Barcoding Analysis Research Articles

Introduction of the South American slipper limpet Crepipatella dilatata in Europe: Secondary spread in the Northeast Atlantic and first records for the Mediterranean Sea

The slipper limpet Crepipatella dilatata, native to Chile and Argentina, was introduced in north-western Spain in 2005 and since then has spread to the Galician and Cantabrian coasts. In this work, we provide the first molecularly validated record of an established population of C. dilatata in central Portugal (Aveiro), and of reproductive individuals (i.e. brooding females) of C. dilatata in the Mediterranean Sea, from three Italian mussel farms (in eastern Sardinia and northern Adriatic). DNA barcoding and phylogenetic analyses based on mitochondrial markers indicate that limpets from Italian farms belong to the C. dilatata ‘clade1’, whereas limpets collected in Aveiro cluster within the C. dilatata ‘clade2’. All these limpets share the same COI haplotypes with introduced populations from Galicia. The arrival of C. dilatata to Europe is undoubtedly due to transoceanic live mussel trade from southern Chile to Galicia. Mussel trade and farming are also the most likely drivers for the post-border dispersal of C. dilatata from Spain to Portugal and to Italy. Several countries have a potential role as a hub for the primary and secondary dispersal of slipper limpets towards and within Europe. This calls the attention for future research focused on assessing the current occurrence and ecological impact of established populations of slipper limpets within and nearby mussel farms along the eastern Atlantic and Mediterranean coasts.

Distribution and taxonomy of two closely related Nychiodes species in southern Italy (Lepidoptera, Geometridae, Ennominae)

We investigated the distribution and species circumscription of Nychiodes obscurariaVillers (1789) and Nychiodes ragusaria Millière (1884). The morphology of male and female genitalia of Calabrian populations (southern Italy) revealed them to belong to N. ragusaria while specimens from Basilicata, Molise and the rest of Italy turned out to be N. obscuraria. Although the DNA barcodes showed a divergence of 1.55% between typical N. obscuraria and N. ragusaria, 9 out of 28 Calabrian specimens of N. ragusaria shared the N. obscuraria haplotype, and sometimes both haplotypes were found at the same site. Genetic introgression appears as the most probable scenario to explain the observed pattern. A correct identification for the investigated species pair should be based on morphology instead of DNA barcode analysis because of the observed mitonuclear discordance at mountain sites. Further studies are needed to clarify the occurrence of this discordance at high elevations only.

Molecular and cytogenetic analyses in Geranium macrorrhizum L. wild Italian plants.

Geranium macrorrhizum L. is a herbaceous species native to southern Europe and was introduced in central Europe and North America. It is also widely distributed in Italy. In this study, molecular and cytogenetic analyses were carried out on 22 wild plants, collected in central and southern Italy, compared with five cultivated plants, with the main purpose to identify those living near the Marmore waterfalls in central Italy, recently described as the new species Geranium lucarinii. Four barcoding markers (rbcL, matK, trnH-psbA intergenic spacer and internal transcribed spacer region) were sequenced and their variability among the plants was evaluated. Chromosome numbers were determined and 45S rDNA was physically mapped by fluorescence in situ hybridization. Moreover, genomic affinity between wild and cultivated plants was evaluated by genomic in situ hybridization. The results of this study supported that all the plants belong to G. macrorrhizum, including the Marmore population. Barcoding analyses showed a close similarity among the wild plants, and a differentiation, although not significant, between the wild plants on one hand and the cultivated plants on the other. Integrated studies focusing on morphological, genetic and ecological characterization of a larger number of wild populations would allow us to know the extent of the variability within the species.

Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi.

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies hadshown thatE. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstratedthat E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.

Confirmation of the valid specific status of Dolichovespulakuami Kim & Yoon, 1996 (Hymenoptera, Vespidae) based on molecular and morphological evidence.

The taxonomic validity of Dolichovespulakuami, especially in relation to D.flora, has been the subject of a long-term debate. Herein, the valid specific status of the former was supported through an integrated analysis of morphological characters and DNA barcodes. The pronotal rugae and male genitalia of the two species are different, and partial mitochondrial genes (cytochrome oxidase subunit I, COI) indicate that they form significantly distinct lineages. The hitherto unknown male of D.kuami is described for the first time, and a brief discussion of the D.maculata species group is provided.

Descriptions of new Hypotrabala Holland, 1893 (Lepidoptera: Lasiocampidae: Lasiocampinae: Selenepherini) in the collections of the African Natural History Research Trust, with notes on allied genera and the description of a new genus

The Afrotropical lappet moth genus Hypotrabala Holland, 1893 is reviewed utilising specimens housed in the African Natural History Research Trust collections. The synthesis of morphological and DNA analyses has reconciled discrepancies in previous treatments of the genus and Hypotrabala is clearly delimited with implications on the nomenclature and taxonomy. The genus Epitrabala Hering, 1932 syn. n. is synonymised with Hypotrabala resulting in the following changes: Hypotrabala argenteoguttata (Aurivillius, 1909) comb. rev. is revived, H. horridula Tams, 1925 stat. rev. is removed from synonymy with Epitrabala nyassana (Aurivillius, 1909) and two taxa, H. horridula seydeli Tams, 1925 syn. n. and Epitrabala argyrostigma Hering, 1932 syn. n. are synonymised with it. Epitrabala nyassana is transferred to the genus Leptometa (comb. n.) as is Leptometa sanguicincta comb. n. (previously assigned to Hypotrabala), while Hypotrabala odonestioides Berio, 1937 is excluded from Hypotrabala although its generic placement remains uncertain. Based on the structures of the male eighth sternite, Hypotrabala fontainei stat. n. is raised to species status and a new genus Megatrabala gen. n. is described for the striking taxon M. regalis (Tams, 1953) comb. n. Furthermore, 19 new species are described: H. aurantiaca, H. cinereamargo, H. exquisita, H. extenuata, H. giustii, H. indefinita, H. igneata, H. lunda, H. lydiae, H. magnimacula, H. obscura, H. ophioglossa, H. pallens, H. pruinosa, H. retorta, H. smithi, H. tabithae, H. tamsi and H. volynkini spp. n. Certain incongruencies between the results of the barcode analyses and species concepts in this genus and Lasiocampidae more generally are discussed.

Abstract 6225: Tumor evolution through selection by ECM stiffness

Abstract In this study, we used experimental evolution to investigate the extent to which the mechanical stiffness of the tumor extracellular matrix (ECM) exerts selection pressure on a genetically diverse population of breast cancer cells. Polyacrylamide hydrogels were fabricated and conjugated with collagen type I to create the ECM with different stiffness. MDA-MB-231 cells were continuously cultured on the hydrogels of the same corresponding stiffness. Growth rate was determined at certain time points by harvesting and counting the cells. Abundance of clonal MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection was identified with sequencing of DNA barcode tags. Cellular phenotypes were characterized through immunofluorescence staining and confocal microscopy. 3D migration of the cancer cells was examined using Boyden chamber assay. Cellular contractility was measured through traction force microscopy. Whole genome transcriptome sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing, and reduced representation bisulfite sequencing were performed for transcriptomic and epigenetic analysis. Sustained culture of MDA-MB-231 breast cancer cells overall three months on soft ECM resulted in an increased proliferation rate and beta1 integrin-dependent spreading area of the cells. Such changes did not occur in clonal cells with low genetic variation and did not occur in ancestral cells on stiff substrates. Barcode analysis revealed two distinct surviving populations post-genetic selection, with one demonstrating robust proliferation on both soft and stiff ECM, and the other exhibiting preferential adaptation to the soft matrix. The soft-selected populations exhibited nuclear localization of the transcriptional regulator yes-associated protein and less wrinkled nuclei compared with ancestral populations when both populations were grown on soft ECM. Aggressive migration was observed in the soft-selected populations, indicating a more malignant selected phenotype. The soft-selected cells showed increased RhoA activity and contractile tension on a soft substrate compared to the ancestral cells. RNA-seq analysis revealed systematic differences between ancestral, stiff-selected, and soft-selected populations corresponding to enriched biological processes related to cell adhesion and ECM organization. Significant differences in DNA methylation and chromatin accessibility were observed in soft selected populations which were correlated with changes in RNA expression. ECM stiffness exerts selection pressure on genetically variable cancer cell populations, selecting specific genetic variants in a cell population optimally adapted for that stiffness. Selected variants on soft ECM tend to be highly migratory, proliferative and generate high traction even on soft ECM. Our results highlight the importance of ECM stiffness-driven evolution in cancer development. Citation Format: Ting-Ching Wang, Suchitaa Sawhney, Daylin Morgan, Richa Rashmi, Marcos R. Estecio, Amy Brock, Irtisha Singh, Richard L. Bennett, Charles F. Baer, Jonathan D. Licht, Tanmay P. Lele. Tumor evolution through selection by ECM stiffness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6225.

