Electrochemical Beacon Method to Quantify 10 Attomolar Nucleic Acids with a Semilog Dynamic Range of 7 Orders of Magnitude.

Change in the dynamics of single-stranded DNA or RNA probes tethered to an Au electrode on immunospecific binding to the analyte is a versatile approach to quantify a variety of molecules, such as heavy metal ions, pesticides, proteins, and nucleic acids (NAs). A widely studied approach is the electrochemical beacon method where the redox of a dye attached to the probe decreases as its proximity to the underlying electrode changes on binding. The limit of quantification (LOQ) defined by the semilog dependence of the signal on target concentration is in the picomolar range. Here, a method was studied where, by differential reflectivity, multiple reactions were measured on a monolith electrode. An alternative contrast mechanism was discovered, which led to an approach to enhance the LOQ to 10 aM and increase the dynamic range to 7 orders of magnitude using similar probes and binding conditions. Quantitative analysis on sequences with the G-C fraction ranging from 37 to 72% was performed. The approach will allow for the development of a label-free, enzyme-free microarray to detect biomolecules including NAs and proteins on a single electrode at quantification from 10 aM to 0.1 nM with high specificity.

Electrochemical Characteristics of a DNA Modified Electrode as a Function of Percent Binding.

Electrochemical characteristics of immobilized double-stranded DNA (dsDNA) on a Au electrode were studied as a function of coverage using a home-built optoelectrochemical method. The method allows probing of local redox processes on a 6 µm spot by measuring both differential reflectivity (SEED-R) and interferometry (SEED-I). The former is sensitive to redox ions that tend to adsorb to the electrode, while SEED-I is sensitive to nonadsorbing ions. The redox reaction maxima, Rmax and Δmax from SEED-R and SEED-I, respectively, are linearly proportional to amperometric peak current, Imax. The DNA binding is measured by a redox active dye, methylene blue, that intercalates in dsDNA, leading to an Rmax. Concomitantly, the absence of Δmax for [Fe(CN)6]4-/3- by SEED-I ensures that there is no leakage current from voids/defects in the alkanethiol passivation layer at the same spot of measurement. The binding was regulated electrochemically to obtain the binding fraction, f, ranging about three orders of magnitude. A remarkably sharp transition, f = fT = 1.25 × 10-3, was observed. Below fT, dsDNA molecules behaved as individual single-molecule nanoelectrodes. Above the crossover transition, Rmax, per dsDNA molecule dropped rapidly as f-1/2 toward a planar-like monolayer. The SEED-R peak at f ∼ 3.3 × 10-4 (∼270 dsDNA molecules) was (statistically) robust, corresponding to a responsivity of ∼0.45 zeptomoles of dsDNA/spot. Differential pulse voltammetry in the single-molecule regime estimated that the current per dsDNA molecule was ∼4.1 fA. Compared with published amperometric results, the reported semilogarithmic dependence on target concentration is in the f > fT regime.

Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile.

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.

Quantitative PCR of Small Nucleic Acids: Size Matters.

Quantitative dysregulation in small nucleic acids (NA), such as microRNA (miRNA), extracted from minimally invasive biopsies, such as, blood, stool, urine, nose, throat, are promising biomarker for diseases diagnosis and management. We quantify the effect of the extra step of poly(A) ligation for cDNA synthesis and small size of the NA on the limit of quantification (LOQ) of quantitative PCR (qPCR), the gold standard to measure copy number. It was discovered that for small NA, the cycle threshold, Ct that is proportional to -log[c], where [c] is the concentration of the target NA exhibits a sharp transition. The results indicate that although the limit of detection (LOD) of qPCR can be in femtomolar range, the LOQ is significantly reduced by well over three orders of magnitude, in picomolar range. Specifically, the study reveals that the PCR product length is the primary reason the limitation on LOQ and is explicitly shown to be an important consideration for primer design for qPCR in general.

Evolution of DDB1-binding WD40 (DWD) in the viridiplantae.

Damaged DNA Binding 1 (DDB1)-binding WD40 (DWD) proteins are highly conserved and involved in a plethora of developmental and physiological processes such as flowering time control, photomorphogenesis, and abiotic stress responses. The phylogeny of this family of proteins in plants and algae of viridiplante is a critical area to understand the emergence of this family in such important and diverse functions. We aimed to investigate the putative homologs of DWD in the viridiplante and establish a deeper DWD evolutionary grasp. The advancement in publicly available genomic data allowed us to perform an extensive genome-wide DWD retrieval. Using annotated Arabidopsis thaliana DWDs as the reference, we generated and characterized a comprehensive DWD database for the studied photoautotrophs. Further, a generic DWD classification system (Type A to K), based on (i) position of DWD motifs, (ii) number of DWD motifs, and (iii) presence/absence of other domains, was adopted. About 72-80% DWDs have one DWD motif, whereas 17-24% DWDs have two and 0.5-4.7% DWDs have three DWD motifs. Neighbor-joining phylogenetic construction of A. thaliana DWDs facilitated us to tune these substrate receptors into 15 groups. Though the DWD count increases from microalgae to higher land plants, the ratio of DWD to WD40 remained constant throughout the viridiplante. The DWD expansion appeared to be the consequence of consistent DWD genetic flow accompanied by several gene duplication events. The network, phylogenetic, and statistical analysis delineated DWD evolutionary relevance in the viridiplante.

Induction of oil accumulation by heat stress is metabolically distinct from N stress in the green microalgae Coccomyxa subellipsoidea C169.

