DNA Barcoding for Industrial Quality Assurance.

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.

Challenges in Medicinal and Aromatic Plants DNA Barcoding-Lessons from the Lamiaceae.

The 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.

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.

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.

Monochromic Radiations Provided by Light Emitted Diode (LED) Modulate Infection and Defense Response to Fire Blight in Pear Trees.

Pathogenesis-related (PR) proteins are part of the systemic signaling network that perceives pathogens and activates defenses in the plant. Eukaryotic and bacterial species have a 24-h 'body clock' known as the circadian rhythm. This rhythm regulates an organism's life, modulating the activity of the phytochromes (phys) and cryptochromes (crys) and the accumulation of the corresponding mRNAs, which results in the synchronization of the internal clock and works as zeitgeber molecules. Salicylic acid accumulation is also under light control and upregulates the PR genes expression, increasing plants' resistance to pathogens. Erwinia amylovora causes fire blight disease in pear trees. In this work, four bacterial transcripts (erw1-4), expressed in asymptomatic E. amylovora-infected pear plantlets, were isolated. The research aimed to understand how the circadian clock, light quality, and related photoreceptors regulate PR and erw genes expression using transgenic pear lines overexpressing PHYB and CRY1 as a model system. Plantlets were exposed to different circadian conditions, and continuous monochromic radiations (Blue, Red, and Far-Red) were provided by light-emitting diodes (LED). Results showed a circadian oscillation of PR10 gene expression, while PR1 was expressed without clear evidence of circadian regulation. Bacterial growth was regulated by monochromatic light: the growth of bacteria exposed to Far-Red did not differ from that detected in darkness; instead, it was mildly stimulated under Red, while it was significantly inhibited under Blue. In this regulatory framework, the active form of phytochrome enhances the expression of PR1 five to 15 fold. An ultradian rhythm was observed fitting the zeitgeber role played by CRY1. These results also highlight a regulating role of photoreceptors on the expression of PRs genes in non-infected and infected plantlets, which influenced the expression of erw genes. Data are discussed concerning the regulatory role of photoreceptors during photoperiod and pathogen attacks.

Effect of antidepressant drugs on the brain sphingolipid system.

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 was 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.

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

A 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 levels.

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

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.

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

PURPOSE: 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.

Sequence-Specific Detection of Aristolochia DNA - A Simple Test for Contamination of Herbal Products.

Herbal medicines are used globally for their health benefits as an alternative therapy method to modern medicines. The market for herbal products has increased rapidly over the last few decades, but this has in turn increased the opportunities for malpractices such as contamination or substitution of products with alternative plant species. In the 1990s, a series of severe renal disease cases were reported in Belgium associated with weight loss treatment, in which the active species Stephania tetrandra was found to be substituted with Aristolochia fangchi. A. fangchi contains toxic aristolochic acids, which have been linked to kidney failure, as well as cancers of the urinary tract. Because of these known toxicities, herbal medicines containing these compounds, or potentially contaminated by these plants, have been restricted or banned in some countries, but they are still available via the internet and in alternate formulations. In this study, a DNA based method based on quantitative real-time PCR (qPCR) was tested to detect and distinguish Aristolochia subg. Siphisia (Duch.) O.C.Schmidt species from a range of medicinal plants that could potentially be contaminated with Aristolochia material. Specific primers were designed to confirm that Aristolochia subg. Siphisia can be detected, even in small amounts, if it is present in the products, fulfilling the aim of offering a simple, cheaper and faster solution than the chemical methods. A synthetic gBlock template containing the primer sequences was used as a reference standard to calibrate the qPCR assay and to estimate the copy number of a target gene per sample. Generic primers covering the conserved 5.8S rRNA coding region were used as internal control to verify DNA quality and also as a reference gene for relative quantitation. To cope with potentially degraded DNA, all qPCR primer sets were designed to generate PCR products of under 100 bp allowing detection and quantification of A. fangchi gBlock even when mixed with S. tetrandra gBlock in different ratios. All proportions of Aristolochia, from 100 to 2%, were detected. Using standards, associating the copy number to each start quantity, the detection limit was calculated and set to about 50 copies.

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

Liriope 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.

Chronic methylphenidate regulates genes and proteins mediating neuroplasticity in the juvenile rat brain.

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 (2.0mg/kg, twice daily for 15days) exposure to young rats (20-25days 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.

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.

TEMPRANILLO is a regulator of juvenility in plants.

Many plants are incapable of flowering in inductive daylengths during the early juvenile vegetative phase (JVP). Arabidopsis mutants with reduced expression of TEMPRANILLO (TEM), a repressor of flowering locus T (FT) had a shorter JVP than wild-type plants. Reciprocal changes in mRNA expression of TEM and FT were observed in both Arabidopsis and antirrhinum, which correlated with the length of the JVP. FT expression was induced just prior to the end of the JVP and levels of TEM1 mRNA declined rapidly at the time when FT mRNA levels were shown to increase. TEM orthologs were isolated from antirrhinum (AmTEM) and olive (OeTEM) and were expressed most highly during their juvenile phase. AmTEM functionally complemented AtTEM1 in the tem1 mutant and over-expression of AmTEM prolonged the JVP through repression of FT and CONSTANS (CO). We propose that TEM may have a general role in regulating JVP in herbaceous and woody species.