Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

Purpose: Methylated cell-free fetal DNA (cffDNA) in maternal plasma can potentially be used as a biomarker for accurate noninvasive prenatal testing (NIPT) of fetal disorders. Recovery and purification of cffDNA are key steps for downstream applications. In this study, we aimed to developed and evaluated different aspects of an optimized method and compared its efficiency with common methods used for extraction of methylated cffDNA. Methods: Single factor experiments, Plackett-Burman (PB) design, and response surface methodology (RSM) were conducted for conventional Triton/Heat/Phenol (cTHP) method optimization. The total cell-free DNA (cfDNA) was extracted from pooled maternal plasma using the optimized method called the Triton/Heat/Phenol/Glycogen (THPG), cTHP method, a column-based kit, and a magnetic bead-based kit. In the next step, methylated cfDNA from the extracted total cfDNA was enriched using a methylated DNA immunoprecipitation (MeDIP) kit. Real-time quantitative polymerase chain reaction was performed on the RASSF1 gene and hyper region to determine the genomic equivalents per milliliter (GEq/ml) values of the methylated cfDNA and cffDNA, respectively. Results: The optimum values of the significant factors affecting cfDNA extraction from 200 μl of plasma were 3% SDS, 1% Triton X-100, 0.9 μg/μl glycogen, and 0.3 M sodium acetate. The GEq/ml values of methylated cffDNA extracted using the THPG method were significantly higher than for the tested extraction methods (p < 0.001). Conclusions: Our results indicate that the THPG method is more efficient than the other tested methods for extraction of low copy number methylated cffDNA from a small volume of maternal plasm

FLP/FRT Recombination from Yeast: Application of a Two Gene Cassette Scheme as an Inducible System in Plants

Phytosensors are plants that are genetically engineered for sensing and reporting the presence of a specific contaminant, including agriculturally important biological agents. Phytosensors are constructed by transforming plants to contain specific biotic- or abiotic-inducible promoters fused to a reporter gene. When such transgenic plants encounter the target biotic or abiotic agent, the specific inducible promoter is triggered and subsequently drives the expression of the reporter gene, which produces a signal for detection. However, several systems lack robustness, rapid induction and promoter strength. Here, we tested the FLP/FRT recombination system in a construct containing a two gene cassette organization and examined its potential in transgenic Arabidopsis and tobacco plants using a β-glucuronidase (GUS) reporter. In this model system, a heat-shock inducible promoter was employed to control the expression of the FLP recombinase gene. Upon heat induction and subsequent active FLP-mediated excision event, the GUS gene was placed in close proximity to the 35S promoter resulting in an active GUS reporter expression. Our results demonstrate that the two gene cassette scheme of inducible FLP/FRT recombination system is functional in tobacco and Arabidopsis, providing additional insights into its possible application in phytosensing such as creating strong readout capabilities

Evaluation of Application Effects of Nitrogen Levels under Drought Stress Condition on Physiological Characteristics and Yield of Kochia (Kochia scoparia) in Saline Agriculture

IntroductionNowadays, the cultivation of plants adapted to adverse conditions, such as drought and salinity, in the country has been considered. Meanwhile, Kochia scoparia, one of the forgotten plants, due to its classification in the group of halophytes, has specific characteristics suitable for cultivation in low-water and saline areas (Salehi, Kafi, &amp; Kiani, 2012). This plant is known as an important annual forage crop, and its grains also have high nutritional value and oil, which can be considered for future industrial applications (Salehi et al., 2012). Studies on the salinity tolerance of the Kochia plant have shown that it is suitable for cultivation in saline areas, and in terms of quantity and quality, can compete with conventional forage plants. The use of natural organic materials, such as humic acid, has received more attention. These materials, as part of soil organic matter, are influenced by physical, chemical, and microbiological changes in biological molecules (Sabzevari &amp; Khazaei, 2009; Dong, C&oacute;rdova-Kreylos, Yang, Yuan, &amp; Scow, 2009). Additionally, nitrogen is the most important element needed for plant growth and development. It is also a key component in many biological compounds, including proteins, nucleic acids, some hormones, and chlorophyll. Nitrogen plays an essential role in photosynthetic processes and the final function of plants (Kaur, Gupta, &amp; Kaur, 2002; Taiz, Zeiger, M&oslash;ller, &amp; Murphy, 2015). As a result of this research, a combination of nitrogen and humic acid can be used as nutritional resources in salt stress conditions.Materials and MethodsThis experiment was conducted in the form of split plots based on the randomized complete block design with three replications in the Saline Research Farm of Ferdowsi University of Mashhad in the 2015 growth season. The main plot included drought stress with a four-week interruption of irrigation at three levels of control (irrigation until the end of the growing season), after establishment (50 days after planting), the beginning of flowering (71 days after planting) and late flowering (82 days after planting) The subplot was included nitrogen application at three levels of zero, 100 and 200 kg.ha-1 from urea fertilizer source. The optimum level of humic acid (2 per thousand) was done as seed at the time of planting for all treatments.Results and DiscussionThe results showed that the drought stress during vegetative and reproductive growth stages had a negative effect on the Kochia plant. However, its effect in the early stages of vegetative growth (after establishment) was greater than the stress at the end of the season (late flowering). Drought stress has a negative effect on Kochia grain yield by reducing the concentration of chlorophyll a, altering the chlorophyll a to b ratio, decreasing carotenoid concentration, and affecting relative leaf water content. However, seed treatments of humic acid and its combination with 100 kg.ha-1 nitrogen level by increasing the concentration of total phenol, soluble carbohydrate concentration, and DPPH free radical scavenging capacity improved photosynthetic pigments and finally kochia grain yield. In general, the most suitable treatment for use in drought stress and saline water source conditions was the combined method of sowing humic acid seeds with 100 kg.ha-1 nitrogen fertilizer.ConclusionIn general, the occurrence of drought stress in vegetative and reproductive growth stages had a negative effect on the kochia plant. However, its effect in the early stages of vegetative growth (after establishment) was greater than the stress at the end of the season (late flowering). The most suitable treatment was using the combined method of seed of humic acid with 100 kg.ha-1 nitrogen fertilizer.

Intra-periaqueductal gray matter administration of orexin-A exaggerates pulpitis-induced anxiogenic responses and c-fos expression mainly through the interaction with orexin 1 and cannabinoid 1 receptors in rats

Different types of trigeminal pains are frequently associated with psychophysiological concerns. Orexin-A and orexin 1 receptor (OX1R) are involved in modulation of both trigeminal pain and anxiety responses. Ventrolateral periaqueductal gray matter (vlPAG), a controlling site for nociception and emotion, receives orexinergic inputs. Here, the role of vlPAG OX1Rs and their interaction with cannabinoid 1 (CB1) receptor was evaluated in anxiety-like behavior following capsaicin-induced dental pulp pain. Rats were cannulated in the vlPAG and orexin-A was injected at the doses of 0.17, 0.35 and 0.51 μg/rat prior to the induction of pain. The elevated plus maze (EPM) and open field (OF) tests were used for assessing the anxiety responses. In addition, the induction of c-fos, in the vlPAG, was investigated using immunofluorescence microscopy. Capsaicin-treated rats displayed significantly higher anxiogenic behavior on EPM and OF tests. Pretreatment with orexin-A (0.51 μg/rat) attenuated capsaicin-mediated nociception, while exaggerated anxiogenic responses (p < 0.05). In addition, orexin-A effects were diminished by the administration of OX1R (SB-334867, 12 μg/rat) and cannabinoid 1 (AM251, 4 μg/rat) receptor antagonists. Intradental capsaicin induced a significant increase in c-fos expression in the vlPAG that was exaggerated by orexin-A (0.51 μg/rat). Blockage of OX1R and CB1 receptors attenuated the effect of orexin-A on c-fos expression in capsaicin-treated rats. In conclusion, the data suggest that manipulation of OX1R and CB1 receptors in the vlPAG alters capsaicin-evoked anxiety like behaviors and c-fos induction in rats