, fly by, or be in the vicinity of extraterrestrial bodies. Consequently, current planetary protection policies require that spacecraft be assembled and readied for launch in controlled clean room environments. To achieve these conditions and maintain compliance with good manufacturing practice regulations, robotic spacecraft components are assembled in ultraclean facilities. Much like facilities in the medical, pharmaceutical, and semiconductor sectors, National Aeronautics and Space Administration (NASA) spacecraft assembly clean rooms (SAC) are kept extremely clean and are maintained to the highest of industry standards (17). Filtered air circulation, controlled temperature and humidity, routine exposure to disinfectants and surfactants, and nutrient-limiting, oligotrophic conditions make it very challenging for microorganisms to persist in such environments, but these measures by no means eradicate biological contaminants entirely (18). Several investigations, both culture based and culture independent, have demonstrated that a variety of bacterial taxa are repeatedly isolated under clean room conditions (18, 24, 26; P. Vaishampayan, S. Osman, G. Andersen, and K. Venkateswaran, submitted for publication). However, despite a growing understanding of the diverse microbial populations present in SAC, predicting the true risk of any such microbes’ compromising the findings of extraterrestrial life detection efforts remains a significant challenge (30). A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination (33).The purification of nucleic acids, subsequent PCR amplification, and shuttling of 16S ribosomal “fingerprint” genes from noncultivable microorganisms into genetically amenable lab strains of Escherichia coli have evolved into a gold standard of molecular means to elucidate the microbial diversity in a given sample. In theory, the cloning and sequencing of 16S ribosomal genes from each and every cell present, regardless of cultivability and inclusive of novel taxa, would result in a comprehensive survey of microbial communities on the surfaces of SAC and colocated spacecraft (24, 26). Unfortunately, the full-length sequencing of all 16S rRNA genes from environmental samples would be prohibitively expensive, making the approach unfeasible for generating comprehensive phylogenetic profiles of complex microbial communities.Attempting to infer population membership from clone libraries limited to hundreds or thousands of sequences has proven to be insufficient for detecting extremely low-abundance organisms. Recent analyses of phylogenetic DNA extracted from soil, water, and air revealed that laboriously derived clone libraries severely under-represent complex bacterial communities compared to very rapid (i.e., requiring only hours) DNA microarray approaches (1, 6, 11, 23, 36). One of the reasons for this is the high sensitivity of PhyloChip methodologies, which are able to detect organisms present in amounts below 10⁻⁴ abundance of the total sample (12). Numerous validation experiments using sequence-specific PCR have confirmed that taxa identified by the microarray were indeed present in the original environmental samples, despite their absence in corresponding clone libraries (3). This highlights the utility of the method compared to classical cloning. Although the analysis of each sample by the PhyloChip provides detailed information on microbial composition, the highly parallel and reproducible nature of this array allows tracking community dynamics over time and treatment. Even without prior sequence information, PhyloChip can identify specific microbial interactions that are key to particular changing environments.A comprehensive census of the microbial communities on the surfaces in three NASA SAC supporting two distinct missions was conducted. To ensure that the maximum diversity of resident microbiota was uncovered, subsamples from each clean room surface sampling were subjected to both DNA microarray protocols and conventional cloning and sequencing of 16S rRNA genes. This study, to our knowledge the first of its kind, focused on comparing the microbial diversity profiles resulting from DNA microarray analyses and conventional cloning and sequencing of 16S rRNA genes arising from a variety of low-biomass surfaces.     

Optimization of the aminopyridopyrazinones class of PDE5 inhibitors: Discovery of 3-[(trans-4-hydroxycyclohexyl)amino]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one

We describe efforts to improve the pharmacokinetic profile of the aminopyridopyrazinone class of PDE5 inhibitors. These efforts led to the discovery of 3-[(trans-4-hydroxycyclohexyl)amino]-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one, a potent and selective inhibitor of PDE5 with an excellent PK profile.     

The phs1-3 mutation in a putative dual-specificity protein tyrosine phosphatase gene provokes hypersensitive responses to abscisic acid in Arabidopsis thaliana

The plant hormone abscisic acid (ABA) controls numerous physiological traits: dormancy and germination of seeds, senescence and resistance to abiotic stresses. In order to get more insight into the role of protein tyrosine phosphatase (PTP) in ABA signalling, we obtained eight homozygous T-DNA insertion lines in Arabidopsis thaliana PTP genes. One mutant, named phs1-3, exhibited a strong ABA-induced inhibition of germination as only 26% of its seeds germinated after 3 days instead of 92% for the Columbia (Col-0) line. Genetic and molecular analyses of phs1-3 showed that it bears a unique T-DNA insertion in the promoter of the gene and that the mutation is recessive. PHS1 expression in the mutant is about half that of the Col-0 line. The upregulation of two ABA-induced genes (At5g06760, RAB18) and the downregulation of two ABA-repressed genes (AtCLC-A, ACL) are enhanced in the phs1-3 mutant compared with the wild-type. The 'in planta' aperture of phs1-3 stomata is reduced and the inhibition of the light-induced opening of stomata by ABA is stronger in phs1-3 leaves than in Col-0 leaves. Finally, PHS1 expression is upregulated in the presence of ABA in both phs1-3 and Col-0 but more intensively in the mutant. Thus, phs1-3 is hypersensitive to ABA. Taken together, these results show that PHS1, which encodes a dual-specificity PTP, is a negative regulator of ABA signalling.     

Crystal structure of Homo sapiens PTD012 reveals a zinc-containing hydrolase fold

The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 A resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an alphabetabetaalpha four-layer topology. A metal ion residing between the central beta-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn(2+). Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn(2+) to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and beta-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate.     

Structure of the Bet3-Tpc6B core of TRAPP: two Tpc6 paralogs form trimeric complexes with Bet3 and Mum2

The transport protein particle (TRAPP) complexes are involved in the tethering process at different trafficking steps of vesicle transport. We here present the crystal structure of a human Bet3-Tpc6B heterodimer, which represents a core sub-complex in the assembly of TRAPP. We describe a conserved patch of Tpc6 with uncharged pockets, forming a putative interaction interface for an anchoring moiety at the Golgi. The structural and functional comparison of the two paralogs Tpc6A and Tpc6B, only found in some organisms, indicates redundancy and added complexity of TRAPP architecture and function. Both iso-complexes, Bet3-Tpc6A and Bet3-Tpc6B, are able to recruit Mum2, a further TRAPP subunit, and we identify the alpha1-alpha2 loop regions as a binding site for Mum2. Our study reveals similar stability of the iso-complexes and similar expression patterns of the tpc6 variants in different mouse organs. These findings raise the possibility that the Tpc6 paralogs might contribute to the formation of two distinct TRAPP complexes that differ in function.     

Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.     

Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.