New data on the clupeoids of lakes Mai-Ndombe and Tumba, western Democratic Republic of the Congo, using an integrated approach combining morphology and barcoding

Morphological and molecular comparisons of clupeoid fishes in lakes Tumba and Mai-Ndombe in the Congo Basin revealed that a large-sized form of a common species in Lake Mai-Ndombe had not been distinguished from the small-sized endemic Mai-Ndombe dwarf sprat Nannothrissa stewarti Poll & Roberts, 1976 in ecological studies. The large-sized form differs from the Mai-Ndombe dwarf sprat in several morphological characters and resembles the smalltoothed pellonula Pellonula leonensis Boulenger, 1916, but in barcoding analysis appears more closely related to the bluntnosed sawtooth pellonuline Potamothrissa obtusirostris (Boulenger, 1909). This large-sized form is probably a species new to science but will remain undescribed for now, and is here referred to as Pellonulinae sp. BIG. A slender clupeoid, also from Lake Mai-Ndombe, has recently been referred to as the Lake Tumba dwarf sprat Nannothrissa parva (Regan, 1917), which would be the first record of this species from that lake. Specimens of a slender clupeoid collected from Lake Mai-Ndombe for this study were identified as the losera fangtooth pellonuline Odaxothrissa losera Boulenger, 1899. This is the first confirmed occurrence of this species in Lake Mai-Ndombe, whereas the presence of N. parva could not be confirmed. The phylogenetic analysis strongly supports the genus Nannothrissa as currently delimited, here barcoded for the first time, but also confirms the previously reported mismatch between morphological classification and molecular data in this group. We further note that the Mai-Ndombe dwarf sprat attains a larger size than the maximum size noted in the original description in 1976, and it is suggested that an intense beach-seine fishery that utilises mosquito nets might have led to a population decline followed by decreased intraspecific competition, enhancing individual growth. For the same reason, Pellonulinae sp. BIG might have been less common previously and so remained undetected until now.

A DNA barcode library of Austrian geometridae (Lepidoptera) reveals high potential for DNA-based species identification.

Situated in the Eastern section of the European Alps, Austria encompasses a great diversity of different habitat types, ranging from alpine to lowland Pannonian ecosystems, and a correspondingly high level of species diversity, some of which has been addressed in various DNA barcoding projects. Here, we report a DNA barcode library of all the 476 species of Geometridae (Lepidoptera) that have been recorded in Austria. As far as possible, species were sampled from different Austrian regions in order to capture intraspecific genetic variation. In total, 2500 DNA barcode sequences, representing 438 species, were generated in this study. For complete coverage of Austrian geometrid species in the subsequent analyses, the dataset was supplemented with DNA barcodes from specimens of non-Austrian origin. Species delimitations by ASAP, BIN and bPTP methods yielded 465, 510 and 948 molecular operational taxonomic units, respectively. Congruency of BIN and ASAP partitions with morphospecies assignments was reasonably high (85% of morphospecies in unique partitions), whereas bPTP appeared to overestimate the number of taxonomic units. The study furthermore identified taxonomically relevant cases of morphospecies splitting and sharing in the molecular partitions. We conclude that DNA barcoding and sequence analysis revealed a high potential for accurate DNA-based identification of the Austrian Geometridae species. Additionally, the study provides an updated checklist of the geometrid moths of Austria.

Molecular Evidence Reveals Taxonomic Uncertainties and Cryptic Diversity in the Neotropical Catfish of the Genus Pimelodus (Siluriformes: Pimelodidae).

Pimelodus is the most speciose genus of the family Pimelodidae, and is amply distributed in the Neotropical region. The species-level taxonomy and phylogenetic relationships within this genus are still poorly resolved, however. These taxonomic problems and the general lack of data have generated major uncertainties with regard to the identification of specimens from different localities. In the present study, we applied a single-locus species delimitation approach to identify the MOTUs found within the genus Pimelodus and provide sound evidence for the evaluation of the species richness of this genus in the different river basins of the Neotropical region. The study was based on the analysis of sequences of the mitochondrial COI gene of 13 nominal species, which resulted in the identification of 24 consensus MOTUs. Only six nominal species were recovered as well-defined molecular entities by both the traditional barcoding analysis and the molecular delimitation methods, while the other seven presented cryptic diversity or persistent taxonomic uncertainties. The lineages identified from the Parnaíba ecoregions, Amazonas Estuary and Coastal Drainages may represent a much greater diversity of Pimelodus species than that recognized currently, although a more detailed study of this diversity will be necessary to provide a more definitive classification of the genus.

Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts.

P16 inactivation is frequently accompanied by telomerase reverse transcriptase (TERT) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. A zinc finger protein-based P16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. P16 methylation drives TERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

Assessment of genetic diversity of freshwater genus Labeo through DNA barcoding and phylogenetic analysis inhabited in the River Indus

ObjectivesGenus Labeo of family Cyprinidae comprising of important group of fish species are known for ornamental purpose, food, extracting oil, medicinal value and for potential viable aquaculture. The present study was conducted to assess the morphometric and molecular identification of freshwater fishes of genus Labeo including Catla catla, Labeo rohita, Labeo calbasu, Labeo gnius, Labeo bata from the three fish resources of River Indus, Punjab, Pakistan. MethodsOut of 75, thirty fish specimen were amplified and sequenced with 650 bp of mitochondrial COI gene for the evaluation of fish biodiversity, management and conservation by using different bioinformatics techniques. ResultsMaximum and minimum genetic divergence between fishes of C. catla, L. calbasu, L. boga, L. gonius and L. rohita, were 0.052 to 0.105, respectively. Highest divergence was found between Labeo bata and Labeo rohita. Interspecific mean distance was found higher than intraspecific distance. In nucleotide base composition, AT contents was higher than GC contents indicated the clear pattern of anti-G bias. Transition rate was k1 = 8.84 for purines and transversional rate was k2 = 15.47 for pyrimidines with transition/transversion bias R = 5.62. Tajima D test and nucleotide diversity was measured D = 1.382883 and π = 0.049981 respectively. Positive value of D indicated a strong selective pressure and lack of population expansion. Thirty species were identified with e-value 0.0 and similar identity 99.85 %–100 % with maximum 600 bp length. Phylogenetic tree revealed two major clades with 95 % high bootstrap values identity showed closet relationship among these species. ConclusionsCurrent study supported the efficacy of the COI gene for the species identification in River Indus and develop the concept for establishing Pakistani fish gene bank which is still needed.

DNA Barcoding Analysis of Trinidad Haemagogus Mosquitoes Reveals Evidence for Putative New Species.

Background: Haemagogus janthinomys is a primary sylvan vector of yellow fever virus and the emerging Mayaro virus. However, despite its medical importance, there is a dearth of data on the molecular taxonomy of this mosquito species. Methods: In this study, DNA barcoding analysis was performed on 64 adult female mosquitoes from Trinidad morphologically identified as Hg. janthinomys. The mitochondrial cytochrome c oxidase I (COI) gene and ribosomal DNA internal transcribed spacer 2 (ITS2) region of the mosquitoes were PCR amplified and sequenced, and molecular phylogenies inferred. Results: The BLASTN analysis showed that only 20% (n = 13/66) of COI sequences had high similarity (>99% identity) to Hg. janthinomys and the remaining sequences had low similarity (<90% identity) to reference GenBank sequences. Phylogenetic analysis of COI sequences revealed the presence of four strongly supported groups, with one distinct clade that did not align with any reference sequences. Corresponding ITS2 sequences for samples in this distinct COI group clustered into three clades. Conclusions: These molecular findings suggest the existence of a putative new Haemagogus mosquito species and underscore the need for further, more in-depth investigations into the taxonomy and classification of the Haemagogus genus.

MBARq: a versatile and user-friendly framework for the analysis of DNA barcodes from transposon insertion libraries, knockout mutants, and isogenic strain populations.

DNA barcoding has become a powerful tool for assessing the fitness of strains in a variety of studies, including random transposon mutagenesis screens, attenuation of site-directed mutants, and population dynamics of isogenic strain pools. However, the statistical analysis, visualization, and contextualization of the data resulting from such experiments can be complex and require bioinformatic skills. Here, we developed mBARq, a user-friendly tool designed to simplify these steps for diverse experimental setups. The tool is seamlessly integrated with an intuitive web app for interactive data exploration via the STRING and KEGG databases to accelerate scientific discovery. The tool is implemented in Python. The source code is freely available (https://github.com/MicrobiologyETHZ/mbarq) and the web app can be accessed at: https://microbiomics.io/tools/mbarq-app.

Three new species of sea star parasite Dendrogaster (Crustacea: Thecostraca) from Japan.

Three new species of ascothoracidan crustaceans, Dendrogaster danni sp. nov., Dendrogaster tanabensis sp. nov., and Dendrogaster jinshomaruae sp. nov. are described from the sea around the Kii Peninsula on the Pacific coast of central Japan. They are found in the coelomic cavities of the sea stars, Neoferdina japonica Oguro & Misaki, 1986., Henricia sp., and Coronaster volsellatus (Sladen, 1889), respectively. Morphological examinations and DNA barcoding analyses of these new species are reported in this study. The emergence of Dendrogaster from their host sea stars is also noted. These findings represent the 11th to 13th species of Dendrogaster that infest Japanese sea stars.

DNA barcoding, identification, and validation of the pufferfish (Order: Tetraodontiformes) in China coastal waters.

The order Tetraodontiformes are one of the most unique groups of teleostean fish, exhibiting highly derived and greatly diversified phenotypes. It is a difficult task for both professionals and nonprofessionals to accurately identify these species only according to morphological characteristics. DNA barcoding can identify species at the molecular level to overcome the limitations of morphological classification. In this study, we collected 616 specimens of pufferfish from the coastal waters of China. According to the morphological characteristics, they were preliminarily identified as 50 species. Further analysis using DNA barcodes identified these specimens as 46 species, belonging to 23 genera, 6 families. According to the species classification results of DNA barcoding, the three species identified by morphology as Takifugu pseudommus, Takifugu chinensis, and Takifugu rubripes should be the same species. Similarly, Lagocephalus wheeleri is the synonym of Lagocephalus spadiceus. Another important discovery of DNA barcoding analysis is that there are closer interspecific genetic distances within the genus Takifugu. If T. rubripes, T. pseudommus, and T. chinensis are taken as one species, the average interspecific to intraspecific genetic distance ratio of Takifugu is only 6.21 times, which does not reach the DNA barcoding threshold of more than 10 times proposed previously. Although the interspecific genetic distance in the genus Takifugu is relatively small, each species can be clustered into independent clades in the NJ tree. In conclusion, this study not only found that there are synonymous phenomena in the order Tetraodontiformes but also provided molecular evidence for the valid species names of Takifugu rubripes and Lagocephalus Spadiceus. The results can provide reliable DNA barcoding information for the identification of pufferfish species, help solve the problem of classification confusion in this order, and provide technical support for the identification of the original components of related commodities on the aquatic product market.

Identification and Toxicity Evaluation of &lt;i&gt;Beauveria&lt;/i&gt; sp. Associated with White Muscardine Disease in Muga Silkworm &lt;i&gt;Antheraea assamensis&lt;/i&gt; Helfer

The present study is an effort to isolate and identify the Beauveria sp. from white muscardine infected Antheraea assamensis Helfer (muga silkworm) larvae collected from rearing fields. The isolate was subjected to morphological identification followed by DNA barcoding analysis by sequencing the ITS1-5.8S-ITS2 region. The isolate was identified morphologically up to genus level. For molecular identification, the DNA was first isolated, amplified by using PCR followed by sequencing the ITS region. A phylogenetic tree was also constructed based on the data obtained from sequencing to trace the evolutionary history of the isolated fungus. The fungal species was identified as Beauveria bassiana with 98.6% sequence similarity with already documented B. bassiana strains. The toxicity of the isolated fungus was also evaluated against A. assamensis larvae. The LC50 value was determined at 96 hr and recorded at 1.1x108 spore/ ml concentration.

Integrating DNA barcoding, microscopic, and chemical analyses for precise identification of Plumbago indica L., A prominent medicinal plant

Plumbago species, particularly P. indica L., P. zeylanica L., and P. auriculata Lam., are extensively utilized in traditional medicine to treat various ailments. The roots of Plumbago plants are rich in plumbagin, a key bioactive compound with reported pharmacological properties. In Thai traditional medicine, P. indica roots, known as “Jettamoon Pleung Deang” crude drugs, are a vital herbal ingredient in traditional formulations. However, due to high demand and limited supply, there have been instances of adulteration and substitution with other Plumbago species. Consequently, implementing reliable authentication methods is crucial for quality control. This study aimed to differentiate P. indica from P. auriculata and P. zeylanica using a combination of microscopic analysis, TLC, and DNA barcoding. Additionally, the authentication of “Jettamoon Pleung Deang” crude drug was examined. Histological characteristics of roots and stems provided clear differentiation of P. indica from P. zeylanica and P. auriculata. TLC profiling under UV at 365 nm highlighted distinct chromatographic patterns among the three Plumbago species. DNA barcoding employed six DNA loci (matK, ITS, ITS2, rbcL, trnH-psbA, and trnL-F) in the three Plumbago species, selecting ITS2 as the potent region for authenticating commercial crude drugs. Utilizing ITS2, the study confirmed that all crude drugs originated from P. indica based on variable site percentages and interspecific divergence. Histological examination also revealed the presence of stems mixed with roots in certain crude drug samples. This integration of microscopy, TLC, and DNA barcoding, particularly utilizing ITS2, proved effective in authenticating Plumbago crude drugs and holds significant promise for quality control within the herbal industry.

DNA barcoding for the identification and authentication of medicinal deer (Cervus sp.) products in China.

Deer products from sika deer (Cervus nippon) and red deer (C. elaphus) are considered genuine and used for Traditional Chinese Medicine (TCM) materials in China. Deer has a very high economic and ornamental value, resulting in the formation of a characteristic deer industry in the prescription preparation of traditional Chinese medicine, health food, cosmetics, and other areas of development and utilization. Due to the high demand for deer products, the products are expensive and have limited production, but the legal use of deer is limited to only two species of sika deer and red deer; other wild deer are prohibited from hunting, so there are numerous cases of mixing and adulteration of counterfeit products and so on. There have been many reports that other animal (pig, cow, sheep, etc.) tissues or organs are often used for adulteration and confusion, resulting in poor efficacy of deer traditional medicine and trade fraud in deer products. To authenticate the deer products in a rapid and effective manner, the analysis used 22 deer products (antler, meat, bone, fetus, penis, tail, skin, and wool) that were in the form of blind samples. Total DNA extraction using a modified protocol successfully yielded DNA from the blind samples that was useful for PCR. Three candidate DNA barcoding loci, cox1, Cyt b, and rrn12, were evaluated for their discrimination strength through BLAST and phylogenetic clustering analyses. For the BLAST analysis, the 22 blind samples obtained 100% match identity across the three gene loci tested. It was revealed that 12 blind samples were correctly labeled for their species of origin, while three blind samples that were thought to originate from red deer were identified as C. nippon, and seven blind samples that were thought to originate from sika deer were identified as C. elaphus, Dama dama, and Rangifer tarandus. DNA barcoding analysis showed that all three gene loci were able to distinguish the two Cervus species and to identify the presence of adulterant species. The DNA barcoding technique was able to provide a useful and sensitive approach in identifying the species of origin in deer products.

Phytoconstituents of Kenyan stinging nettle (Urtica species) and their molecular docking interactions revealed anti-inflammatory potential as cyclooxygenase-2 inhibitors

Inflammation is a complex, natural protective response towards different stimuli characterized by the dilation and permeation of the blood vessels with a surge in leukocytes in the tissues. The current treatment involves the use of anti-inflammatory drugs, corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs), which have been associated with adverse side effects especially gastrointestinal ulcers. Therefore, there is a growing need to explore alternative sources from medicinal plants. In the present study we investigated anti-inflammatory activities of the leaves of the Kenyan stinging nettle using in vivo and in silico molecular docking. Molecular identification of plant sample was carried out based on DNA barcoding. Crude extracts were prepared using water and methanol: dichloromethane (1:1) and preliminary identification of total phenolic and flavonoids was carried out using Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. The classical model of Carrageenan- induced paw edema was used to test the in vivo anti-inflammatory activity of the extracts. The extract was screened using Laser Raman Spectroscopy and Liquid Chromatography Mass Spectroscopy (LC-MS) and the molecular interaction between the identified compounds within the binding site of Cyclooxygenase-2 (COX-2) performed through molecular docking as the confirmatory tool of in vivo experiments. Based on DNA barcoding analysis, the plant sample was identified as Urtica species. The total phenolic content of the aqueous and methanol: dichloromethane extracts were 3.75 mg gallic acid equivalents (GAE)/g dry sample and 6.26 mg GAE/g dry sample while total flavonoid content were 0.3872 mg quercetin/g dry sample and 1.76 mg quercetin/g dry sample, respectively. The aqueous extract significantly (p < 0.05) reduced the paw edema in the Carrageenan model of inflammation. LC-MS confirmed the presence of 19 phytochemicals, of which 10 and 9 were phenolic and flavonoid compounds, respectively. From these identified compounds quercetin attained the lowest binding energy when complexed with COX-2, followed by rhamnetin, quercetin rhamnoside, epigallocatechin gallate and chlorogenic acid. Molecular docking studies supported the in vivo findings and confirmed the anti-inflammatory potential of Urtica sp. These findings suggest Urtica sp. is a potential source of bioactive compounds that could be employed in the future development of anti-inflammation drugs. Further studies can attempt to assess the individual isolated active compound(s) for their anti-inflammatory activity.