PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be added to a PCR assay; There are not likely to be contaminants in ISSA recycled water that would inhibit PCR resulting in false-negative results; The TaqMan PCR product detection system is the most promising method for developing a rapid, highly automated gene-based microbial monitoring system. The method is inherently quantitative. NASA and other government agencies have invested in other technologies that, although potentially could lead to revolutionary advances, are not likely to mature in the next 5 years into working systems; PCR-based methods cannot distinguish between DNA or RNA of a viable microorganism and that of a non-viable organism. This may or may not be an important issue with reclaimed water on the ISSA. The recycling system probably damages the capacity of the genetic material of any bacteria or viruses killed during processing to serve as a template in a PCR desinged to amplify a large segment of DNA (less than 650 base pairs). If necessary, vital dye staining could be used in addition to PCR, to enumerate the viable cells in a water sample; The quality control methods have been developed to insure that PCR's are working properly, and that reactions are not contaminated with PCR carryover products which could lead to the generation of false-positive results; and The sequences of the small rRNA subunit gene for a large number of microorganisms are known, and they consititue the best database for rational development of the oligonucleotide reagents that give PCR its great specificity. From those gene sequences, sets of oligonucleotide primers for PCR and Taqman detection that could be used in a NASA microbial monitor were constructed using computer based methods. In addition to space utilization, a microbial monitior will have tremendous terrestrial applications. Analysis of patient samples for microbial pathogens, testing industrial effluent for biofouling bacteria, and detection biological warfare agents on the battlefield are but a few of the diverse potential uses for this technology. Once fully developed, gene-based microbial monitors will become the fundamental tool in every lab that tests for microbial contaminants, and serve as a powerful weapon in mankind's war with the germ world

A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes

<p>Abstract</p> <p>Background</p> <p>Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, <it>Mollicutes</it>. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins.</p> <p>Results</p> <p>Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the <it>Mollicutes</it>. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling.</p> <p>Conclusions</p> <p>We describe novel features of PARCELs (<b>P</b>alindromic <b>A</b>mphipathic <b>R</b>epeat <b>C</b>oding <b>EL</b>ements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches.</p

Sexually-dimorphic targeting of functionally-related genes in COPD

Background: There is growing evidence that many diseases develop, progress, and respond to therapy differently in men and women. This variability may manifest as a result of sex-specific structures in gene regulatory networks that influence how those networks operate. However, there are few methods to identify and characterize differences in network structure, slowing progress in understanding mechanisms driving sexual dimorphism. Results: Here we apply an integrative network inference method, PANDA (Passing Attributes between Networks for Data Assimilation), to model sex-specific networks in blood and sputum samples from subjects with Chronic Obstructive Pulmonary Disease (COPD). We used a jack-knifing approach to build an ensemble of likely networks for each sex. By adapting statistical methods to compare these network ensembles, we were able to identify strong differential-targeting patterns associated with functionally-related sets of genes, including those involved in mitochondrial function and energy metabolism. Network analysis also identified several potential sex- and disease-specific transcriptional regulators of these pathways. Conclusions: Network analysis yielded insight into potential mechanisms driving sexual dimorphism in COPD that were not evident from gene expression analysis alone. We believe our ensemble approach to network analysis provides a principled way to capture sex-specific regulatory relationships and could be applied to identify differences in gene regulatory patterns in a wide variety of diseases and contexts. Electronic supplementary material The online version of this article (doi:10.1186/s12918-014-0118-y) contains supplementary material, which is available to authorized users

Hybrid corrosion protection of a prestressed concrete bridge

The Kyle of Tongue Bridge in Sutherland, Northern Scotland opened in 1970, has an overall span of 184m of 18 approximately equal spans and carries a single lane dual carriageway. The bridge was repaired in 1989 due to chloride induced corrosion. However, inspections from 1999 onwards reported on-going corrosion and structural deterioration. A refurbishment contract was let in 2011 to extend the service life of the structure for a 20 year period by providing corrosion arrest and prevention. This paper describes how hybrid corrosion protection was used to offer protection to the prestressed concrete beams of the bridge. The results indicate that hybrid anodes provide an attractive alternative to other corrosion protection systems as they can be targeted to specific areas of need. They offer a temporary energising phase to arrest corrosion, followed by a permanent galvanic mode phase which is particularly beneficial for prestressed concrete structures in order to reduce the risk of hydrogen embrittlement

Constraints and barriers to the application of geosynthetics

This paper outlines the current state of practice in the UK with regard to the use of geosynthetics in construction projects. It examines the awareness that geotechnical engineers possess about the technical and sustainability (i.e. reduced CO2) advantages that geosynthetics can provide over traditional construction techniques, and the barriers to their increased use. A survey of UK International Geosynthetic Society (IGS) Corporate Sponsors was carried out to understand the perceived constraints and barriers to increased early stage inclusion of geosynthetic based design solutions, and to obtain views on the current awareness of the UK construction industry of the sustainability benefits. The findings from the survey highlight a lack of clarity in the guidance on fill material provided in the literature. The survey results also showed that main barriers to the use of geosynthetics were education and the conservative approach of consultants

Measuring Distances Using Infrared Surface Brightness Fluctuations

Surface brightness fluctuations (SBFs) are much brighter in the IR than they are at optical wavelengths, making it possible to measure greater distances using IR SBFs. We report new K' (2.1 micron) SBF measurements of 9 galaxies in the Fornax and Eridanus clusters using a 1024^2-pixel HgCdTe array. We used improved analysis techniques to remove contributions from globular clusters and background galaxies, and we assess the relative importance of other sources of residual variance. We applied the improved methodology to Fornax and Eridanus images and to previously published Virgo cluster data. Apparent fluctuation magnitudes were used in conjunction with Cepheid distances to M31 and the Virgo cluster to calibrate the K' SBF distance scale. We find the absolute fluctuation magnitude MK'= -5.61+/-0.12, with an intrinsic scatter to the calibration of 0.06 mag. No statistically significant change in MK' is detected as a function of (V-I). Our calibration is consistent with constant age and metallicity stellar population models. The lack of a correlation with (V-I) in the context of the stellar population models implies that elliptical galaxies bluer than (V-I)=1.2 have SBFs dominated by younger (5-8 Gyr) populations. K' SBFs prove to be a reliable distance indicator as long as the residual variance from globular clusters and background galaxies is properly removed. Also, it is important that a sufficiently high S/N ratio be achieved to allow reliable sky subtraction because residual spatial variance can bias the measurement of the SBF power spectrum. (abridged)Comment: Accepted for publication in ApJ, 44 pages, 10 Postscript figure

Color Transformations for the 2MASS Second Incremental Data Release

Transformation equations are presented to convert colors and magnitudes measured in the AAO, ARNICA, CIT, DENIS, ESO, LCO (Persson standards), MSSSO, SAAO, and UKIRT photometric systems to the photometric system inherent to the 2MASS Second Incremental Data Release. The transformations have been derived by comparing 2MASS photometry with published magnitudes and colors for stars observed in these systems. Transformation equations have also been derived indirectly for the Bessell & Brett (1988) and Koornneef (1983) homogenized photometric systems.Comment: To appear in AJ, May 200