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The discovery of non-coding RNA (ncRNA) has been mainly limited to laboratory model systems and human pathogenic bacteria. In this study, we begin to explore the ncRNA diversity in four recently sequenced environmental Vibrio species (Vibrio alginolyficus 40B, Vibrio communis 1DA3, Vibrio mimicus VM573 and Vibrio campbellii BAA-1116) by performing in silico searches using Infernal and Rfam for the identification of putative ncRNA-encoding genes. This search method resulted in the identification of 31-38 putative ncRNA genes per species and the total ncRNA catalogue spanned an assortment of regulatory mechanisms (riboswitches, cis-encoded ncRNAs, trans-encoded ncRNAs, modulators of protein activity, ribonucleoproteins, transcription termination ncRNAs and unknown). We chose to experimentally validate the identifications for V. campbellii BAA-1116 using a microarray-based expression profiling strategy. Transcript hybridization to tiled probes targeting annotated V. campbellii BAA-1116 intergenic regions revealed that 21 of the 38 predicted ncRNA genes were expressed in mid-exponential-phase cultures grown in nutrient-rich medium. The microarray findings were confirmed by testing a subset of three highly expressed (6S, tmRNA and TPP-2) and three moderately expressed (CsrB, GcvB and purine) ncRNAs via reverse transcription PCR. Our findings provide new information on the diversity of ncRNA in environmental vibrios while simultaneously promoting a more accurate annotation of genomic intergenic regions. DOI 10.1099/mic.0.039149-0

Proanthocyanidins (PACs) are an abundant class of compounds found in a variety of plant materials. Here we demonstrate the application of these materials as capture molecules for the removal of bacterial lipopolysaccharide (LPS) from solution. PACs from whole cranberries, grape juice, black tea, and cranberry juice were purified and immobilized onto thiol-activated Sepharose beads. This material was used in pull-down type assays for the capture of LPS. The binding of LPS by PACs has been shown to compete with that of polymyxin B which is known to bind the lipid A component of LPS. Assays conducted in the presence of lipid A verified that at least some component of the LPS binding activity of the PACs is via the lipid A moiety. Molar comparison of polymyxin B to proanthocyanidins indicated that the Sepharose immobilized PACs have a binding affinity for LPS similar to that of polymyxin B.

Rapid and accurate differential determination of viable versus nonviable microbes is critical for formulation of an appropriate response after pathogen detection. Sensors for rapid bacterial identification can be used for applications ranging from environmental monitoring and homeland defense to food process monitoring, but few provide viability information. This study combines the rapid screening capability of the array biosensor using an immunoassay format with methods for determination of viability. Additionally, cells captured by the immobilized antibodies can be cultured following fluorescence imaging to further confirm viability and for cell population expansion for further characterization, e.g., strain identification or antibiotic susceptibility testing. Finally, we demonstrate analysis of captured bacteria using the polymerase chain reaction (PCR). PCR results for wavegnide-captured cells were 3 orders of magnitude more sensitive than the fluorescence immunoassay and can also provide additional genetic information on the captured microbes. These approaches can be used to rapidly detect and distinguish viable versus nonviable and pathogenic versus nonpathogenic captured organisms, provide culture materials for further analysis on a shorter time scale, and assess the efficacy of decontamination or sterilization procedures.

Despite the success of the adenovirus vaccine administered to US military trainees, acute respiratory disease (ARD) surveillance still detected breakthrough infections (respiratory illnesses associated with the adenovirus serotypes specifically targeted [...]

Identification of genetic variations of influenza viruses is essential for epidemic and pandemic outbreak surveillance and determination of vaccine strain selection. In this study, we combined a random amplification strategy [...]

The expression of recombinant antibody fragments on the surface of mammalian cells has recently emerged as a therapeutic strategy, particularly in the treatment of a number of cancers. Screening technologies that allow for the facile characterization of fragments expressed on the cell surface would hasten the identification and isolation of reagents to be used as therapeutics. In this report, we describe a cellular microarray-based platform for the comparative functional analysis of single-chain antibodies (scFvs) expressed on the plasma membrane of mammalian cells. Using the anti-fluorescein monoclonal antibody 4-4-20 as a model system, the native binding site and three mutants were expressed as scFvs on the membrane of HEK 293T/17 cells in a microarray format. Collectively, the equilibrium dissociation constants of the soluble forms of the wild-type scFv and the three mutants spanned nearly 3 orders of magnitude. Expression of the scFvs on the surface of mammalian cells was achieved by the deposition of plasmid DNAs in micrometer-sized spots onto the surface of a glass microscope slide. The addition of cells to the printed array resulted in the expression of the scFvs in clusters of cells in spatially discrete locations. Ligand binding assays performed with a fluorescein--bovine serum albumin conjugate demonstrated the ability of the transfected cell microarray to differentiate the relative binding affinities of the expressed scFvs. Further, the apparent affinities of the membrane-displayed scFvs were within 10-fold of those reported for the soluble forms of the scFvs. The assays described herein demonstrate the potential for cellular microarrays to be used for the high-throughput screening of potential therapeutic reagents. More generally, our work details the utility of transfected cell microarrays in mediating the functional characterization of expressed membrane receptor proteins.

Outbreaks of infection with chikungunya virus (CHIKV), an alphavirus that is transmitted by bites of infected Aedes spp. mosquitoes, were frequent in sub-Saharan Africa and southern and Southeast Asia during [...]

Research into the control of the expression of glucose-6-phosphatase (G6Pase), the vital enzyme in glucose homeostasis, has led to the identification of three activation elements (AEs) within the nucleotide -234 to +3 region of the G6Pase promoter. These AEs are vital for optimal and liver-specific expression of the G6Pase gene. The promoter of G6Pase seems to be complex, permitting it to maintain tight regulation of glucose homeostatis in response to various stimuli.

Abstract Background Resource-limited tropical countries are home to numerous infectious pathogens of both human and zoonotic origin. A capability for early detection to allow rapid outbreak containment and prevent spread to non-endemic regions is severely impaired by inadequate diagnostic laboratory capacity, the absence of a “cold chain” and the lack of highly trained personnel. Building up detection capacity in these countries by direct replication of the systems existing in developed countries is not a feasible approach and instead requires “leapfrogging” to the deployment of the newest diagnostic systems that do not have the infrastructure requirements of systems used in developed countries. Methods A laboratory for molecular diagnostics of infectious agents was established in Bo, Sierra Leone with a hybrid solar/diesel/battery system to ensure stable power supply and a satellite modem to enable efficient communication. An array of room temperature stabilization and refrigeration technologies for reliable transport and storage of reagents and biological samples were also tested to ensure sustainable laboratory supplies for diagnostic assays. Results The laboratory demonstrated its operational proficiency by conducting an investigation of a suspected avian influenza outbreak at a commercial poultry farm at Bo using broad range resequencing microarrays and real time RT-PCR. The results of the investigation excluded influenza viruses as a possible cause of the outbreak and indicated a link between the outbreak and the presence of Klebsiella pneumoniae. Conclusions This study demonstrated that by application of a carefully selected set of technologies and sufficient personnel training, it is feasible to deploy and effectively use a broad-range infectious pathogen detection technology in a severely resource-limited setting.

Abstract Background Spatial epidemiology is useful but difficult to apply in developing countries due to the low availability of digitized maps and address systems, accurate population distributions, and computational tools. A community-based mapping approach was used to demonstrate that participatory geographic information system (PGIS) techniques can provide information helpful for health and community development. Results The PGIS process allowed for the rapid determination of sectional (neighborhood) boundaries within the city of Bo, Sierra Leone. When combined with data about hospital laboratory visits, a catchment area for one hospital in Bo could be established. A survey of households from within the catchment area determined that the average population per household (about 6 individuals) was similar to that found in the 2004 census. However, we also found that the average house was inhabited by more than one household, for an average of 17.5 inhabitants per residential building, which is critical information to know when estimating population size using remote imagery that can detect and enumerate buildings. Conclusions The methods developed in this paper serve as a model for the involvement of communities in the generation of municipal maps and their application to community and health concerns.

Abstract Background The detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids. This selective removal can be performed using a magnetic bead-based solid phase with covalently immobilized capture probes. One of the requirements to attain efficient host background nucleic acids subtraction is the capture probe characteristics. Findings In this study we investigate how various capture probe characteristics influence the subtraction efficiency. While the primary focus of this report is the impact of probe length, we also studied the impact of probe conformation as well as the amount of capture probe attached to the solid phase. The probes were immobilized on magnetic microbeads functionalized with a phosphorous dendrimer. The subtraction efficiency was assessed by quantitative real time PCR using a single-step capture protocol and genomic DNA as target. Our results indicate that short probes (100 to 200 bp) exhibit the best subtraction efficiency. Additionally, higher subtraction efficiencies with these probes were obtained as the amount of probe immobilized on the solid phase decreased. Under optimal probes condition, our protocol showed a 90 - 95% subtraction efficiency of human genomic DNA. Conclusions The characteristics of the capture probe are important for the design of efficient solid phases. The length, conformation and abundance of the probes determine the capture efficiency of the solid phase.

Abstract Background The inhibition of Escherichia coli growth in the presence of Vaccinium macrocarpon has been extensively described; however, the mechanisms of this activity are not well characterized. Findings Here, E. coli was grown in media spiked with cranberry juice. The growth rate and media pH were monitored over more than 300 generations. The pH of the growth media was found to decrease during cell growth. This result was unique to media spiked with cranberry juice and was not reproduced through the addition of sugars, proanthocyanidins, or metal chelators to growth media. Conclusion This study demonstrated that factors other than sugars or proanthocyanidins in cranberry juice result in acidification of the growth media. Further studies are necessary for a complete understanding of the antimicrobial activity of cranberry products.

Abstract Background Febrile respiratory illness (FRI) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. A particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (HRV) and enteroviruses (HEV) which are frequent causes of FRI. Resequencing Pathogen Microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking. Results Using HRV and HEV as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. A minimal number of probe sequences (26 for HRV and 13 for HEV), which were potentially capable of detecting all serotypes of HRV and HEV, were determined and implemented on the Resequencing Pathogen Microarray RPM-Flu v.30/31 (Tessarae RPM-Flu). The specificities of designed probes were validated using 34 HRV and 28 HEV strains. All strains were successfully detected and identified at least to species level. 33 HRV strains and 16 HEV strains could be further differentiated to serotype level. Conclusion This study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse RNA viruses with a minimal number of prototype sequences. The results demonstrated that the newly designed RPM-Flu v.30/31 can provide comprehensive and specific analysis of HRV and HEV samples which implicates that this design strategy will be applicable for other genetically diverse viruses.

1. Introduction The detection and analysis of low-abundance nucleic acids (NAs) in complex host-pathogen mixtures requires the enrichment of the target of interest prior to any downstream process which means [...], The abundance of mammalian 18S and 28S ribosomal RNA can decrease the detection sensitivity of bacterial or viral targets in complex host-pathogen mixtures. A method to capture human RNA in a single step was developed and characterized to address this issue. For this purpose, capture probes were covalently attached to magnetic microbeads using a dendrimer linker and the solid phase was tested using rat thymus RNA (mammalian components) with Escherichia coli RNA (bacterial target) as a model system. Our results indicated that random capture probes demonstrated better performance than specific ones presumably by increasing the number of possible binding sites, and the use of a tetrame-thylammonium-chloride (TMA-Cl-) based buffer for the hybridization showed a beneficial effect in the selectivity. The subtraction efficiency determined through real- time RT-PCR revealed capture-efficiencies comparable with commercially available enrichment kits. The performance of the solid phase can be further fine tuned by modifying the annealing time and temperature.