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

Comparative kinetic modeling of growth and molecular hydrogen overproduction by engineered strains of \u3ci\u3eThermotoga maritima \u3c/i\u3e

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H2 production limit. The study generated a kinetic model explaining H2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equation-based model predicted that H2 production by Tma200 (0.217 mol-H2 g–1-biomass) and Tma100 (0.147 mol-H2 g–1-biomass) were higher than wild type (0.096 mol-H2 g–1 -biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h–1) and Tma100 (0.495 h–1) are compared with the wild type (0.663 h–1). A higher maintenance energy along with growth and non-growth H2 coefficients corroborate the higher H2 productivity of the engineered strains. The modeled data established a continuous fermentation system for the sustainable H2 production. (Inludes 2 supplemental figures

Comparative kinetic modeling of growth and molecular hydrogen overproduction by engineered strains of \u3ci\u3eThermotoga maritima \u3c/i\u3e

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H2 production limit. The study generated a kinetic model explaining H2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equation-based model predicted that H2 production by Tma200 (0.217 mol-H2 g–1-biomass) and Tma100 (0.147 mol-H2 g–1-biomass) were higher than wild type (0.096 mol-H2 g–1 -biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h–1) and Tma100 (0.495 h–1) are compared with the wild type (0.663 h–1). A higher maintenance energy along with growth and non-growth H2 coefficients corroborate the higher H2 productivity of the engineered strains. The modeled data established a continuous fermentation system for the sustainable H2 production. (Inludes 2 supplemental figures

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 25ÊC, 30ÊC, 32ÊC, 33ÊC, 34ÊC, 35ÊC, and 36ÊC. Lipid and starch accumulation was less extreme than N stress. Growth was reduced above 25ÊC, but heat stress induced lipid droplet synthesis was negatively correlated with growth only past a demonstrated threshold temperature above 32ÊC. The optimal temperature for lipid accumulation was 35ÊC, 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 \u3c1 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

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 magnitud

Journal of Oral Health and Craniofacial Science

ABSTRACT Clowning is a form of humour. It is an art form that invites play, interaction, and laughter. Clown Care is a programme in hospitals and medical centres involving visits from specially trained hospital clowns. Clowning helps patients to focus on something other than their illness. Olsson et al. and Spitzer suggested that clown care could create a warm climate, promote good interpersonal relationships, and relieve feelings of frustration, anxiety, or hostility. Hospital clowns work worldwide as a health humanization resort, providing interplay with patients, family and staff creating a positive emotional state that fosters affi rmative environmental conditions. This type of activity varies greatly in terms of professionalism, accountability and artistic methods. Promotion of emotional and psychosocial well-being of patients transcends opportunities for oral health promotion activities in hospitals, schools and community. Previous research reports on clown training refl ects attitudebuilding potential for the healthcare students provided that it is performed in a deep, essential, strict and continuous fashion in a facultative manner rather than mandatory allowing the student to build his own artistic, professional and personal path. Thus, the prospect of introducing training curriculum of this underrated nontechnical skill for dental students in Indian dental education system needs to be harnessed

Environmental effects on growth, neutral lipid and taurine biosynthesis in microalgae

Microalgae are photosynthetic, fast growing microorganisms that are found in a wide variety of environments including harsh conditions, and have been commonly exploited for producing food, biofuel and bioproducts. This research focuses on using microalgae for increasing neutral lipid and taurine biosynthesis in biodiesel production and as a supplement in aquaculture feedstock industry, respectively. The major issue with batch cultivation of microalgae for biodiesel production is the inverse relationship between biomass and neutral lipid accumulation under nitrogen deprivation. In the present study, the problem is addressed by continuous addition of the precise limited amount of nitrogen source, which not only was found to produce neutral lipid in growing microaglae, but also led to oscillatory patterns in lipid productivity. Further, the physiological significance of these lipid synthesis oscillations is presented. The kinetic modeling along with the stability analysis provided physiologically significant parameters in lipid biosynthesis. A nonequilibrium thermodynamic coupling approach was used to formulate the reaction-diffusion process in microalgae growth and lipid accumulation. Taurine is a free amino acid commonly present in almost all animals and has shown its nutritional significance. Using the high sensitivity of LC-MS/MS methodology, selected microalgae from marine as well as fresh water were found to synthesize taurine, but at very low levels and varying with lineage. An attempt at creating a synthetic pathway for taurine synthesis in the chloroplast of Chlamydomonas reinhardtii was pursued by trying to integrate the fused codon optimized cysteine dioxygenase and cysteine sulfinic acid decarboxylase genes designed using animal sequences. Genotyping combined with northern, western and analytical analysis concluded that the first step (cysteine to cysteine sulfinic acid) was functionally present. Further, based on bioinformatic analysis and supplementation experiments, a new pathway for taurine synthesis termed the serine/sulfate or Animal II pathway was proposed in microalgae. The overall study concluded that the influence of environmental factors and thermodynamic coupling have a high impact on the growth, lipid accumulation and taurine production in microalgae

Corrigendum to “Heavy metal ion detection on a microspot electrode using an optical electrochemical probe” [Electrochem. Commun. 86 (2018) 94–98]

The authors regret to inform an error. Ravi F. Saraf of this pub- lication discloses financial interest in Vajra Instrument as its founder and President. In accordance with its Conflict of Interest policy, the University of Nebraska-Lincoln’s Conflict of Interest in Research Committee has determined that this must be disclosed. The authors would like to apologise for any inconvenience caused

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

<div><p>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 <i>Arabidopsis thaliana</i> 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 <i>A</i>. <i>thaliana</i> 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.</p></div