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

Genomic tools for identification of medicinal plants

DNA barcoding of herbal medicines has raised awareness of species substitution and adulteration, highlighting issues surrounding their safety and quality. Regulation of herbal medicines is a pressing issue for regulatory agencies and, in response, DNA barcodes have recently been incorporated into the British Pharmacopoeia. Previous studies have found that DNA barcoding to species-level may be impaired by evolutionary mechanisms. This thesis investigates evolutionary relationships of genus Berberis and their impacts on DNA barcoding. Phylogenetic relationships within genus Berberis in the Himalayas and the Hengduan Mountains are studied using whole plastid genomes and hundreds of nuclear loci. The phylogenies reveal pronounced biogeographic structures in the Sino-Himalayan region and suggest that the relatively recent orogeny of the Hengduan Mountains has a strong impact on in situ diversification of Berberis species. Low phylogenetic resolution at species-level may be explained by incomplete lineage sorting. The phylogenies suggest that evolutionary mechanisms hinder DNA barcoding to species-level and, therefore, a method is devised for identifying evolutionary lineages. A strategy for generating DNA barcodes based on diagnostic nucleotides using whole plastid genomes is presented. These barcodes are tested on commercial samples, and their utility for regulatory purposes outlined. Furthermore, species substitution and adulteration in global trade are evaluated with two different specimen identification methods. The first uses the phylogenetic placements of commercial samples of Berberis for specimen identification. The second approach applies DNA metabarcoding to commercial samples of Phyllanthus amarus. The results of these analyses show that congeneric species are in trade and further reveal a high congruence between species in global and local markets, emphasizing the dependency of global medicinal plant trade on local trade systems. Finally, sequencing data from genus Arabidopsis is analysed to identify the effect of assembling nuclear loci that belong to paralogous clusters on phylogenomic inference. Read mapping from cognate paralogues in Arabidopsis has little to no effect on outcomes from phylogenomic inference

Challenges and insights in the exploration of the low abundance human ocular surface microbiome.

PURPOSE The low microbial abundance on the ocular surface results in challenges in the characterization of its microbiome. The purpose of this study was to reveal factors introducing bias in the pipeline from sample collection to data analysis of low-abundant microbiomes. METHODS Lower conjunctiva and lower lid swabs were collected from six participants using either standard cotton or flocked nylon swabs. Microbial DNA was isolated with two different kits (with or without prior host DNA depletion and mechanical lysis), followed by whole-metagenome shotgun sequencing with a high sequencing depth set at 60 million reads per sample. The relative microbial compositions were generated using the two different tools MetaPhlan3 and Kraken2. RESULTS The total amount of extracted DNA was increased by using nylon flocked swabs on the lower conjunctiva. In total, 269 microbial species were detected. The most abundant bacterial phyla were Actinobacteria, Firmicutes and Proteobacteria. Depending on the DNA extraction kit and tool used for profiling, the microbial composition and the relative abundance of viruses varied. CONCLUSION The microbial composition on the ocular surface is not dependent on the swab type, but on the DNA extraction method and profiling tool. These factors have to be considered in further studies about the ocular surface microbiome and other sparsely colonized microbiomes in order to improve data reproducibility. Understanding challenges and biases in the characterization of the ocular surface microbiome may set the basis for microbiome-altering interventions for treatment of ocular surface associated diseases

Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node.

Dendritic and monocytic cells co-operate to initiate and shape adaptive immune responses in secondary lymphoid tissue. The complexity of this system is poorly understood, also because of the high phenotypic and functional plasticity of monocytic cells. We have sequenced mononuclear phagocytes in mesenteric lymph nodes (LN) of three adult cows at the single-cell level, revealing ten dendritic-cell (DC) clusters and seven monocyte/macrophage clusters with clearly distinct transcriptomic profiles. Among DC, we defined LN-resident subsets and their progenitors, as well as subsets of highly activated migratory DC differing in transcript levels for T-cell attracting chemokines. Our analyses also revealed a potential differentiation path for cDC2, resulting in a cluster of inflammatory cDC2 with close transcriptional similarity to putative DC3 and monocyte-derived DC. Monocytes and macrophages displayed sub-clustering mainly driven by pro- or anti-inflammatory expression signatures, including a small cluster of cycling, presumably self-renewing, macrophages. With this transcriptomic snapshot of LN-derived mononuclear phagocytes, we reveal functional properties and differentiation trajectories in a "command center of immunity", and identify elements that are conserved across species

Ecological and genetic differentiation of two subspecies of Saussurea alpina in the Western Alps

In the Western Alps, two subspecies of Saussurea alpina are found in a partly overlapping distribution area; however, they prefer distinct habitats. While Saussurea alpina ssp. alpina is distributed throughout the Alps and beyond, S. alpina ssp. depressa is endemic to the region ranging from the Maritime Alps (France) to the Valais (Switzerland). The sympatric occurrence of closely related taxa raises general questions which factors drive speciation. In order to investigate the genetic and ecological differentiation of the two subspecies, we applied Amplified Fragment Length Polymorphisms and analyzed the habitat preferences via soil pH measurements and vegetation relevés. Overall, we studied 12 populations (five populations of S. alpina ssp. alpina and seven of S. alpina ssp. depressa). The populations were found to be genetically highly differentiated (F ST=0.42). Nevertheless, a weak, but significant genetic differentiation between the subspecies could be detected (F CT=0.04) and the results of the ecological analyses showed a clear differentiation in habitat types. While S. alpina ssp. alpina occurs in alpine grasslands, S. alpina ssp. depressa occurs mainly on calcareous scree with a significantly higher soil pH (median pH=7 and median of pH=8.14 for S. alpina ssp. alpina and S. alpina ssp. depressa, respectively) and different surrounding vegetation. We conclude from the clear difference in ecology that ecological plant speciation is a major factor in establishing and maintaining a reproductive barrier. We explain the weak genetic differentiation with a recent separation of the two evolutionary units during the last glaciation cycles or even in postglacial periods

Authentication of Iceland Moss (Cetraria islandica) by UPLC-QToF-MS chemical profiling and DNA barcoding

The lichen Cetraria islandica or Iceland Moss is commonly consumed as tea, food ingredients (e.g. in soup or bread) and herbal medicines. C. islandica, which has two chemotypes, can be difficult to distinguish from the sister species Cetraria ericetorum. They are collectively referred to as the Cetraria islandica species complex. This study aimed to use an UPLC-QToF-MS chemical profiling together with DNA barcoding to distinguish species and chemotypes of the C. islandica species complex. Our results show that the two chemotypes of C. islandica are clearly distinguishable from each other and from C. ericetorum by the chemometric approach. The RPB2 barcode was able to differentiate C. islandica from C. ericetorum with a barcode gap, but the widely used nrITS barcode failed. Neither of them could discriminate chemotypes of C. islandica. In conclusion, this integrative approach involving chemical profiling and DNA barcoding could be applied for authentication of Iceland Moss materials

Boundary States for GS superstrings in an Hpp wave background

We construct the boundary states preserving half the global supersymmetries in string theory propagating on a Hpp background.Comment: 13 pages, latex2e with JHEP3.0 class, no figures. V1: clarified the status of branes not sitting at the origin and added a constraint which boundary states must satisf

Bacterial tolerance to host-exuded specialized metabolites structures the maize root microbiome.

Plants exude specialized metabolites from their roots, and these compounds are known to structure the root microbiome. However, the underlying mechanisms are poorly understood. We established a representative collection of maize root bacteria and tested their tolerance against benzoxazinoids (BXs), the dominant specialized and bioactive metabolites in the root exudates of maize plants. In vitro experiments revealed that BXs inhibited bacterial growth in a strain- and compound-dependent manner. Tolerance against these selective antimicrobial compounds depended on bacterial cell wall structure. Further, we found that native root bacteria isolated from maize tolerated the BXs better compared to nonhost Arabidopsis bacteria. This finding suggests the adaptation of the root bacteria to the specialized metabolites of their host plant. Bacterial tolerance to 6-methoxy-benzoxazolin-2-one (MBOA), the most abundant and selective antimicrobial metabolite in the maize rhizosphere, correlated significantly with the abundance of these bacteria on BX-exuding maize roots. Thus, strain-dependent tolerance to BXs largely explained the abundance pattern of bacteria on maize roots. Abundant bacteria generally tolerated MBOA, while low abundant root microbiome members were sensitive to this compound. Our findings reveal that tolerance to plant specialized metabolites is an important competence determinant for root colonization. We propose that bacterial tolerance to root-derived antimicrobial compounds is an underlying mechanism determining the structure of host-specific microbial communities

Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node

Dendritic and monocytic cells co-operate to initiate and shape adaptive immune responses in secondary lymphoid tissue. The complexity of this system is poorly understood, also because of the high phenotypic and functional plasticity of monocytic cells. We have sequenced mononuclear phagocytes in mesenteric lymph nodes (LN) of three adult cows at the single-cell level, revealing ten dendritic-cell (DC) clusters and seven monocyte/macrophage clusters with clearly distinct transcriptomic profiles. Among DC, we defined LN-resident subsets and their progenitors, as well as subsets of highly activated migratory DC differing in transcript levels for T-cell attracting chemokines. Our analyses also revealed a potential differentiation path for cDC2, resulting in a cluster of inflammatory cDC2 with close transcriptional similarity to putative DC3 and monocyte-derived DC. Monocytes and macrophages displayed sub-clustering mainly driven by pro- or anti-inflammatory expression signatures, including a small cluster of cycling, presumably self-renewing, macrophages. With this transcriptomic snapshot of LN-derived mononuclear phagocytes, we reveal functional properties and differentiation trajectories in a “command center of immunity”, and identify elements that are conserved across species

The lactonase BxdA mediates metabolic specialisation of maize root bacteria to benzoxazinoids.

Root exudates contain specialised metabolites that shape the plant's root microbiome. How host-specific microbes cope with these bioactive compounds, and how this ability affects root microbiomes, remains largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound in the maize rhizosphere MBOA (6-methoxybenzoxazolin-2(3H)-one) and formed AMPO (2-amino-7-methoxy-phenoxazin-3-one). AMPO forming bacteria were enriched in the rhizosphere of benzoxazinoid-producing maize and could use MBOA as carbon source. We identified a gene cluster associated with AMPO formation in microbacteria. The first gene in this cluster, bxdA encodes a lactonase that converts MBOA to AMPO in vitro. A deletion mutant of the homologous bxdA genes in the genus Sphingobium, did not form AMPO nor was it able to use MBOA as a carbon source. BxdA was identified in different genera of maize root bacteria. Here we show that plant-specialised metabolites select for metabolisation-competent root bacteria. BxdA represents a benzoxazinoid metabolisation gene whose carriers successfully colonize the maize rhizosphere and thereby shape the plant's chemical environmental footprint