Search Results (8)

Chemical probing, for decades, has been one of the most popular tools for studying the secondary structure of RNA molecules. Recently, protocols for simultaneous analysis of multiple RNAs have been developed, enabling in vivo transcriptome-wide interrogation of the RNA structure dynamics. One of the most popular methods is the selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). In this study, we describe the evaluation of this protocol by addressing the influence of the reverse transcription enzymes, buffer conditions, and chemical probes on the properties of the cDNA library and the quality of mutational profiling-derived structural signals. Our results reveal a SuperScript IV (SSIV) reverse transcriptase as a more efficient enzyme for mutational profiling of SHAPE adducts and shed new light on the role of Mn²⁺ cations in the modulation of SSIV readthrough efficiency. Full article

Cereal crops have starch in their endosperm, which has provided calories to humans and livestock since the dawn of civilization to the present day. Starch is one of the important biological factors which is contributing to the yield of cereal crops. Starch is synthesized by different enzymes, but starch structure and amount are mainly determined by the activities of starch synthase enzymes (SS) with the involvement of starch branching enzymes (SBEs) and debranching enzymes (DBEs). Six classes of SSs are found in Arabidopsis and are designated as soluble SSI-V, and non-soluble granule bound starch synthase (GBSS). Soluble SSs are important for starch yield considering their role in starch biosynthesis in cereal crops, and the activities of these enzymes determine the structure of starch and the physical properties of starch granules. One of the unique characteristics of starch structure is elongated glucan chains within amylopectin, which is by SSs through interactions with other starch biosynthetic enzymes (SBEs and DBEs). Additionally, soluble SSs also have conserved domains with phosphorylation sites that may be involved in regulating starch metabolism and formation of heteromeric SS complexes. This review presents an overview of soluble SSs in cereal crops and includes their functional and structural characteristics in relation to starch synthesis. Full article

Starch is a major ingredient in rice, and the amylose content of starch significantly impacts rice quality. OsSS (starch synthase) is a gene family related to the synthesis of amylose and amylopectin, and 10 members have been reported. In the present study, a synteny analysis of a novel family member belonging to the OsSSIV subfamily that contained a starch synthase catalytic domain showed that three segmental duplications and multiple duplications were identified in rice and other species. Expression data showed that the OsSS gene family is involved in diverse expression patterns. The prediction of miRNA targets suggested that OsSS are possibly widely regulated by miRNA functions, with miR156s targeted to OsSSII-3, especially. Haplotype analysis exhibited the relationship between amylose content and diverse genotypes. These results give new insight and a theoretical basis for the improved amylose content and eating quality of rice. Full article

At the end of year 2019 the first reports appeared of a new coronavirus and on 31st December 2019 WHO declared a public health emergency of international concern. To date (as of 6:08 pm CET, 24th November 2020) according to WHO the new coronavirus, now called severe acute respiratory syndrome (SARS)-CoV-2, has infected 58,900,547 people and killed 1,393,305 people worldwide. It is extremely important to develop means for express diagnostics to ensure prompt action to limit the spread of infection. One of the diagnostic approaches, is the detection of viral particles in swabs. This approach can be realized using a biosensor with specific ligands, based on peptide molecules complementary to surface viral proteins. The concept of the so-called Systems of Conjugated Ionic-Hydrogen Bonds (abbreviated—SSIVS, CIHBS) implemented in the Protein-3D computer program, was applied to analyze the spatial structures of the bonds between the SARS-CoV-2 spike protein and the ACE-2 (Angiotensin converting enzyme 2) receptor, in order to reveal the perspective peptide sequences. There are two clearly marked areas of contact of the spike with the cell receptor—upper and lower, which are visualized in the SSIVS form, and the complex formed at this site is strong enough to ensure its attachment to the coronavirus spike and can compete for binding with the ACE-2 receptor. Two peptides were developed that form a spatial structure complementary to the coronavirus spike: of eight (No. one) and of 15 (No. two) amino acid residues. The peptides were covalently bound to biochip platforms via neutral linkers to form sites with peptides No. one and No. two. The third site has a neutral hydrophilic surface to serve as a reference. The platform was integrated with a microfluidic channel and was used as a flow through device. The detection of bound viral particles was carried out using UV excitation and direct registration of viral proteins fluorescence. The preliminary laboratory tests demonstrated the efficiency of the biosensor. Full article

A substantial increase in yield of food crops is crucial to feeding the burgeoning global population. There is a need to introduce new breeding strategies that will accelerate the average phenotypic values of crop plants. The use of induced mutations coupled with modern genomics tools is an effective strategy for identifying and manipulating genes for crop improvement. High-throughput TILLING (Targeting Induced local Lesions IN Genomes) methodology, detects mutations in mutagenized populations, and EcoTILLING identifies single nucleotide polymorphisms (SNPs) within a natural population and associates these variations with traits of breeding interest. The main advantage of these techniques as a “reverse genetics” strategy is that they can be applied to any species regardless of genome size and ploidy level. In cereals, several space-induced and EMS-induced mutant populations have been used to identify mutants with important traits including salinity tolerance, grain size, and recombinant crossovers via TILLING by sequencing (TbyS). Genes such as TaSSIV, which plays an important role in starch granule formation, and Pin a and Pin b, which have been associated with kernel hardness in wheat, have been exploited in cereals via the EcoTILLING approach. This review focused on the functions and challenges of TILLING and the relation of TILLING to next-generation sequencing (NGS) technologies which help to exploit the induced mutations and their potential applications in cereal crops. Full article

Image velocimetry has proven to be a promising technique for monitoring river flows using remotely operated platforms such as Unmanned Aerial Systems (UAS). However, the application of various image velocimetry algorithms has not been extensively assessed. Therefore, a sensitivity analysis has been conducted on five different image velocimetry algorithms including Large Scale Particle Image Velocimetry (LSPIV), Large-Scale Particle Tracking Velocimetry (LSPTV), Kanade–Lucas Tomasi Image Velocimetry (KLT-IV or KLT), Optical Tracking Velocimetry (OTV) and Surface Structure Image Velocimetry (SSIV), during low river flow conditions (average surface velocities of 0.12–0.14 m s ${}^{-1}$ , Q60) on the River Kolubara, Central Serbia. A DJI Phantom 4 Pro UAS was used to collect two 30-second videos of the surface flow. Artificial seeding material was distributed homogeneously across the rivers surface, to enhance the conditions for image velocimetry techniques. The sensitivity analysis was performed on comparable parameters between the different algorithms, including the particle identification area parameters (such as Interrogation Area (LSPIV, LSPTV and SSIV), Block Size (KLT-IV) and Trajectory Length (OTV)) and the feature extraction rate. Results highlighted that KLT and SSIV were sensitive to changing the feature extraction rate; however, changing the particle identification area did not affect the surface velocity results significantly. OTV and LSPTV, on the other hand, highlighted that changing the particle identification area presented higher variability in the results, while changing the feature extraction rate did not affect the surface velocity outputs. LSPIV proved to be sensitive to changing both the feature extraction rate and the particle identification area. This analysis has led to the conclusions that for surface velocities of approximately 0.12 m s ${}^{-1}$ image velocimetry techniques can provide results comparable to traditional techniques such as ADCPs. However, LSPIV, LSPTV and OTV require additional effort for calibration and selecting the appropriate parameters when compared to KLT-IV and SSIV. Despite the varying levels of sensitivity of each algorithm to changing parameters, all configuration image velocimetry algorithms provided results that were within 0.05 m s ${}^{-1}$ of the ADCP measurements, on average. Full article

Wheat is a staple food commodity grown worldwide, and wheat starch is a valuable source of energy and carbon that constitutes 80% of the grain weight. Manipulation of genes involved in starch synthesis significantly affects wheat grain weight and yield. TaSSIV plays an important role in starch synthesis and its main function is granule formation. To mine and stack more favorable alleles, single nucleotide polymorphisms (SNPs) of TaSSIV-A, B, and D were investigated across 362 wheat accessions by Ecotype-Targeting Induced Local Lesions IN Genome (EcoTILLING). As a result, a total of 38 SNPs in the amplified regions of three TaSSIV genes were identified, of which 10, 15, and 13 were in TaSSIV-A, B, and D, respectively. These 38 SNPs were evaluated by using KASP and six SNPs showed an allele frequency >5% whereas the rest were <5%, i.e., considered to be minor alleles. In the Chinese mini core collection, three haplotypes were detected for TaSSIV–A and three for TaSSIV–B. The results of an association study in the Chinese mini core collection with thousand grain weight (TGW) and spike length (SPL) showed that Hap-2-1A was significantly associated with TGW and Hap-3-1B with SPL. Allelic frequency and geographic distribution indicated that the favored haplotype (Hap-2-1A) has been positively selected in Chinese wheat breeding. These results suggested that the Kompetitive Allele Specific PCR (KASP) markers can be applied in starch improvement to ultimately improve wheat yield by marker assisted selection in wheat breeding. Full article

The function of a wheat starch regulator 1 (TaRSR1) in regulating the synthesis of grain storage starch was determined using the barley stripe mosaic virus—virus induced gene-silencing (BSMV-VIGS) method in field experiments. Chlorotic stripes appeared on the wheat spikes infected with barley stripe mosaic virus-virus induced gene-silencing- wheat starch regulator 1 (BSMV-VIGS-TaRSR1) at 15 days after anthesis, at which time the transcription levels of the TaRSR1 gene significantly decreased. Quantitative real-time PCR was also used to measure the transcription levels of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-TaRSR1-silenced wheat plants at 20, 27, and 31 days after anthesis. The results showed that the transcription levels of some starch synthesis-related enzyme genes were markedly induced at different sampling time points: TaSSI, TaSSIV, TaBEIII, TaISA1, TaISA3, TaPHOL, and TaDPE1 genes were induced at each of the three sampling time points and TaAGPS1-b, TaAGPL1, TaAGPL2, TaSSIIb, TaSSIIc, TaSSIIIb, TaBEI, TaBEIIa, TaBEIIb, TaISA2, TaPHOH, and TaDPE2 genes were induced at one sampling time point. Moreover, both the grain starch contents, one thousand kernel weights, grain length and width of BSMV-VIGS-TaRSR1-infected wheat plants significantly increased. These results suggest that TaRSR1 acts as a negative regulator and plays an important role in starch synthesis in wheat grains by temporally regulating the expression of specific starch synthesis-related enzyme genes. Full article