ABSTRACT
Monitoring of the introduction of unapproved genetically modified (GM) events is required because the approval status of a GM event may differ from country to country. The on-site methods such as loop-mediated isothermal amplification (LAMP) offer a technological answer for the rapid GM detection beyond the laboratories. Real-time LAMP assays detecting one GM target were reported earlier. To increase the efficiency of the assay, a multiplex real-time LAMP simultaneously targeting Figwort Mosaic Virus promoter (P-FMV) that constructs region between the Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac) and neomycin phosphotransferase II (nptII) marker gene was developed. The assay could detect as low as 0.1% for each GM target within 45 min. To the best of our knowledge, multiplexing in real-time LAMP using the Genie II system with applicability in GM detection has been reported herein for the first time. The developed method provides rapid, on-site, and real-time GM detection in seeds and food products.
Subject(s)
Nucleic Acid Amplification Techniques , Plants, Genetically Modified , Seeds , Nucleic Acid Amplification Techniques/methods , Plants, Genetically Modified/genetics , Seeds/genetics , Seeds/chemistry , Caulimovirus/genetics , Promoter Regions, Genetic , Molecular Diagnostic TechniquesABSTRACT
Ulotaront, a trace amine-associated receptor 1 (TAAR1) and serotonin 5-HT1A receptor agonist without antagonist activity at dopamine D2 or the serotonin 5-HT2A receptors, has demonstrated efficacy in the treatment of schizophrenia. Here we report the phase 1 translational studies that profiled the effect of ulotaront on brain responses to reward, working memory, and resting state connectivity (RSC) in individuals with low or high schizotypy (LS or HS). Participants were randomized to placebo (n = 32), ulotaront (50 mg; n = 30), or the D2 receptor antagonist amisulpride (400 mg; n = 34) 2 h prior to functional magnetic resonance imaging (fMRI) of blood oxygen level-dependent (BOLD) responses to task performance. Ulotaront increased subjective drowsiness, but reaction times were impaired by less than 10% and did not correlate with BOLD responses. In the Monetary Incentive Delay task (reward processing), ulotaront significantly modulated striatal responses to incentive cues, induced medial orbitofrontal responses, and prevented insula activation seen in HS subjects. In the N-Back working memory task, ulotaront modulated BOLD signals in brain regions associated with cognitive impairment in schizophrenia. Ulotaront did not show antidepressant-like biases in an emotion processing task. HS had significantly reduced connectivity in default, salience, and executive networks compared to LS participants and both drugs reduced this difference. Although performance impairment may have weakened or contributed to the fMRI findings, the profile of ulotaront on BOLD activations elicited by reward, memory, and resting state is compatible with an indirect modulation of dopaminergic function as indicated by preclinical studies. This phase 1 study supported the subsequent clinical proof of concept trial in people with schizophrenia.Clinical trial registration: Registry# and URL: ClinicalTrials.gov NCT01972711, https://clinicaltrials.gov/ct2/show/NCT01972711.
ABSTRACT
Flaxseed/linseed is an important oilseed crop having applications in the food, nutraceutical, and paint industry. Seed weight is one of the most crucial determinants of seed yield in linseed. Here, quantitative trait nucleotides (QTNs) associated with thousand-seed weight (TSW) have been identified using multi-locus genome-wide association study (ML-GWAS). Field evaluation was carried out in five environments in multi-year-location trials. SNP genotyping information of the AM panel of 131 accessions comprising 68,925 SNPs was employed for ML-GWAS. From the six ML-GWAS methods employed, five methods helped identify a total of 84 unique significant QTNs for TSW. QTNs identified in ≥ 2 methods/environments were designated as stable QTNs. Accordingly, 30 stable QTNs have been identified for TSW accounting up to 38.65% trait variation. Alleles with positive effect on trait were analyzed for 12 strong QTNs with r 2 ≥ 10.00%, which showed significant association of specific alleles with higher trait value in three or more environments. A total of 23 candidate genes have been identified for TSW, which included B3 domain-containing transcription factor, SUMO-activating enzyme, protein SCARECROW, shaggy-related protein kinase/BIN2, ANTIAUXIN-RESISTANT 3, RING-type E3 ubiquitin transferase E4, auxin response factors, WRKY transcription factor, and CBS domain-containing protein. In silico expression analysis of candidate genes was performed to validate their possible role in different stages of seed development process. The results from this study provide significant insight and elevate our understanding on genetic architecture of TSW trait in linseed.
ABSTRACT
Early flowering, maturity, and plant height are important traits for linseed to fit in rice fallows, for rainfed agriculture, and for economically viable cultivation. Here, Multi-Locus Genome-Wide Association Study (ML-GWAS) was undertaken in an association mapping panel of 131 accessions, genotyped using 68,925 SNPs identified by genotyping by sequencing approach. Phenotypic evaluation data of five environments comprising 3 years and two locations were used. GWAS was performed for three flowering time traits including days to 5%, 50%, and 95% flowering, days to maturity, and plant height by employing five ML-GWAS methods: FASTmrEMMA, FASTmrMLM, ISIS EM-BLASSO, mrMLM, and pLARmEB. A total of 335 unique QTNs have been identified for five traits across five environments. 109 QTNs were stable as observed in ≥2 methods and/or environments, explaining up to 36.6% phenotypic variance. For three flowering time traits, days to maturity, and plant height, 53, 30, and 27 stable QTNs, respectively, were identified. Candidate genes having roles in flower, pollen, embryo, seed and fruit development, and xylem/phloem histogenesis have been identified. Gene expression of candidate genes for flowering and plant height were studied using transcriptome of an early maturing variety Sharda (IC0523807). The present study unravels QTNs/candidate genes underlying complex flowering, days to maturity, and plant height traits in linseed.
ABSTRACT
BACKGROUND: Insect resistant and herbicide tolerant genetically modified (GM) events have been approved in many countries. Screening methods could facilitate preliminary testing to check the GM status, which may target control elements, transgenes, and marker genes or construct regions. Among these, methods targeting the construct region, i.e., the junction between two genetic elements of a transgenic cassette are more specific. OBJECTIVE: Loop-mediated isothermal amplification (LAMP) assays targeting three construct regions were developed; between Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac), cry2Ab2 gene and nos terminator (cry2Ab2-tnos), and cp4-epsps gene and nos terminator (cp4epsps-tnos). METHOD: LAMP assays were performed by incubation at constant temperatures for selected targets. Positive amplification was detected as a change in color from orange to green on addition of SYBR® Green dye in visual LAMP and as real-time amplification curves in real-time LAMP. RESULTS: These assays showed acceptable specificity and sensitivity. Visual LAMP was found to be sensitive enough to detect as low as 0.005%, equivalent to two target copies. Real-time LAMP assays were able to detect as low as four copies of the target within 40 min, making them suitable for rapid on-site testing for GM organisms (GMO). Practical utility was also verified using spiked test samples. CONCLUSIONS: These assays could be employed to address some of the biosafety or post-release monitoring issues, as well as to check for approved and unapproved GM events in a country. HIGHLIGHTS: LAMP assays targeting three construct regions have been developed, enabling screening for approved or unapproved GMO.
Subject(s)
Crops, Agricultural , Herbicides , Animals , Herbicides/pharmacology , Insecta , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Plants, Genetically Modified/genetics , Sensitivity and SpecificityABSTRACT
A plausible mechanism underlying flavonoid-associated cognitive effects is increased cerebral blood flow (CBF). However, behavioural and CBF effects following flavanone-rich juice consumption have not been explored. The aim of this study was to investigate whether consumption of flavanone-rich juice is associated with acute cognitive benefits and increased regional CBF in healthy, young adults. An acute, single-blind, randomised, cross-over design was applied with two 500-ml drink conditions - high-flavanone (HF; 70·5 mg) drink and an energy-, and vitamin C- matched, zero-flavanone control. A total of twenty-four healthy young adults aged 18-30 years underwent cognitive testing at baseline and 2-h after drink consumption. A further sixteen, healthy, young adults were recruited for functional MRI assessment, whereby CBF was measured with arterial spin labelling during conscious resting state at baseline as well as 2 and 5 h after drink consumption. The HF drink was associated with significantly increased regional perfusion in the inferior and middle right frontal gyrus at 2 h relative to baseline and the control drink. In addition, the HF drink was associated with significantly improved performance on the Digit Symbol Substitution Test at 2 h relative to baseline and the control drink, but no effects were observed on any other behavioural cognitive tests. These results demonstrate that consumption of flavanone-rich citrus juice in quantities commonly consumed can acutely enhance blood flow to the brain in healthy, young adults. However, further studies are required to establish a direct causal link between increased CBF and enhanced behavioural outcomes following citrus juice ingestion.
Subject(s)
Cerebrovascular Circulation , Citrus paradisi/chemistry , Citrus sinensis/chemistry , Cognition Disorders/prevention & control , Flavanones/therapeutic use , Fruit and Vegetable Juices/analysis , Nootropic Agents/therapeutic use , Adult , Breakfast , Cerebral Angiography , Cognition , Cognition Disorders/diagnostic imaging , Cohort Studies , Cross-Over Studies , England , Flavanones/administration & dosage , Flavanones/analysis , Functional Food/analysis , Humans , Magnetic Resonance Angiography , Nootropic Agents/administration & dosage , Nootropic Agents/analysis , Prefrontal Cortex/blood supply , Prefrontal Cortex/diagnostic imaging , Single-Blind Method , Task Performance and Analysis , Young AdultABSTRACT
Bipolaris sorghicola (Lefebvre and Sherwin) is a well known and economically important seed-borne pathogen with the specific species of sorghum (Sorghum bicolor [L] Moench) as host. Thirty-two strains were obtained from different geographical area of sorghum growing places in India. Molecular characterization using three marker systems i.e., universal rice primers (URP), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) was carried out. Molecular marker work revealed differences along with geographical origin clustering of various B. sorghicola strains which could not be revealed through conventional method of characterization. Out of 13 URPs, 20 ISSR and 50 RAPD primers screened, 8 primers each from URP and ISSR, and 10 primers from RAPD marker were found to result in reproducible banding pattern. One hundred per cent of polymorphic bands was recorded in all three molecular markers. Total number of bands was recorded 1986 with average of 248.25 in URP marker, and 2026 bands with average of 253.25 in ISSR marker and 2158 bands with average of 215.80 in RAPD markers. Maximum heterozygosity (Hn) was revealed by URP 17R (0.40), ISSR 10 (0.41) and RAPD marker OPC-5 (0.34). The polymorphism information content (PIC) values ranged between 5.89 to 8.28 in URP, 4.57 to 8.79 in ISSR and 4.44 to 9.64 in RAPD marker profiles. Maximum cophenetic correlation was found in URP (r = 0.910) followed by ISSR (r = 0.904) and RAPD (r = 0.870). The combined analysis of all three marker systems showed high cophenetic correlation (r = 0.911), which indicated a very good fit of the data for genetic diversity analysis. To best of our knowledge, this is a first report of genetic characterization of B. sorghicola. Hence, combined use of three marker systems would be more sensitive and reliable in characterizing genetic variability in B. sorghicola strains.
Subject(s)
Ascomycota/genetics , Genetic Variation , Plant Diseases/microbiology , Sorghum/microbiology , DNA, Fungal/genetics , Genetic Markers , India , PhylogenyABSTRACT
RATIONALE: There has recently been increasing interest in the potential of flavanols, plant-derived compounds found in foods such as fruit and vegetables, to ameliorate age-related cognitive decline. Research suggests that cocoa flavanols improve memory and learning, possibly as a result of their anti-inflammatory and neuroprotective effects. These effects may be mediated by increased cerebral blood flow (CBF), thus, stimulating neuronal function. OBJECTIVES: The present study employed arterial spin labelling functional magnetic resonance imaging to explore the effect of a single acute dose of cocoa flavanols on regional CBF. METHODS: CBF was measured pre- and post-consumption of low (23 mg) or high (494 mg) 330 ml equicaloric flavanol drinks matched for caffeine, theobromine, taste and appearance according to a randomized counterbalanced crossover double-blind design in eight males and ten females, aged 50-65 years. Changes in perfusion from pre- to post-consumption were calculated as a function of each drink. RESULTS: Significant increases in regional perfusion across the brain were observed following consumption of the high flavanol drink relative to the low flavanol drink, particularly in the anterior cingulate cortex and the central opercular cortex of the parietal lobe. CONCLUSIONS: Consumption of cocoa flavanol improves regional cerebral perfusion in older adults. This provides evidence for a possible acute mechanism by which cocoa flavanols are associated with benefits for cognitive performance.
Subject(s)
Cacao/chemistry , Cerebrovascular Circulation/drug effects , Flavonols/pharmacology , Rest/physiology , Adult , Aged , Beverages , Cerebral Cortex/blood supply , Cerebral Cortex/drug effects , Cross-Over Studies , Double-Blind Method , Female , Healthy Volunteers , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Parietal Lobe/blood supply , Parietal Lobe/drug effects , Polyphenols , TheobromineABSTRACT
OBJECTIVES: Diffusion tensor tractography (DTT) was performed to quantify diffuse axonal injury (DAI) in major white matter (WM) fiber bundles (FBs) of patients with frontal lobe injury and to correlate these changes with neuropsychological tests (NPT) at 6 month follow-up. PATIENTS AND METHODS: DTT was performed in 21 patients with moderate traumatic brain injury (TBI) within week and after 6 month follow-up, and in controls. DTI indices were calculated from the entire FBs in patients as well as controls. Bonferroni multiple comparisons Post hoc test was performed for determining the changes in DTI indices. Paired t-test was performed between DTI indices at baseline and follow-up. Pearson's correlation was performed between NPT scores and DTI indices. RESULTS: Significant changes in DTI indices were observed in some of the FBs as compared to controls which incompletely recovered at 6 month follow-up. DTI indices of different WM FBs correlated significantly with some of the NPT. CONCLUSION: We conclude that DTT based quantification helps in assessment of DAI in patients with moderate frontal lobe injury. Some of the FBs recover partially at 6 month follow-up and correlate with NPT scores.
Subject(s)
Brain Injuries/pathology , Brain Injuries/psychology , Diffusion Tensor Imaging/methods , Frontal Lobe/injuries , Neuropsychological Tests , Adolescent , Adult , Anisotropy , Brain Injuries/surgery , Female , Follow-Up Studies , Functional Laterality/physiology , Humans , Image Processing, Computer-Assisted , Male , Middle Aged , Neurosurgical Procedures , Young AdultABSTRACT
This study aimed to demonstrate age-related and gender-related changes in diffusion tensor imaging (DTI) indices of deep grey matter (GM) nuclei of the normal human brain. DTI was performed on 142 subjects (age: 10-52 years). Regions of interest were placed on the caudate nucleus (CN), putamen, globus pallidus, frontal white matter (WM), occipital WM, anterior and posterior limb of internal capsule, genu of the corpus callosum and splenium in all participants. The quadratic regression model was used to describe age-related and gender-related changes in DTI indices for GM and WM. We observed increased fractional anisotropy (FA) values with age up to adulthood in GM, and a rise up to the third decade of life followed by a decrease in FA for WM. We observed higher FA values in males compared to females in CN and all WM regions. Decreased mean diffusivity with age was observed in GM and WM irrespective of gender. This normative data may be valuable in the diagnosis of neurodegenerative diseases.