Algae are often promoted as feedstock organisms to produce a sustainable petroleum fossil fuel alternative. However, to induce lipid accumulation most often requires a severe stress that is difficult to induce in large batch cultures. The objective of this study is to analyze and mathematically model heat stress on growth, chlorophyll content, triacylglyceride, and starch synthesis in algae. We initially screened 30 algal species for the most pronounced induction of lipid droplets from heat stress using confocal microscopy and mass spectroscopy techniques. One species, Coccomyxa subellipsoidea C169, was selected and subjected to further biochemical analyses using a jacketed bioreactor amended with 1% CO2 at 25°C, 30°C, 32°C, 33°C, 34°C, 35°C, and 36°C. Lipid and starch accumulation was less extreme than N stress. Growth was reduced above 25°C, but heat stress induced lipid droplet synthesis was negatively correlated with growth only past a demonstrated threshold temperature above 32°C. The optimal temperature for lipid accumulation was 35°C, which led to 6% of dry weight triglyceride content and a 72% reduction from optimal growth after 5 days. Fatty acid influx rates into triglycerides and 15N labeling of amino acids and proteins indicate that heat stress is mechanistically distinct from N stress. Thus, this study lends support to a novel hypothesis that lipid droplet triglycerides result from a redistribution of carbon flux as fatty acids to neutral storage lipids over membrane or other lipids.

Quantitative Electrochemical DNA Microarray on a Monolith Electrode with Ten Attomolar Sensitivity, 100% Specificity, and Zero Background.

Circulating microRNA are promising diagnostic and prognostic biomarkers of disease in quantitative blood tests. A label-free, PCR-free, electrochemical microarray technology on a monolith electrode is described, with 10 attomolar (aM) sensitivity and responsiveness to binding of <1 zeptomole of target to immobilized ssDNA probes with zero background. Specificity is 100% in a mixture with five nonspecific miRNA each with a 103-fold higher concentration. Direct measurement on plasma-derived miRNA without cDNA conversion and PCR demonstrated multiplexing and near-ideal quantitative correlation with an equivalent pure sample. The dynamic range is a target concentration ranging from 10-2 to 103 femtomolar (fM). This PCR-free novel technology can be applied as a test for cancer diagnosis/prognosis to detect 103 copies of a miRNA sequence in RNA extracted from 100 µL of plasma.

Pathogenicity and transmission of triple reassortant H3N2 swine influenza A viruses is attenuated following Turkey embryo propagation.

Genetic lineages of swine influenza A viruses (SIVs) have recently been established in Turkeys in the United States. To identify molecular determinants that are involved in virulence and transmission of SIVs to Turkeys, we sequentially passaged two triple reassortant H3N2 SIV isolates from Minnesota in ten day old specific-pathogen free (SPF) Turkey embryos and tested them in seven-day old Turkey poults. We found that SIV replication in Turkey embryos led to minimal mutations in and around the receptor binding and antigenic sites of the HA molecule, while other gene segments were unchanged. The predominant changes associated with Turkey embryo passage were A223V, V226A and T248I mutations in the receptor-binding and glycosylation sites of the HA molecule. Furthermore, Turkey embryo propagation altered receptor specificity in SIV strain 07-1145. Embryo passaged 07-1145 virus showed a decrease in α2, 6 sialic acid receptor binding compared to the wild type virus. Intranasal infection of wild type SIVs in one-week-old Turkey poults resulted in persistent diarrhea and all the infected birds seroconverted at ten days post infection. The 07-1145 wild type virus also transmitted to age matched in-contact birds introduced one-day post infection. Turkeys infected with embryo passaged viruses displayed no clinical signs and were not transmitted to in-contact poults. Our results suggest that Turkey embryo propagation attenuates recent TR SIVs for infectivity and transmission in one week old Turkeys. Our findings will have important implications in identifying molecular determinants that control the transmission and virulence of TR SIVs in Turkeys and other species.

Modeling of rhythmic behavior in neutral lipid production due to continuous supply of limited nitrogen: mutual growth and lipid accumulation in microalgae.

The relative effects of three precise nitrogen limitation regimes on green micro-algae were assessed using the Trebouxiophycean alga Coccomyxa subellipsoidea grown in a chemostatic bioreactor system. The data provides further evidence that growth and triglyceride (TAG) accumulation are concurrent and independently proportional to the degree of nitrogen limitation in algae. Additionally, TAG accumulation was observed to proceed via oscillations with respect to time and percent dry weight quantity. The predator-prey model was applied to fit the experimental data and to obtain the physiological significance of these oscillations. The results determine the conditions of maximum neutral lipid productivity with respect to nitrate stress and highlight an area of potential future research.

Kinetic modeling of photoautotropic growth and neutral lipid accumulation in terms of ammonium concentration in Chlamydomonas reinhardtii.

This study focuses on the cell growth and the neutral lipid production modeling of Chlamydomonas reinhardtii in terms of different ammonium concentrations. Autotrophy was maintained during growth in a double walled bioreactor, using Tris Phosphate (TP medium) with only CO(2) and NH(4)Cl as sole sources of carbon and nitrogen, respectively. Nitrogen depletion results in an increase in neutral lipid production with an indirect effect on the growth of algal cells. Modified Baranyi-Roberts and logistic equations were used to describe the cell growth whereas Luedeking-Piret equation was used for neutral lipid production kinetics. Sensitivity analysis shows that the model equations satisfactorily predict the cell growth and lipid production. Based on the mathematical model predictions, growing algal cells in higher ammonium containing medium initially and switching to low ammonium containing medium in a later stage may result in elevated amounts of lipid production, which may be used for scale up and commercialization.