A novel barcode system for rapid identification of rice (Oryza sativa L.) varieties using agro-morphological descriptors and molecular markers.

Rice varietal identification is a crucial aspect in breeding, seed production and trade in order to protect the interests of the farmers and consumers. As the number of varieties released is rising every year, the need to identify them unambiguously also increases. Here, we developed a novel barcode system to identify 62 rice genotypes using agro-morphological descriptors and molecular markers. In all, 62 rice genotypes, for 22 agro-morphological traits were recorded. In addition, 19 molecular markers were used for developing genotype-specific DNA fingerprints. The descriptor notes of 10 essential agro-morphological traits and allele codes of the polymorphic markers were used to generate two-dimensional (2-D) barcodes for the rice genotypes. Using agro-morphological traits, 31 rice genotypes were unambiguously distinguished while, with the polymorphic markers we were able to distinguish all rice genotypes except BPT2295 and Jaya. However, using both agro-morphological descriptors and molecular markers in combination, it was possible to distinguish all the rice genotypes used in the present study. These agro-morphological notes and allele codes from the molecular marker data together were used to develop QR (Quick Response) codes for rapid identification of rice genotypes as they facilitate storage of more data. In the present investigation, we have demonstrated the potentiality of agro-morphological traits and molecular markers in distinguishing rice genotypes. The novel QR code system proposed in the present study can also be extended to other crops not only for varietal identification but also for germplasm management and trade.

Study of COVID-19 Infection, its Severity and Outcome in COVID-19 Vaccinated People at Tertiary Health Care Center, North West Rajasthan.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of corona virus-induced disease 19 (COVID-19) that was declared as a global pandemic in March 2020 by the world health organization (WHO). Two vaccines were granted for emergency use by the Central Drugs Standard Control Organization (CDSCO) in India, Covishield® (AstraZeneca's vaccine manufactured by Serum Institute of India) and Covaxin® (manufactured by Bharat Biotech Limited). Sputnik - V has been granted EUA in the month of April 2021. The purpose of this study is to determine the association of COVID-19 infection, its severity and outcome in COVID-19 vaccinated people. METHODS: This was a hospital based prospective cohort study done between March to June 2021 at PBM Associated Group of Hospitals (AGH), Bikaner, Raj. Total 1028 COVID suspected cases consulted in COVID OPD or hospitalized under department of medicine, out of which 146 satisfied the inclusion and exclusion criteria, out of these 146, first 100 cases who gave consent for part of study were selected. RESULTS: Among 100 COVID-19 infected cases, 49 received first dose while rest got both doses. After first dose of vaccination 42.86% had mild and 32.65% had severe clinical infection while after both doses 80.39% had mild and 11.76% had severe clinical infection. On evaluation of HRCT Chest, after first dose 8.16% had normal 40.82% were in severe category while those who got both doses it was 52.82% 3.92% respectively. Among 49 who got first dose, 10.20% recovered on just home based treatment without any need of hospitalization, while 89.8% got admitted in dedicated COVID hospital out of which 73.47% got recovered and 16.33% died. Among 51 who got both the doses, 66.67% recovered on just home based treatment, while 33.33% required hospitalization out of which 25.49% got recovered and 7.84% died. CONCLUSION: After 2nd dose of vaccine there is a significant risk reduction in need of hospitalization and getting severe infection and mortality when compared with first dose only.

Novel (phenylethynyl)pyrene-LNA constructs for fluorescence SNP sensing in polymorphic nucleic acid targets.

We describe fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids. Furthermore, we prove the utility of these probes for the effective detection of single-nucleotide polymorphisms in natural nucleic acids. High-affinity hybridization of the probes and excellent fluorescence responses to single-base mismatches in DNA/RNA targets are demonstrated in model dual-probe and doubly labeled probe formats. This stimulated us to develop two diagnostic systems for the homogeneous detection of a drug-resistance-causing mutation in HIV-1 protease cDNA and RNA gene fragments. Target sequences were obtained by analysis of 200 clinical samples from patients currently receiving anti-HIV/AIDS combination therapy at the Russian Federal AIDS Center. Using these fluorescent oligonucleotides, we were able to detect the target mutation despite all the challenges of the natural targets, that is, the presence of additional mutations, neighboring sequence variation, and low target concentration, which typically reduce binding and effectiveness of sensing by fluorescent oligonucleotides.

A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity.

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

Novel insights into the use of Glowing LNA as nucleic acid detection probes--influence of labeling density and nucleobases.

Appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA (locked nucleic acid) display large increases in fluorescence intensity and remarkably high quantum yields upon hybridization with nucleic acid targets. Thermal denaturation and fluorescence spectroscopy studies on ONs modified with known thymine monomer X and novel 5-methylcytosine monomer Y provide new insights into the design principles and mechanism of these Glowing LNA nucleic acid detection probes.

Improved silencing properties using small internally segmented interfering RNAs.

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19-27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10-12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo.

The sugar conformation governs (6-4) photoproduct formation at the dinucleotide level.

The LNA dinucleotide mimic of TpT whose two-sugar puckers are locked in the C3'-endo conformation selectively produces the corresponding cyclobutane pyrimidine dimer under 254 nm irradiation. In the natural series (TpT) the sugar puckers are in a major C2'-endo sugar conformation and the (6-4) photoproduct is also produced. Consequently, this study demonstrates that the C2'-endo conformation of the sugar pucker is necessary for (6-4) photoproduct formation.

Chemically modified oligonucleotides with efficient RNase H response.

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.

Pyrene-perylene as a FRET pair coupled to the N2'-functionality of 2'-amino-LNA.

Detection of nucleic acid hybridization via fluorescence resonance energy transfer (FRET) using pyren-1-ylmethyl and perylen-3-ylmethyl N2'-functionalized 2'-amino-LNA nucleosides incorporated into oligonucleotides exhibited a clear distance dependence of the FRET efficiency, ranging from below 10% when the fluorophores were approximately 40A apart to approximately 90% when the fluorophores were in close proximity.

Sensitive SNP dual-probe assays based on pyrene-functionalized 2'-amino-LNA: lessons to be learned.

A homogenous fluorescence dual-probe assay containing 2'-N-(pyren-1-ylmethyl)-2'-amino-LNA (locked nucleic acid) building blocks has been developed for effective mismatch-sensitive nucleic acid detection. The pyrene units, which are connected to the rigid bicyclic furanose derivative of 2'-amino-LNA through a short linker, are positioned at the 3' and 5' ends of a dual-probe system. Whereas hybridization with complementary DNA/RNA results in very strong excimer signals, as the pyrene units are in close proximity to one another in the ternary complex, exposure to most singly mismatched DNA/RNA targets results in significantly lower excimer emission intensity. The mechanism that underlies this excellent optical discrimination of singly mismatched targets is clarified by comparison of the thermal-denaturation profiles and fluorescence properties of the dual probe and a covalently linked analogue. Optical discrimination of singly mismatched targets arises from a decrease in excimer emission intensity due to a failure to form a ternary complex (a decrease in thermal stability) and/or local mismatch-induced changes in the helix geometry, depending on the position of the mismatched base pair. The devised dual-probe assay constitutes a simple and sensitive system for the detection of single-nucleotide polymorphism and highlights that conformational restriction combined with the use of short probes conveys favorable properties to dual-probe constructs.

Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities.

Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5'-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37 degrees C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life approximately 10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations approximately 0.1 microM for acridine-conjugated TFO/LNA (or approximately 2 microM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated.

Triplex-forming ability of modified oligonucleotides.

We present our studies on the ability of several different nucleotide analogs as triplex-forming oligonucleotides. The modifications tested include 4'-C-hydroxymethyl, LNA, 2'-amino-LNA and N2'-functionalized 2'-amino-LNA. Triplexes containing monomers of N2'-glycyl-functionalized 2'-amino-LNA are particularly stable.

Dual-probe system using pyrenylmethyl-modified amino-LNA for mismatch detection.

A dual-probe containing pyrenylmethyl amino-LNA has been developed for sensitive mismatch detection. While hybridization with complementary DNA/RNA results in very strong excimer signals, exposure to singly mismatched DNA/RNA targets results in significantly decreased excimer emission.

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets.

BACKGROUND: DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. RESULTS: We investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and alpha-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two alpha-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1). CONCLUSION: The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

Optimized DNA targeting using N,N-bis(2-pyridylmethyl)-beta-alanyl 2'-amino-LNA.

Incorporation of N,N-bis(2-pyridylmethyl)-beta-alanyl 2'-amino-LNA (bipyridyl-functionalized 2'-amino locked nucleic acid) monomers into DNA strands enables high-affinity targeting of complementary DNA with excellent Watson-Crick selectivity in the presence of divalent metal ions. Positioning of bipyridyl-functionalized 2'-amino-LNA monomers in two complementary DNA strands in a "3'-end zipper" constitution allows modulation of duplex stability, i.e., a strong stabilizing effect with one equivalent of divalent metal ion per bipyridyl pair, or a strong destabilizing effect with an excess of divalent metal ions.

DNA self-assembling systems: branched oligonucleotides as building blocks and monitoring by pyrene excimer band formation.

The construction of a DNA self-assembling system created by four Y-shaped branched oligodeoxynucleotide building blocks has been studied. The assembly was verified by changes in the fluorescence emission spectra and revealed an additive effect in pyrene excimer band formation during DNA self-assembly.

Long term stability following genioplasty: a cephalometric study.

BACKGROUND: A receding chin associated with an orthognathic mandible is a common situation and surgical changes in chin position are often required to improve the overall harmony of the face. Genioplasty is one such procedure. Stability of hard and soft tissue changes following genioplasty on a long term basis needs to be assessed. Studies on the stability of hard and soft tissue changes following genioplasty on a short term basis have revealed it as a procedure with good stability. This study is done to assess the stability of hard and soft tissue changes following genioplasty on a long term basis. MATERIALS AND METHODS: Pre-surgical, postsurgical and long term post-surgical cephalograms of 15 cases treated by vertical reduction augmentation genioplasty were obtained. Paired t-test was used to compare the changes between pre-surgical, postsurgical and long term postsurgical cephalograms. RESULTS: Findings of this study demonstrated that genioplasty is a stable procedure. After long term follow-up period, there was a relapse of 1.5 mm at the pogonion accounting for 24% of the surgical advancement. This is attributed to the remodeling that occurs at the surgical site, but not the instability due to the surgical procedure. CONCLUSION: With the present study, it can be concluded that vertical reduction and advancement genioplasty can be considered as an adjunctive procedure that produces predictable results and the bony and soft tissue stability were generally very good.

A structure-activity study of the inhibition of HIV-1 Tat-dependent trans-activation by mixmer 2'-O-methyl oligoribonucleotides containing locked nucleic acid (LNA), alpha-L-LNA, or 2'-thio-LNA residues.

The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

Interstrand communication between 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomers in nucleic acid duplexes: directional control and signalling of full complementarity.

Very efficient interstrand communication systems in nucleic acid duplexes, based on pyrene excimer formation between 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomers, demonstrate the versatility of functionalized 2'-amino-LNA monomers for Angstrom-scale chemical engineering.

Ovipositional responses of Chilo partellus (Swinhoe) (Lepidoptera: pyralidae) to natural products from leaves of two maize (Zea mays L.) Cultivars.

Ovipositional responses of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) to hexane extracts of leaves of two maize (Zea mays L.) cultivars, one resistant (Kisan) and one susceptible (Basilocal), were studied in two-choice bioassays. Gravid females laid a significantly higher percentage of eggs on substrates smeared with extract of Basilocal leaves (HEBL) (69%) than on those smeared with extracts of Kisan leaves (HEKL) (31%). Several chemicals were isolated from HEKL, three of which were characterized as dotriacontanol, heptadecanol, and nonadecanol. These chemicals were either absent or were present in very small amounts in HEBL, but in HEKL they were detected in much larger amounts. Each isolated chemical was tested for its effect on C. partellus oviposition in two-choice bioassays. Maximum ovipositional deterrence (90%) was observed for the compound MR-22a, followed in decreasing order by nonadecanol, MR-7, and heptadecanol. The identity of the remaining compounds is being investigated. The results indicate that the relative resistance of Kisan maize compared to Basilocal is partly due to the presence of certain ovipositional deterrents in its leaves.