Browsing by Author "Sgamma, Tiziana"
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Item Open Access Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages(International Journal of Nanomedicine - Dove Medical Press, 2019-03-28) Fulgione, Andrea; Ianniello, Flora; Papaianni, Marina; Contaldi, Felice; Sgamma, Tiziana; Giannini, Cinzia; Pastore, Stella; Velotta, Raffaele; Della Ventura, Bartolomeo; Roveri, Norberto; Lelli, Marco; Capuano, Federico; Capparelli, RosannaPurpose: 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.Item Open Access Building a DMU e-Biology resource for health sciences’ students.(International Academy of Technology, Education and Development (IATED), 2017-11-18) Pena-Fernandez, A.; Sgamma, Tiziana; Young, Christopher N. J.; Randles, Michael J.; del Aguila, C.; Hurtado, C.; Evans, M. D.; Potiwat, N.; Izquierdo, F.; Pena, M. A.; Coope, J.; Armstrong, M.; Bhambra, Avninder S.The BSc Biomedical Science (BMS) programme at De Montfort University (DMU, Leicester, UK) is accredited by the Institute of Biomedical Science (IBMS). Students enrolled within this programme acquire highly sought after skills related with human health sciences to work in: pathology departments in hospitals; research institutions; biotechnology and pharmaceutical industries; and the education sector to name a few. The degree recruits a large number of students with currently around 600 students enrolled on this programme at DMU. Despite pre-entry requirements of knowledge of subjects related to human biology, biology or chemistry, we have noted that first year students require basic support in STEM subjects (biology, chemistry and mathematics) in modules such as “Basic Microbiology”, “Basic Anatomy and Physiology” and “Chemistry for the Biosciences”. This support is especially necessary for students that come from non-traditional routes such as Business and Technology Education Council (BTEC) routes. Moreover, usually topics related with microbiology and human diseases are challenging for students, often causing stress impacting their overall performance and experience. A group of BMS academics at DMU in conjunction with universities in the European Union (EU; e.g. University of San Pablo CEU, Spain) have started to design, create and develop a series of e-learning resources or units in human biology and BMS for undergraduate students that study health sciences degrees in the EU. These units are being uploaded onto the DMU web server (http://parasitology.dmu.ac.uk/) and will be only accessible for students from participating universities during the first phase of this project (2017/18 course) in which comprehensive feedback will be collected. This web server space has three sections or modules (theoretical section, virtual laboratory and microscope) in which the new e-learning resources will be preliminary accommodated. These units will be interactive and easy to follow, and will cover basic human biology (e.g. cells, cell structure), human anatomy and physiology, histology and basic microbiology, which will be embedded in a theoretical module named DMU e-Biology within the above URL link. They will include formative assessments and case studies throughout each unit. In addition, a series of practical units are being developed which describe routine practical elements in any biomedical laboratory such as laboratory materials, pipetting, molecular techniques (e.g. PCR), cell culture (e.g. use of biological safety cabinet) and histological techniques (e.g. use of microtome, staining techniques). The development of this teaching and learning resource will cover a gap in the traditional teaching and learning methods that are currently used and provided in the participating universities. The DMU e-Biology will aid to our undergraduate students to gain knowledge in human biology and microbiology by promoting self-learning. We consider that the DMU e-Biology will help overcome spatiotemporal, equipment and resource barriers. Additionally, it may help student retention as currently about a 10% of our first year students fail to continue BMS at DMU. Finally, the creation of the DMU e-Biology will also provide support to the DMU Student Retention and Attainment Strategy 2016-2020 through the DMU Student Learning Hub, which is currently under development.Item Open Access Building on-line materials for teaching parasitology to health sciences’ students: initial impressions.(2017-06-05) Pena-Fernandez, A.; Ollero, M.; Fenoy, S.; Magnet, A.; Mackenzie, S.; Pena, M. A.; Izquierdo, F.; Hurtado, C.; Ioannou, M.; Bornay, F.; Halliwell, R.; Acosta, L.; Torrus, D.; Singh, Harprit; Sgamma, Tiziana; Evans, M.; Bhambra, Avninder S.; Baho, S.; del Aguila, C.Background: It is widely recognised that the use of web-based teaching resources is an increasingly important method for delivering education, and it will be particularly important in the near future due to the progressively increasing number of health science students and the current number of academics in the “European Higher Education Area”. The study of parasitology and infectious diseases is essential to build professionals in the health sector with the key knowledge and skills to face global public health threats such as food-, water- or vector-borne infectious diseases outbreaks. However, the current time dedicated to the teaching of this discipline in all health sciences degrees at De Montfort University (DMU, Leicester, UK) is very little or non-existent depending on the degree/master. Methods: An innovative teaching group at DMU is trying to fill this gap in the currently available teaching offer in line with new trends in global health education, the large number of students enrolled in any health degree and the increasing number of students that would like to study this discipline (but due to different commitments do not have enough time or resources to study on a full time basis). Thus, an innovative teaching group from different EU Universities (DMU and the Spanish universities: University of San Pablo CEU, University of Alcalá, and University Miguel Hernández de Elche) and clinicians (University Hospitals of Leicester, UK) have started to design, create and develop a complete on-line package in Parasitology for undergraduate and postgraduate students that study health sciences. Results: The e-Parasitology package will be accessible through the DMU website (http://parasitology.dmu.ac.uk) in 2017 and will be focused on infection, prevention and treatment of major and emerging parasitological diseases. Conclusions: This teaching resource will aid our undergraduate and postgraduate students to gain a significant knowledge in parasitology by promoting self-learning and internationalization. This poster will explore one of the first mini-modules developed so far related with Toxocara, a helminthiasis with prevalence rates that can reach as high as 40% or more in parts of the world, and the challenges for its development.Item Open Access Case study: Co-creating a flipped feed-in approach to a virtual biochemistry lab assessment: increasing student achievement and engagement in a large, diverse cohort(Portland Press, 2022-03-16) Cassambai, Shabana; Bridge, Hannah; Gill, Palak K.; Shobaloju, Similoluwa; Sgamma, Tiziana; Rushworth, Jo V.More inclusive, authentic assessments are required to address awarding gaps and to ensure that bioscience students can apply their knowledge to relevant work-based scenarios. In this case report, we present a co-created approach to designing a more inclusive, virtual biochemistry lab assessment for a diverse cohort of ∼270 first-year students. The assignment was to write up an inclusive clinical case study as a one-page journal article. A flipped classroom approach taught the relevant skills, along with simulated labs from Learning Science Ltd. Student Lecturers co-created the assessment, including the marking rubric and the unexemplars. We replaced traditional feedback with a flipped, feed-in approach where students were able to engage in a formative assessment with peer marking and unexemplars. Whilst the summative assessment was marked anonymously, a dialogue-based approach was employed, where students could request personalised audio feed-forward from the tutor. The high pass rate (97%) and student satisfaction score (88%) suggest that this approach is an effective way to challenge, engage and support a large, diverse cohort of students. First-year students who participated in the flipped feed-in experience obtained a significantly higher summative mark (56.7% cf. 50.9%) than those who did not. Interestingly, students in receipt of learning adjustments scored higher marks on average in the summative assessment (59.3% cf. 54.3%), suggesting that we have reversed the disability-based attainment gap. Further investigation into whether a co-created, flipped feed-in approach can reduce attainment gaps based on ethnicity, gender and age is warranted.Item Open Access Challenges in Medicinal and Aromatic Plants DNA Barcoding—Lessons from the Lamiaceae(MDPI, 2022-01-05) Sgamma, Tiziana; Nazar, Nazia; Howard, Caroline; Adrian, SlaterThe potential value of DNA barcoding for the identification of medicinal plants and authentication of traded plant materials has been widely recognized; however, a number of challenges remain before DNA methods are fully accepted as an essential quality control method by industry and regulatory authorities. The successes and limitations of conventional DNA barcoding are considered in relation to important members of the Lamiaceae. The mint family (Lamiaceae) contains over one thousand species recorded as having a medicinal use, with many more exploited in food and cosmetics for their aromatic properties. The family is characterized by a diversity of secondary products, most notably the essential oils (EOs) produced in external glandular structures on the aerial parts of the plant that typify well-known plants of the basil (Ocimum), lavender (Lavandula), mint (Mentha), thyme (Thymus), sage (Salvia) and related genera. This complex, species-rich family includes widely cultivated commercial hybrids and endangered wild-harvested traditional medicines, and examples of potential toxic adulterants within the family are explored in detail. The opportunities provided by next generation sequencing technologies to whole plastome barcoding and nuclear genome sequencing are also discussed with relevant examples.Item Open Access Chronic methylphenidate regulates genes and proteins mediating neuroplasticity in the juvenile rat brain(Elsevier, 2017-06-17) Quansah, Emmanuel; Sgamma, Tiziana; Jaddoa, Estabraq; Zetterstrom, T. S. C.Methylphenidate (MPH) is the front-line psychostimulant medication prescribed for alleviating the symptoms associated with attention deficit hyperactivity disorder (ADHD) in children. Here, we investigated the effects of chronic MPH (2.0 mg/kg, twice daily for 15 days) exposure to young rats (20-25 days old at start of treatment) on the expression of genes and proteins associated with neuroplasticity, such as activity regulated cytoskeleton-associated protein (Arc), insulin receptor substrate protein 53 (IRSp53), cell division control protein 42 (Cdc42), and actin-related protein 2 (Arp2). Chronic MPH increased Arc expression in areas of the cerebrum including, the striatum, nucleus accumbens and hippocampus. In addition, chronic MPH also increased the expression of IRSp53 in the striatum, while Cdc42 and Arp2 were specifically increased in the nucleus accumbens. Conversely, chronic MPH decreased Arc and IRSp53 protein expression in the cerebellum, indicating differential effects of the drug in cerebral areas relative to the cerebellum. Overall, our results indicate that chronic MPH treatment increases expression of genes and proteins associated with dendritic spine formation and neuronal plasticity in target areas of the cerebrum while it decreases the expression in the cerebellum.Item Metadata only Comparison of different Rhodiola species using NMR-metabolomics, HPTLC and DNA barcoding techniques(2016-07) Booker, Anthony; Zhai, Lixiang; Gkouva, C.; Li, Shuyuan; Frommenwiler, Debora; Reich, Eike; Howard, Caroline; Sgamma, Tiziana; Williams, Sarah; Slater, A.; Heinrich, M.The fast developing international trade of products based on local and traditional knowledge and their associated value chains have become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products (HMPs) has been overlooked in quality issues in transnational trade. Medicinal Rhodiola species, including R. rosea L. and R. crenulata (Hook.f. & Thomson) H.Ohba, abbreviate genus have been widely used as traditional herbal medicines with numerous claims for their therapeutic effects [1]. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. This study compares the phytochemical differences among Rhodiola raw materials available on the market and provides a practical method for Rhodiola authentication. Nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography (HPTLC) techniques were used to analyse the phytochemical differences between five Rhodiola species. We compared the phytochemical composition of collected Rhodiola samples to authenticated samples. Rosavin and rosarin were mainly present in R. rosea whereas an unknown compound was only present in R. crenulata. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. 7% of the raw-material samples were not labelled properly and the species information was not clearly illustrated. Both 1H-NMR and HPTLC methods provided an integrated analysis of the phytochemical differences between the species studied. This study provided an identification method for R. rosea and R. crenulata and provided further analytical data that could form the basis for the identification of other species. The integrated identification approach combining these two analytical platforms offers an innovative and practical way of assessing the chemical variability along the value chains of medicinal plants.Item Open Access Creating a model module for the novel resource DMU e-Parasitology.(International Academy of Technology, Education and Development (IATED), 2017-11-18) Pena-Fernandez, A.; Ollero, M. D.; Fenoy, S.; Magnet, A.; Izquierdo, F.; Pena, M. A.; Bornay, F.; Acosta, L.; Parker, L. A.; Sgamma, Tiziana; del Aguila, C.The study of parasitology has become essential to build future health care professionals with skills to respond to public health threats such as the recent outbreaks due to Cryptosporidium spp. or Giardia in the United Kingdom (UK). To facilitate the teaching of parasitology, which negligible across the different undergraduate and taught masters degrees at De Montfort University (DMU, Leicester, UK), a group from different EU Universities [DMU and the Spanish universities: University of San Pablo CEU (USP-CEU) and University Miguel Hernández], clinicians and practising Biomedical Scientists from the UK National Health Service are developing an on-line package for teaching and learning parasitology named DMU e-Parasitology. This package will have three sections or modules: a theoretical module with mini e-learning units to study major human parasites such as Leishmania spp. or malaria; a virtual laboratory module with units to enhance the learning and study of parasitic diseases such as culture of parasites, staining and use of light microscope to identify these organisms or molecular techniques for the identification of parasites to species level; and a virtual microscope with a real slide collection of clinical samples of parasites. The e-Parasitology resource is being created for undergraduate/postgraduate human health science students, with corresponding degrees of difficulty. Units will include a tool to assess the learning process of the students, in form of a quiz, activity and/or exam, and several formative activities throughout each mini-module. The development of this teaching resource will cover a gap in the traditional teaching and learning methods that are currently used and provided in the participating Universities. The DMU e-Parasitology will aid to our undergraduate students to gain a significant knowledge in parasitology by promoting self-learning. A unit related with Toxocara, a helmintiasis with prevalence rates that can reach as high as 40% or more in different parts of the world, was firstly developed to use as a model for the development of the DMU e-Parasitology. Three undergraduate students that studied parasitology during the first term in 2016/17 [n=27; 6 European Credit Transfer and Accumulation System credits (ECTS); 3rd year module] from the bilingual Pharmacy and Biotechnology degree at USP-CEU were voluntarily recruited to provide comprehensive feedback for this model unit at the beginning of the second term. This unit was tested with these students because of their comprehensive knowledge of parasitology. Students described it as interactive and presenting the appropriate content and resources to study the parasitic disease addressed (toxocariasis). Limitations were the poor navigability in the formative exercise section and the excessive information provided in some slides that could hinder their understanding. The team has addressed these limitations and is using this unit as a model to build the DMU e-Parasitology, which will be accessible through the DMU website (http://parasitology.dmu.ac.uk) in 2018. We consider that this teaching and learning resource will overcome barriers of time, space, equipment and resources; and may help students and scientists around the world in the diagnostic of different parasitic diseases that impact human health.Item Metadata only Detection of Extended-Spectrum Beta-Lactamases E. coli in Animal Faeces Collected in Urban Parks in Leicester, UK(2017-06-02) Adeyemi, J.; Reid, R.; Baho, S.; Hoosen, H.; del Aguila, C.; Fenoy, S.; Pena, M. A.; Izquierdo, F.; Magnet, A.; Sgamma, Tiziana; Ollero, M. D.; Hurtado, C.; Pena-Fernandez, A.Background: The presence and distribution of antibiotic resistance bacteria in the environment could constitute an emerging public health concern. Different studies have described these bacteria in a range of animals and their possible role in the contamination of the environment, however very little studies have determined these bacteria in urban ecosystems. Recovery and remediation of affected environments with these biological hazards, and the establishment of protection interventions, constitute a challenge that requires a collaborative international response to protect the public, especially in urban ecosystems. A preliminary study carried out by our research group have detected Extended-Spectrum β-Lactamases (ESBL) genes for Gram-negative bacteria in animal faecal samples collected in different urban parks in the city centre of Leicester (United Kingdom). Methods: This study investigated the presence of ESBL-producing genes (blaCTX-M, blaSHV, blaTEM and blaOXA) within Escherichia coli (E. coli) due to its implications for human health. 30 faecal samples were collected in the same parks between August and September of 2016. A veterinarian identified the animal species as follow: 22 avian (18 waterfowls, 4 pigeons), 4 dogs, 3 cats and 1 fox. After appropriate treatment of the samples, CTX-M-1-type producing E. coli was detected by molecular analysis in 5 of the analysed samples (17%); all of them from waterfowls. Results: The results described here, although preliminary, might indicate that waterfowls might be carriers of ESBL E. coli producers in Leicester. Waterfowls may have a possible role in the spread of CTX-M-1 producing E. coli in urban ecosystems although more research is required prior to implementing intervention programs in the monitored environment. Conclusions: Possible control measures may be cleaning frequently urban parks, roads and pavement from animal faeces, especially avian faeces; or banning exposure practices such as feeding these animals, activity that is very popular in the monitored city.Item Open Access Development of a virtual environment for teaching and learning biomedical techniques and equipment for the study of human pathogens.(IATED, 2018-07-04) Pena-Fernandez, A.; Fenoy, S.; Evans, M. D.; Sgamma, Tiziana; Hurtado, C.; Izquierdo, F.; Randles, M.; Young, Christopher N. J.; Acosta, L.; del Aguila, C.An international innovative teaching group from different EU Universities (De Montfort University, Leicester, UK; University of San Pablo CEU, Madrid, Spain; University of Miguel Hernandez, Elche, Spain) and biomedical scientists registered by the Health and Care Professions Council (HCPC, UK) are developing a complete e-learning package in medical parasitology for undergraduate and postgraduate students that study Health Sciences. This package, named DMU e-Parasitology, is accessible through the DMU website (http://parasitology.dmu.ac.uk) and will present different modules including a virtual laboratory module for the study of traditional and novel biomedical laboratory techniques and equipment for detecting, identifying and studying human pathogens, specifically parasites. These techniques could also be potentially used to study other pathogens such as bacteria or viruses. The virtual biomedical laboratory is under development, but is available in the DMU website here: http://parasitology.dmu.ac.uk/learn/laboratory.htm. To develop this new module of the DMU e-Parasitology, we are using Storyline 360 software and the scaffolding and methods used to build the theoretical module (Peña-Fernández et al., 2017) [1]. To facilitate the navigation, study and comprehension of the final user, we have divided the virtual laboratory into a series of sub-sections that include different units; the sub-sections so far are: microscopes (with units such as the electron microscope); molecular biology (e.g. polymerase chain reaction and gel electrophoresis); biological safety cabinets and cell/parasite culture; biochemical and immunological techniques (e.g. magnetic immunoseparation); histology (e.g. microtome) and staining techniques (e.g. Kinyoun staining). The virtual laboratory units are highly interactive and present short videos of academics and/or technicians working in real conditions with the different laboratory equipment such as a thermocycler, a microtome, or a biological safety cabinet, as well as performing a specific technique such as a staining to determine pathogens. Therefore, the user of this virtual environment will receive a complete and “real” experience of the work in a biomedical laboratory. The DMU e-Parasitology package, and specifically its virtual laboratory environment, could help technicians and students across the world to learn how to work in a biomedical laboratory as well as to perform techniques to identify and diagnose human pathogens such as microsporidia or Plasmodium spp. Thus, the virtual resource is supported by a virtual library that includes a real collection of clinical slides that will provide the user with the functionality of a light and/or an immunofluorescence microscope. In conclusion, the virtual laboratory may serve as a high quality and reliable on-line environment for the learning of techniques and equipment. These resources can be used to improve the learning of undergraduate and postgraduate students of human health sciences as well as to develop CPD training. Moreover, the virtual laboratory module may impact in the teaching of laboratory techniques and skills in developing countries due to their limited resources. This communication will explore the design and development of the virtual laboratory environment that will be publicly accessible by the end of 2018.Item Open Access DNA Authentication of Raw Herbal Drugs for Industrial Quality Assurance(Thieme, 2015-11) Howard, Caroline; Cayrou, Caroline; Scheyhing, U.; Schotz, K.; Williams, S.; Koch, E.; Slater, A.; Sgamma, TizianaMany plant-based medicines are still prepared from plants collected from the wild, requiring routine testing to ensure their correct identity. Quality control of wild-harvested plant materials has typically involved morphological and chemical analysis, but both approaches have their limitations. DNA-based authentication assays could complement these techniques and are currently under development for incorporation into industrial quality assurance procedures for a number of commercial products. A general strategy for the design of a robust DNA authentication assay for routine testing has been established. Specimens of the commercial plant species and its potential adulterants are collected and a DNA barcode sequence library created. PCR primers are designed to informative sequence strings that can be used to distinguish a target species from others in the dataset. Primers designed to generate short amplicons can be optimized for multiplex PCR, quantitative PCR and high resolution melt curve (HRM) assays. Rhodiola rosea is a one such target for DNA test development. Raw material for R. rosea containing products still derives mainly from collection in the wild, with almost a dozen closely related species growing in the same habitat. Positive identification of the correct plant species is not obvious and misidentifications or even adulterations are common. Although rosavines are considered to be characteristic constituents of R. rosea, there is some doubt about their use as chemical markers. Informative sequence differences between the DNA barcodes of different Rhodiola species have been used to design a specific qPCR assay. This allows the quantitation of the target species DNA, but cannot detect unknown adulterant species. Conversely, an HRM assay can detect unknown species in mixed samples, but only at relatively high levels of contamination. Lessons learned from these and other examples will be discussed.Item Open Access DNA Authentication of St John’s Wort (Hypericum perforatum L.) Commercial Products Targeting the ITS Region(Multidisciplinary Digital Publishing Institute, 2019-04-09) Howard, Caroline; Hill, Eleanor; Kreuzer, Marco; Mali, Purvi; Masiero, Eva; Sgamma, Tiziana; Slater, A.There 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.Item Open Access DNA Barcoding for Industrial Quality Assurance(Thieme, 2017-10) Sgamma, Tiziana; Lockie-Williams, C.; Kreuzer, M.; Williams, S.; Scheyhing, U.; Koch, E.; Slater, A.; Howard, C.DNA barcoding methods originally developed for the identification of plant specimens have been applied to the authentication of herbal drug materials for industrial quality assurance. These methods are intended to be complementary to current morphological and chemical methods of identification. The adoption of these methods by industry will be accelerated by the introduction of DNA-based identification techniques into regulatory standards and monographs. The introduction of DNA methods into the British Pharmacopoeia is described, along with a reference standard for use as a positive control for DNA extraction and polymerase chain reaction (PCR). A general troubleshooting chart is provided to guide the user through the problems that may be encountered during this process. Nevertheless, the nature of the plant materials and the demands of industrial quality control procedures mean that conventional DNA barcoding is not the method of choice for industrial quality control. The design of DNA barcode-targeted quantitative PCR and high resolution melt curve tests is one strategy for developing rapid, robust, and reliable protocols for high-throughput screening of raw materials. The development of authentication tests for wild-harvested Rhodiola rosea L. is used as a case study to exemplify these relatively simple tests. By way of contrast, the application of next-generation sequencing to create a complete profile of all the biological entities in a mixed herbal drug is described and its potential for industrial quality assurance discussed.Item Open Access Effect of antidepressant drugs on the brain sphingolipid system(2020-05-14) Zetterstrom, Tyra; Jaddoa, Estabraq; Masania, Jinit; Masiero, Eva; Sgamma, Tiziana; Arroo, R. R. J.; Sillence, Daniel J.Background: Major depression is a common mood disorder and the central sphingolipid system has been identified as a possible drug target of this condition. Here we investigated the action of antidepressant drugs on sphingolipid levels in rat brain regions, plasma and in cultured mouse macrophages. Methods: Two antidepressant drugs were tested; the serotonin reuptake inhibitor paroxetine and the noradrenaline reuptake inhibitor desipramine, either following acute or chronic treatments. Content of sphingosine and ceramide were analysed using LC-MS or HPLC-UV respectively. This from samples of brain, plasma and cultured mouse macrophages. Antidepressant induced effects on mRNA expression for two key genes of the sphingolipid pathway, SMPD1 and ASAH1 were also measured by using quantitative Real-Time PCR. Results: Chronic but not acute administration of paroxetine or desipramine reduced sphingosine levels in the prefrontal cortex and hippocampus (only paroxetine) but not in the striatum. Ceramide levels were also measured in the hippocampus following chronic paroxetine and likewise to sphingosine this treatment reduced its levels. The corresponding collected plasma samples from chronically treated animals did not show any decrease of sphingosine compared to the corresponding controls. Both drugs failed to reduce sphingosine levels from cultured mouse macrophages. The drug-induced decrease of sphingolipids coincided with reduced mRNA expression of two enzymes of the central sphingolipid pathway, i.e. acid sphingomyelinase (SMPD1) and acid ceramidase (ASAH1). Conclusions: This study supports the involvement of brain sphingolipids in the mechanism of action by antidepressant drugs and for the first time highlights their differential effects on brain versus plasma levels.Item Embargo Ethylene inhibitor silver nitrate enhances regeneration and genetic transformation of Prunus avium (L.) cv Stella(Springer Netherlands, 2014-08-22) Sgamma, Tiziana; Thomas, Brian; Muleo, RosarioThe effects of the ethylene inhibitors, silver nitrate (AgNO3) and aminoethoxyvinylglycine (AVG), together with Agrobacterium co-cultivation were investigated for improving both regeneration and genetic transformation of leaf explants of the recalcitrant sweet cherry cultivar Stella. Co-cultivated young leaf explants with A. tumefaciens strain EHA105 harbouring a binary vector pROK containing both the phytochrome B of Arabidopsis thaliana and the nptII genes, were cultured on M2 MS-modified medium supplemented with either AVG (2.54 µM) or AgNO3 (296.08 µM) for 21 days in the dark, and then transferred onto ethylene-free inhibitors medium in the light. Efficiency, in terms of frequency on shoot organogenesis, was observed in explants subjected to co-cultivation with Agrobacterium and not co-cultivated in presence of each ethylene inhibitors. Ethylene inhibitors helped in the morphogenesis process and mass production of shoots. An increase in regeneration efficiency was observed in ethylene-inhibitor-free medium, which was almost four times greater than the control leaf explants. When ethylene inhibitors were added to the medium, a further regeneration efficiency enhancement for AVG (45%) and AgNO3 (63.3%) was observed. Whereas, in explants not co-cultivated with Agrobacterium, the resulting regeneration efficiency was 25% for AVG and 23.3%, for AgNO3. The regeneration frequency from leaf explants was also enhanced in treatments with AVG when combined with Agrobacterium co-cultivation, with 139 shoots regenerated from a total of 27 regenerating explants. The AgNO3 also contributed to control Agrobacterium growth in the post-co-cultivation phase. Only the lines originating from AgNO3-treated explants showed kanamycin resistance, 9 out of 141 regenerated lines, and polymerase chain reaction analysis confirmed both the insertion of the genes into the plant genome and the absence of bacterial contamination of the transgenic shoots.Item Open Access Evaluation of the plastid and nuclear DNA barcodes in genus Ocimum towards quality assurance in herbal industry(Elsevier, 2024-12-27) Amit Kumar; Vereena Rodrigues; Akanksha Saxena; Priyanka Mishra; Ashutosh K. Shukla; Ajit Kumar Shasany; Nazar, Nazia; Sgamma, Tiziana; Slater, A.; Velusamy SundaresanRationale Species of the genus Ocimum are of immense value and are in high demand in trade which leads to unscrupulous adulteration of both crude drugs as well as formulations. Traditional method-based authentication is difficult in case of incomplete or damaged samples and in dried herbal bulk material. High degree of morphological similarity, issues of polyploidy, and the possibility of inter- and intraspecific hybridization have plagued the Ocimum market. Hence, there is an immediate need for developing DNA barcodes for fast and accurate identification of the species. Objective In this study, three plastid regions (matK, rbcL, and trnH-psbA) and two nuclear regions (ITS and ITS2) have been evaluated for their performance as DNA barcodes to check the delineation of Ocimum species. Materials and Methods Five widely accepted markers were assessed for their efficiency using methods like Nearest genetic distance, Wilcoxon test, Best Match (BM), and Neighbor-Joining (NJ) tree methods. Conclusion Our study suggests that discrimination rate amongst single barcodes is the highest for trnH-psbA with the NJ analysis (92 %). By combining two or more barcodes, no significant changes were observed. A reliable and commercially viable DNA barcoding system has been developed for accurate species identification of various Ocimum species. The varied approaches used in the investigation had different species identification potential. The identification success rates of plastid DNA and nuclear DNA barcodes were comparable using pairwise genetic distance (PWG-distance), BM, and NJ methods. Despite having high inter-specific and the lowest intra-specific genetic distance, trnH-psbA failed to discriminate Ocimum species having hybrid origin, indicating the need to develop more suitable barcode loci in future.Item Open Access Genus-Specific Real-Time PCR and HRM Assays to Distinguish Liriope from Ophiopogon Samples(Multidisciplinary Digital Publishing Institute, 2017-10-26) Masiero, Eva; Banik, Dipanwita; Abson, John; Greene, Paul; Slater, A.; Sgamma, TizianaLiriope 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.Item Open Access In vitro plant culture system induces phase transition in fruit-bearing plants(Acta Horticulturae, 2016-02-25) Sgamma, Tiziana; Cirilli, M.; Caboni, E.; Maurizio, M.; Thomas, B.; Muleo, RosarioThe juvenile to adult switch is the most important post-embryonic transition. In woody plants the juvenile phase can last many years with a great economic impact. In Arabidopsis, the small RNAs miR156 and miR172 play a crucial but opposite role in the regulation of this process. miR156 maintains juvenility, negatively regulating SPLs genes, while miR172 promotes adult transition, targeting the floral repressors AP2-like transcription factors. In this work, peach (Prunus persica L. Batch) orthologs of Arabidopsis epigenetic and genetic factors involved in the juvenility to adult phase transition were studied. In peach, higher levels of ppa-miR156 were detected in seedlings, in vitro and extra vitro plants than in adult plants. Also, PpSPLs were more expressed in adult plants, confirming a possible role for the miR156-SPL pathway in promoting juvenile-like characteristics. ppa-miR172 expression level was low in seedlings and in vitro plants but an increase was observed in the adult donor plant, corresponding to lower expression of PpAP2-like genes. In Arabidopsis, flower induction is also promoted by activation of the FLOWERING LOCUS T (FT) gene. In peach leaf tissue, low levels of PpFT-like expression in rejuvenated plants and seedlings were detected. We propose that, in peach, conserved key genes present in herbaceous plants and woody species are involved in juvenile to adult and adult to juvenile-like phase transitions.Item Open Access Integrating DNA Barcoding Within an OrthogonalApproach for Herbal Product Authentication:A Narrative Review(Wiley, 2024-11-12) Nazar, Nazia; Saxena, Akanksha; Sebastian, Anu; Slater, Adrian; Sundaresan, Velusamy; Sgamma, TizianaIntroduction: Existing methods for morphological, organoleptic, and chemical authentication may not adequately ensure the accurate identification of plant species or guarantee safety. Herbal raw material authentication remains a major challenge in herbal medicine. Over the past decade, DNA barcoding, combined with an orthogonal approach integrating various testing methods for quality assurance, has emerged as a new trend in plant authentication. Objective: The review evaluates DNA barcoding and common alternative testing in plant-related sectors to enhance quality assurance and accurate authentication. Method: Studies were selected based on their relevance to the identification, quality assurance, and safety of herbal products. Inclusion criteria were peer-reviewed articles, systematic reviews, and relevant case studies from the last two decades focused on DNA barcoding, identification methods, and their applications. Exclusion criteria involved studies lacking empirical data, those not peer-reviewed, or those unrelated to the main focus. This ensured the inclusion of high-quality, pertinent sources while excluding less relevant studies. Results: An orthogonal approach refers to the use of multiple, independent methods that provide complementary information for more accurate plant identification and quality assurance. This reduces false positives or negatives by confirming results through different techniques, combining DNA barcoding with morphological analysis or chemical profiling. It enhances confidence in results, particularly in cases of potential adulteration or misidentification of plant materials. Conclusion: This study highlights the persistent challenges in assuring the quality, purity, and safety of plant materials. Additionally, it stresses the importance of incorporating DNA-based authentication alongside traditional methods, to enhance plant material identification.Item Embargo Juvenility(Elsevier Ltd., 2016-10-01) Sgamma, Tiziana