Potentiometric sensor for the nanoscale monitoring of Ni²⁺ ion in environmental samples by the fabrication of coated pyrolytic graphite electrode based on a novel C–C-coupled compound

<p>Novel ligand 5,5ʹ-((3-nitrophenyl)methylene)bis(2,6-diaminopyrimidin-4(3H)-one) (L) was synthesised and characterised. Preliminary studies on L have showed that it has more affinity towards the Ni²⁺ ion. Thus, the L was used as the electroactive material in the fabrication of poly(vinyl chloride) (PVC)-based membrane sensors such as coated graphite electrode (CGE) and coated pyrolytic graphite electrode (CPGE). Several polymeric membranes were fabricated by incorporating L as ionophore, NaTPB as anion excluders and BA, 1-CN, DBP, DOP and <i>o</i>-NPOE as solvent mediators and their effect on potentiometric response studied. Comparative electroanalytical studies performed on the CGE and CPGE depict that the CPGE with optimised membrane composition of L:PVC:<i>o</i>-NPOE:NaTPB in the ratio of 7:33:58:2 (w/w, mg) exhibited the best response in terms of wide working concentration range from 2.0 × 10⁻⁸ to 1.0 × 10⁻¹ mol L⁻¹, (3.64 µgL⁻¹ –18.2 g L⁻¹) lower detection limit of 8.1 × 10^–⁹ mol L⁻¹ (1.47 µg L⁻¹) with Nernstian compliance of 29.4 ± 0.2 mV decade⁻¹ of activity of Ni²⁺ ion in the pH range of 3.5–9.0. The sensor can work satisfactorily in water–acetonitrile and water–methanol mixtures. It can tolerate 30% acetonitrile and 20% methanol content in the mixtures. The sensor showed fast response time of 8 s and could be used successfully for a period of 4 months. The sensor reflects its utility in the quantification of Ni²⁺ ion in real samples and has been successfully employed as an indicator electrode in the potentiometric titration of Ni²⁺ ion with EDTA.</p

Expression levels of the selected miRNAs in developmental stages of <i>S. litura</i>.

<p>The absolute expression levels of the selected miRNAs with respect to U6snRNA (endogenous control) were analyzed by TaqMan miRNA assays using the total RNA isolated from each stage of the insect, <i>S. litura</i>. The bar graphs represent the expression level of each miRNA at a particular stage in comparison to the expression level in 1^st instar larvae. The statistical significance of the qRT analysis was determined by P value < 0.05 while the error bars represent the SD.</p

Gene ontology classification of the target genes for the selected miRNAs.

<p>Gene ontology (GO) term was assigned to each target gene based on the annotation and were summarized into three main GO categories (biological process, cellular component, molecular function) while only the top 10 sub categories are presented.</p

Analysis of novel miRNAs identified in the library.

<p>(A) Similarity between the precursor sequences of a miRNA family identified in the population. (B) (i) The largest miRNA cluster identified from the library using <i>Sf</i> draft genome assembly as a reference. (ii) Similarity in the nucleotide composition of the mature miRNA sequences present in the cluster, especially the seed region.</p

Homology analysis of <i>Spodoptera</i> miRNAs.

<p>(A) (i) Identity in the seed region of the <i>Spodoptera</i> miRNA with respect to its counterpart from other insects. (ii) Sequence conservation of the <i>Sf</i> miRNA, including the seed region over a wide range of insect species. (B) Similarity between the precursor sequences of <i>Sf</i> miRNA with its counterpart from <i>B. mori</i>. The nucleotide differences are only outside the mature region but in the stem loop sequence.</p

Northern validation of selected miRNAs.

<p>Both the known and novel miRNAs were checked by northern analysis for their expression in Sf21 cells using the total RNA isolated from them. All the miRNAs exhibited the expected sizes from the library.</p

Tissue distribution of selected miRNAs in <i>S. litura</i>.

<p>The absolute expression levels of the selected miRNAs with respect to U6snRNA (endogenous control) were analyzed by TaqMan miRNA assays using total RNA isolated from respective tissue parts of the larvae. The bar graphs (A, C) represent the expression level of each miRNA in a particular tissue part but in comparison to that of miR-305-5p. (B) The bar graph represents the relative distribution of expression levels of each miRNA in three segments of the whole gut, i.e., fore gut, mid gut and hind gut. The statistical significance of the qRT analysis was determined by P value < 0.05 while the error bars represent the SD.</p

Analysis of small RNA reads and conservation of homologous miRNAs.

<p>(A) The sequence length distribution of small RNA read tags in Sf21 cells showed majority of the population at 20 nt, a typical length of mature miRNAs followed by the read lengths around 27 nt which could be putative piRNA sequences. (B) Percentage of known miRNAs from <i>Spodoptera frugiperda</i> with homologs from other insects; <i>B. mori, D. melanogaster, T. castenum, A. aegypti, A. gambie</i> and <i>C. elegans</i>.</p

Mapping QTLs for Salt Tolerance in Rice (<i>Oryza sativa</i> L.) by Bulked Segregant Analysis of Recombinant Inbred Lines Using 50K SNP Chip

<div><p>Soil salinity is a major constraint to rice production in large inland and coastal areas around the world. Modern high yielding rice varieties are particularly sensitive to high salt stress. There are salt tolerant landraces and traditional varieties of rice but with limited information on genomic regions (QTLs) and genes responsible for their tolerance. Here we describe a method for rapid identification of QTLs for reproductive stage salt tolerance in rice using bulked segregant analysis (BSA) of bi-parental recombinant inbred lines (RIL). The number of RILs required for the creation of two bulks with extreme phenotypes was optimized to be thirty each. The parents and bulks were genotyped using a 50K SNP chip to identify genomic regions showing homogeneity for contrasting alleles of polymorphic SNPs in the two bulks. The method was applied to ‘CSR11/MI48’ RILs segregating for reproductive stage salt tolerance. Genotyping of the parents and RIL bulks, made on the basis of salt sensitivity index for grain yield, revealed 6,068 polymorphic SNPs and 21 QTL regions showing homogeneity of contrasting alleles in the two bulks. The method was validated further with ‘CSR27/MI48’ RILs used earlier for mapping salt tolerance QTLs using low-density SSR markers. BSA with 50K SNP chip revealed 5,021 polymorphic loci and 34 QTL regions. This not only confirmed the location of previously mapped QTLs but also identified several new QTLs, and provided a rapid way to scan the whole genome for mapping QTLs for complex agronomic traits in rice.</p></div