Smartphone multiplex microcapillary diagnostics using Cygnus: Development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.     

An active precursor in assembly of yeast nuclear ribonuclease P

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.     

Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction

To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.     

Characterization of conserved sequence elements in eukaryotic RNase P RNA reveals roles in holoenzyme assembly and tRNA processing

RNase P is a ubiquitous endoribonuclease responsible for cleavage of the 5' leader of precursor tRNAs (pre-tRNAs). Although the protein composition of RNase P holoenzymes varies significantly among Bacteria, Archaea, and Eukarya, the holoenzymes have essential RNA subunits with several sequences and structural features that are common to all three kingdoms of life. Additional structural elements of the RNA subunits have been found that are conserved in eukaryotes, but not in bacteria, and might have functions specifically required by the more complex eukaryotic holoenzymes. In this study, we have mutated four eukaryotic-specific conserved regions in Saccharomyces cerevisiae nuclear RNase P RNA and characterized the effects of the mutations on cell growth, enzyme function, and biogenesis of RNase P. RNase P with mutations in each of the four regions tested is sufficiently functional to support life although growth of the resulting yeast strains was compromised to varying extents. Further analysis revealed that mutations in three different regions cause differential defects in holoenzyme assembly, localization, and pre-tRNA processing in vivo and in vitro. These data suggest that most, but not all, eukaryotic-specific conserved regions of RNase P RNA are important for the maturation and function of the holoenzyme.     

Resource Holding Potential and the Outcome of Aggressive Interactions between Paired Male <Emphasis Type="Italic">Aegus chelifer chelifer</Emphasis> (Coleoptera: Lucanidae) Stag Beetles

Interactions between male stag beetles usually involve aggressive behavior using their long mandibles as weapons to compete with rival males over females. Considerable variation exists within populations in male body size, and may affect their behavior and the outcome of male-male contests. We investigated the aggressive interactions between male Aegus chelifer chelifer, a small tropical stag beetle species. Morphological traits in relation to aggressiveness and the outcome of fights were examined in laboratory-reared beetles. The fight-engagement ratios of major and minor morph males were not significantly different and analyses revealed that the size of body parts had more effect on the fighting success than the weapon part (mandibles). The probability of winning a contest was higher in males with a larger head width (HW), and so HW was considered as the resource holding potential (RHP). No effects of the trait size on the initiation of fights or aggressive intensity was found. Relationships between the fight duration and RHP were not significantly consistent with any assessment strategies, but were close to the mutual assessment model.     

Haplotype variation and phylogeography of Rhizoctonia solani AG1-IA strains based on rDNA5.8S-ITS and ß-actin gene sequence analyses

The haplotype variation and phylogeography of Rhizoctonia solani AG1-IA strains were estimated using rDNA5.8S-ITS and ß-actin gene sequences. Two haplotypes of ITS sequences were revealed, designated as ITSa and ITSb. Thirty-four SNPs from the ß-actin gene, which displayed more allele discriminations than rDNA-ITS, were identified. The SNPs were used to classify R. solani into 12 haplotypes, designated as H1–H12. Most SNPs occurred at the third codon position and resulted as silent mutations. Three SNPs occurred at the first codon position and one SNP occurred at the second position, which these four SNPs causing non-synonymous mutations. According to the translational amino acid sequences, the 12 nucleotide-inferred haplotypes were further classified into 5 groups, designated as ITRL, VARI, VARL, VTRI, and VAMI. Analysis of the geographical distribution of 12 haplotypes, showed that H1 and H9 were widely distributed in all studied locations and existed simultaneously in the strains collected in most areas. The results lead to the speculation that H1 and H9 could be the founder haplotypes of ß-actin gene, and other haplotypes might be derived from them.     

Identification and mapping of genetic loci affecting the free-threshing habit and spike compactness in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Recombinant inbred lines of the International Triticeae Mapping Initiative (ITMI) mapping population were used to localize genetic loci that affect traits related to the free-threshing habit (percent threshability, glume tenacity, and spike fragility) and to spike morphology (spike length, spikelet number, and spike compactness) of wheat (Triticum aestivum L.). The ITMI population was planted in three environments during 1999 and 2000, and phenotypic and genotypic data were used for composite interval mapping. Two quantitative trait loci (QTL) that consistently affected threshability-associated traits were localized on chromosomes 2D and 5A. Coincident QTL on the short arm of 2D explained 44% of the variation in threshability, 17% of the variation in glume tenacity, and 42% of the variation in rachis fragility. QTL on chromosomes 2D probably represent the effect of Tg, a gene for tenacious glumes. Coincident QTL on the long arm of 5A explained 21% and 10% of the variation in glume tenacity and rachis fragility, respectively. QTL on 5A are believed to represent the effect of Q. Overall, free-threshing-related characteristics were predominantly affected by Tg and to a lesser extent by Q. Other QTL that were significantly associated with threshability-related traits in at least one environment were localized on chromosomes 2A, 2B, 6A, 6D, and 7B. Four QTL on chromosomes 1B, 4A, 6A, and 7A consistently affected spike characteristics. Coincident QTL on the short arm of chromosome 1B explained 18% and 7% of the variation in spike length and spike compactness, respectively. QTL on the long arm of 4A explained 11%, 14%, and 12% of the variation in spike length, spike compactness, and spikelet number, respectively. A QTL on the short arm of 6A explained 27% of the phenotypic variance for spike compactness, while a QTL on the long arm of 7A explained 18% of the variation in spikelet number. QTL on chromosomes 1B and 6A appear to affect spike dimensions by modulating rachis internode length, while QTL on chromosomes 4A and 7A do so by affecting the formation of spikelets. Other QTL that were significantly associated with spike morphology-related traits, in at least one environment, were localized on chromosomes 2B, 3A, 3D, 4D, and 5A.Communicated by J. Dvorak     

Development of novel fluorescent probe 3-perylene diphenylphosphine for determination of lipid hydroperoxide with fluorescent image analysis

A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, (1)H NMR, and (13)C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials.