Regulation of juvenility in Antirrhinum majus

Floral initiation is regulated by an elaborate network of signalling pathways, including the photoperiodic pathway. In Arabidopsis, flowering is promoted through this pathway by activation of FLOWERING LOCUS T (FT) by CONSTANS (CO) in long days. During juvenility plants are incapable of flowering in response to environmental conditions that would normally be favourable. This project studies the molecular basis of floral incompetence during juvenility in the model annual species, Antirrhinum majus and the important commercial tree species, Olea europaea, which has an extended juvenile phase. Photoperiod transfer experiments were used to measure the length of juvenility in plants grown in controlled environment cabinets at different Daily Light Integrals. Analysis of Antirrhinum FT (AmFT) expression during development showed that AmFT expression is minimal during juvenility and increases in all leaves following the end of the juvenile phase. The photoperiodic pathway was shown to be active during juvenility, suggesting that an additional mechanism involving the repression of FT could be involved in the regulation of juvenility. Full length Antirrhinum and Olive cDNAs representing homologues of the Arabidopsis FT repressors TEMPRANILLO 1 (AtTEM1) and AtTEM2, which act antagonistically with CO, were isolated. Molecular and phylogenetic analyses revealed high amino acid identities between Antirrhinum (AmTEM) and Olive (OeTEM) TEM-like proteins and AtTEM1 & 2. AmTEM and OeTEM proteins contain AP2 and B3 domains, consistent with AtTEM1 and AtTEM2, and can be classified as Class I members of the RAV sub-family of B3 transcription factors. AmTEM and OeTEM expression levels were shown to be higher during juvenility suggesting a potential role for TEM in controlling juvenility. A reciprocal relationship between expression levels of AmTEM/AtTEM1 and AmFT/AtFT was revealed in both Antirrhinum and Arabidopsis. Analysis of expression across development showed that AmTEM/AtTEM1 levels decline at around the time juvenility ends corresponding to when AmFT/AtFT levels start to increase. Arabidopsis tem1 mutants over-expressing AmTEM, OeTEM or AtTEM1 exhibited delayed flowering compared to the tem1 mutant, which demonstrated their role in regulating flowering time. Over-expression of AmTEM was shown to increase the length of the juvenile phase, delay the induction of AtCO and AtFT expression and reduce the overall levels of AtFT expression. Conversely, the juvenile phases of tem1 single and tem1/2 double mutants were shown to be shorter than in wild-type plants, with the induction of AtCO and AtFT expression occurring earlier. These findings are consistent with a role for TEM in regulating juvenility, which occurs through the down-regulation of FT and CO, and results in the inability to proceed to reproductive growth

High luminosity interaction region design for collisions with detector solenoid

An innovatory interaction region has been recently conceived and realized on the Frascati DA{\Phi}NE lepton collider. The concept of tight focusing and small crossing angle adopted until now to achieve high luminosity in multibunch collisions has evolved towards enhanced beam focusing at the interaction point with large horizontal crossing angle, thanks to a new compensation mechanism for the beam-beam resonances. The novel configuration has been tested with a small detector without solenoidal field yielding a remarkable improvement in terms of peak as well as integrated luminosity. The high luminosity interaction region has now been modified to host a large detector with a strong solenoidal field which significantly perturbs the beam optics introducing new design challenges in terms of interaction region optics design, beam transverse coupling control and beam stay clear requirementsComment: 3 pages, 4 figures, presented to the IPAC10 conferenc

Molecular Verification of the UK National Collection of Cultivated Liriope and Ophiopogon Plants

open access articleA collection of cultivated Liriope and Ophiopogon plants was established in 1996–1998 and subsequently hosted at a horticultural college. Uncertainties about the identification of the accessions, compounded by potential errors in propagation and labelling have led to waning confidence in the identities of the plants in the collection. The potential for using DNA barcoding to determine the species identities of the accessions was investigated. The DNA barcode regions of the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) and nuclear ribosomal internal transcribed spacer (nrITS) were amplified. DNA sequence analysis allowed the sequences of the accessions to be compared to reference sequences in public databases. A simple haplotype map of the characteristic polymorphic positions in the rbcL regions was used to clearly distinguish between the two genera and assign Ophiopogon accessions to individual species or sub-groups of species. The ITS sequence data confirmed these genus and species assignations and provided greater resolution to distinguish between closely related species. The combination of two DNA barcodes allowed most of the accessions to be assigned to individual species. This molecular verification confirmed the identity of about 70% of the accessions, with the remaining 30% demonstrating a range of mistaken identities at the species and genus level

DNA Authentication of St John’s Wort (Hypericum perforatum L.) Commercial Products Targeting the ITS Region

open access articleThere is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region was adapted to screen commercial products for the presence of Hypericum perforatum L. material. DNA differing widely in amount and extent of fragmentation was detected in a number of product types. Two assays were designed to further analyse this DNA using a curated database of selected Hypericum ITS sequences: A qPCR assay based on a species-specific primer pair spanning the ITS1 and ITS2 regions, using synthetic DNA reference standards for DNA quantitation and a Next Generation Sequencing (NGS) assay separately targeting the ITS1 and ITS2 regions. The ability of the assays to detect H. perforatum DNA sequences in processed medicines was investigated. Out of twenty different matrices tested, both assays detected H. perforatum DNA in five samples with more than 103 ITS copies µL−1 DNA extract, whilst the qPCR assay was also able to detect lower levels of DNA in two further samples. The NGS assay confirmed that H. perforatum was the major species in all five positive samples, though trace contaminants were also detected

Regulation of juvenility in Antirrhinum majus

Floral initiation is regulated by an elaborate network of signalling pathways, including the photoperiodic pathway. In Arabidopsis, flowering is promoted through this pathway by activation of FLOWERING LOCUS T (FT) by CONSTANS (CO) in long days. During juvenility plants are incapable of flowering in response to environmental conditions that would normally be favourable. This project studies the molecular basis of floral incompetence during juvenility in the model annual species, Antirrhinum majus and the important commercial tree species, Olea europaea, which has an extended juvenile phase. Photoperiod transfer experiments were used to measure the length of juvenility in plants grown in controlled environment cabinets at different Daily Light Integrals. Analysis of Antirrhinum FT (AmFT) expression during development showed that AmFT expression is minimal during juvenility and increases in all leaves following the end of the juvenile phase. The photoperiodic pathway was shown to be active during juvenility, suggesting that an additional mechanism involving the repression of FT could be involved in the regulation of juvenility. Full length Antirrhinum and Olive cDNAs representing homologues of the Arabidopsis FT repressors TEMPRANILLO 1 (AtTEM1) and AtTEM2, which act antagonistically with CO, were isolated. Molecular and phylogenetic analyses revealed high amino acid identities between Antirrhinum (AmTEM) and Olive (OeTEM) TEM-like proteins and AtTEM1 & 2. AmTEM and OeTEM proteins contain AP2 and B3 domains, consistent with AtTEM1 and AtTEM2, and can be classified as Class I members of the RAV sub-family of B3 transcription factors. AmTEM and OeTEM expression levels were shown to be higher during juvenility suggesting a potential role for TEM in controlling juvenility. A reciprocal relationship between expression levels of AmTEM/AtTEM1 and AmFT/AtFT was revealed in both Antirrhinum and Arabidopsis. Analysis of expression across development showed that AmTEM/AtTEM1 levels decline at around the time juvenility ends corresponding to when AmFT/AtFT levels start to increase. Arabidopsis tem1 mutants over-expressing AmTEM, OeTEM or AtTEM1 exhibited delayed flowering compared to the tem1 mutant, which demonstrated their role in regulating flowering time. Over-expression of AmTEM was shown to increase the length of the juvenile phase, delay the induction of AtCO and AtFT expression and reduce the overall levels of AtFT expression. Conversely, the juvenile phases of tem1 single and tem1/2 double mutants were shown to be shorter than in wild-type plants, with the induction of AtCO and AtFT expression occurring earlier. These findings are consistent with a role for TEM in regulating juvenility, which occurs through the down-regulation of FT and CO, and results in the inability to proceed to reproductive growth.EThOS - Electronic Theses Online ServiceUniversity of WarwickGBUnited Kingdo

Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce

Background: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results. Results: In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T (FT) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A-1 (PROTEIN PHOSPHATASE 2A-1), LsPP2AA3 (PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3) and LsTIP41 (TAP42-INTERACTING PROTEIN OF 41 kDa), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A-1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A-1, LsPP2AA3 and LsTIP41, would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile. Conclusions: Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments

Fatigue assessment of a FSAE car rear upright by a closed form solution of the critical plane method

Material fatigue is extensively discussed and researched within scientific and industrial communities. Fatigue damage poses a significant challenge for both metallic and non-metallic components, often resulting in unexpected failures of in-service parts. Within multiaxial fatigue assessment, critical plane methods have gained importance due to their ability to characterize a component's critical location and detect early crack propagation. However, the conventional approach to calculate critical plane factors is time-consuming, making it primarily suitable for research purposes or when critical regions are already known. In many real-world scenarios, identifying the critical area of a component is difficult due to complex geometries, varying loads, or time limitations. This challenge becomes particularly crucial after topological optimization of components and in the context of lightweight design. Recently, the authors proposed an efficient method for evaluating critical plane factors in closed form, applicable to all cases that necessitate the maximization of specific parameters based on stress and strain components or their combination. This paper presents and validates the proposed methodology, with reference to a rear upright of a FSAE car, which is characterized by a complex geometry and is subjected to non-proportional loading conditions. The efficient algorithm demonstrated a substantial reduction in computation time compared to the standard plane scanning method, while maintaining solution accuracy

Genus-Specific Real-Time PCR and HRM Assays to Distinguish Liriope from Ophiopogon Samples

open access articleLiriope and Ophiopogon species have a long history of use as traditional medicines across East Asia. They have also become widely used around the world for ornamental and landscaping purposes. The morphological similarities between Liriope and Ophiopogon taxa have made the taxonomy of the two genera problematic and caused confusion about the identification of individual specimens. Molecular approaches could be a useful tool for the discrimination of these two genera in combination with traditional methods. Seventy-five Liriope and Ophiopogon samples from the UK National Plant Collections of Ophiopogon and Liriope were analyzed. The 5′ end of the DNA barcode region of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLa) was used for the discrimination of the two genera. A single nucleotide polymorphism (SNP) between the two genera allowed the development of discriminatory tests for genus-level identification based on specific PCR and high-resolution melt curve (HRM) assays. The study highlights the advantage of incorporating DNA barcoding methods into plant identification protocols and provides simple assays that could be used for the quality assurance of commercially traded plants and herbal drugs

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages

open access articlePurpose: The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on. Materials and methods: Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage–HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen. Results: Our data highlighted that the biomimetic HA nanocrystal–bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions. Conclusion: Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin