DNA Barcoding Technology Research Articles

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Non-invasive gene delivery across the blood-brain barrier: present and future perspectives.

Rapid Identification of Common Poisonous Plants in China Using DNA Barcodes

Toxic plants have been a major threat to public health in China. However, identification and tracing of poisoned species with traditional methods are unreliable due to the destruction of plant morphology by cooking and chewing. DNA barcoding is independent of environmental factors and morphological limitations, making it a powerful tool to accurately identify species. In our study, a total of 83 materials from 26 genera and 31 species of 13 families were collected and 13 plant materials were subjected to simulated gastric fluid digestion. Four markers (rbcL, trnH-psbA, matK, and ITS) were amplified and sequenced for all untreated and mock-digested samples. The effectiveness of DNA barcoding for the identification of toxic plants was assessed using Basic Local Alignment Search Tool (BLAST) method, PWG-Distance method, and Tree-Building (NJ) method. Except for the matK region, the amplification success rate of the remaining three regions was high, but the sequencing of trnH-psbA and ITS was less satisfactory. Meanwhile, matK was prone to be more difficult to amplify and sequence because of simulated gastric fluid. Among the three methods applied, BLAST method showed lower recognition rates, while PWG-Distance and Tree-Building methods showed little difference in recognition rates. Overall, ITS had the highest recognition rate among individual loci. Among the combined loci, rbcL + ITS had the highest species recognition rate. However, the ITS region may not be suitable for DNA analysis of gastric contents and the combination of loci does not significantly improve species resolution. In addition, identification of species to the genus level is sufficient to aid in the clinical management of most poisoning events. Considering primer versatility, DNA sequence quality, species identification ability, experimental cost and speed of analysis, we recommend rbcL as the best single marker for clinical identification and also suggest the BLAST method for analysis. Our current results suggest that DNA barcoding can rapidly identify and trace toxic species and has great potential for clinical applications. In addition, we suggest the creation of a proprietary database containing morphological, toxicological and molecular information to better apply DNA barcoding technology in clinical diagnostics.

Unraveling Tumor Heterogeneity by Using DNA Barcoding Technologies to Develop Personalized Treatment Strategies in Advanced-Stage PDAC.

Simple SummaryPancreatic cancer is one of the hardest-to-treat cancers. This is mainly due to its heterogeneity, where subsets of cancer cells possess distinct properties and abilities that determine if and how they metastasize or respond to therapy. DNA barcoding technologies have emerged as a powerful tool to study this heterogeneity, as they allow labeling of individual tumor cells within a cancer cell pool and follow their cellular states and fates during metastasis or upon therapy. The aim of this review was to provide an overview of the various levels of tumor heterogeneity in pancreatic cancer, the obstacles these levels of heterogeneity can cause for effective personalized treatment strategies, and how different barcoding approaches can be applied to study these important questions.Tumor heterogeneity is a hallmark of many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), and an inherent consequence of the clonal evolution of cancers. As such, it is considered the underlying concept of many characteristics of the disease, including the ability to metastasize, adapt to different microenvironments, and to develop therapy resistance. Undoubtedly, the high mortality of PDAC can be attributed to a high extent to these properties. Despite its apparent importance, studying tumor heterogeneity has been a challenging task, mainly due to its complexity and lack of appropriate methods. However, in recent years molecular DNA barcoding has emerged as a sophisticated tool that allows mapping of individual cells or subpopulations in a cell pool to study heterogeneity and thus devise new personalized treatment strategies. In this review, we provide an overview of genetic and non-genetic inter- and intra-tumor heterogeneity and its impact on (personalized) treatment strategies in PDAC and address how DNA barcoding technologies work and can be applied to study this clinically highly relevant question.

Application of DNA Molecular Identification Method to Distinguish Ejiao and its Adulterants

To identify Ejiao and its adulterants at the DNA level by using DNA molecula marker. Ejiao (Asini Corii Colla) is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the morphological characteristics of Asini Corii Colla and its adulterants, traditional identification techniques often complex and professional, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Asini Corii Colla and its adulterants accurately, this study identified and its adulterant samples based on the CytB sequence. Sequence characteristics, Basic Local Alignment Search Tool (BLAST) application, genetic distance, construction of phylogenetic tree showed the CytB sequence to accurately identify Asini Corii Colla from its adulterants. Furthermore, in this study, we designed a specific primer, based on the CytB sequence, and established a PCR detection system for rapid, sensitive, and specific identification of Asini Corii Colla. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Asini Corii Colla.

First record of traumatic myiasis obtained from forest musk deer (Moschus berezovskii)

Myiasis is an infestation of maggots on living tissue in humans and animals all over the world. It is known to occur in wild animals, while no information is reported in forest musk deer (Moschus berezovskii). During our research on the conservation of forest musk deer, we found a new record of traumatic myiasis of an injured forest musk deer. The flies are likely Lucilia caesar (Linnaeus, 1758) according to the results of DNA barcoding technology. We report traumatic myiasis of forest musk deer for the first time, which expands the information on parasite and myiasis of forest musk deer and confirms the potential risk of traumatic myiasis of forest musk deer.

Abstract LB226: Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death

Abstract The role of maternal microbiome transmitted at birth in cancer control is poorly understood. We have developed the first germfree B6 mouse model of breast cancer to investigate the role of the maternal microbiome in controlling oncogenic/metastatic frequencies of pro-oncogenic mammary cells. In this model, a DNA barcoded, primitive normal mouse mammary epithelial cell encoding MMTV-PyMT oncogene, was transplanted in large numbers into conventional or germfree B6 mice. Next-gen sequencing analysis of the DNA barcodes in tissues enabled us to clonally track millions of cells and measure the frequency and growth dynamics of clones at the primary site and their systemic distribution in circulation and all vital organs, generating an unprecedented high-definition map of cancer progression. Our results show that in conventional B6 mice with maternal microbial transmission at-birth, a small fraction (~0.01%) of transplanted cells transform de novo and produce slow growing, late-onset benign tumors (mouse median survival of > 1 year). In contrast, in germfree B6 mice, a >10-fold higher frequency of cells transform de novo and generate early-onset, highly aggressive metastatic clones, and are frequently associated with features leading to early euthanasia or endpoint (i.e. sudden death, intracardiac metastasis, paralysis, swollen abdomen, early multiorgan aggressive metastasis) (median survival of ~4 months; p<0.005 vs conventional mice). However, postnatal intervention with healthy fecal implants reconstituted gut microbiota in germfree B6 mice and significantly restored protective effects against cancer progression and sudden death, as well as improved quality/duration of life (median survival of >1 year; p<0.005 vs germfree mice; p=ns vs conventional mice). We found maternal microbial transmission fully protected against systemic metastasis. GC-MS analysis identified significantly distinct primary tumor metabolome associated with conventional (non-metastatic) mice vs germfree (metastatic) mice. Metagenomic analysis of fecal samples from a subset of tumor bearing conventional (non-metastatic) mice vs fecal implanted (late-onset, less aggressive metastatic) germfree mice identified significant association between metastasis and altered microbial communities. These findings using novel germfree mouse model of metastasis and cellular DNA barcoding technology provide first direct evidence that diverse microbial communities transmitted at birth play a critical role in protecting the host from clonal de novo mammary cell transformation and progression to systemic disease and thereby improving quality and duration of life. Citation Format: Nagarajan Kannan, Syed Mohammed Musheer Aalam, Xiaojia Tang, Krishna Kalari, Purna Kashyap, Jun Chen, Stephen Johnson, Anguraj Sadanandam, Mark Sherman. Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB226.

The Role of Aire in the selection of Regulatory T cells in Type 1 Diabetes

Abstract In autoimmune diseases like T1D, it is proposed that failure in central tolerance mechanisms towards pancreatic islet self-antigens leads to the escape of pathogenic clones and loss of critical immunosuppressive antigen-specific Tregs. The importance of Aire in maintaining central tolerance against self-reactive antigens is evidenced by the multiorgan autoimmune disease seen in individuals with mutation the Aire gene. When Aire-expression is specifically ablated in mouse mTECs, there is a dramatic increase in the number of self-reactive T cells that escape negative selection, in addition, a dramatic loss of Tregs. Utilizing novel high-throughput platforms and single-cell DNA barcoding technology has allowed us to assess several thousand CD4+ T-cell clones. The use of FoxP3-GFP reporter mice permits us to identify individual FoxP3+ Tregs that have undergone Aire-dependent selection in the thymus and compare the TCR repertoire to mice that have had Aire specifically ablated from the mTECs. Identification of unique clones and gene signatures within our large datasets has allowed us to start to qualitatively assess the functional role of the identified TCRs within the model of T1D. GLIPH2 clustering using our extensive insulin-specific TCRs have identified a number of motifs shared within our TCR dataset, as well as a unique V-gene usage that may predict TCR affinity. Our proposed study aims to expand our knowledge on TCR repertoire of Aire-dependent FoxP3+ Tregs and the antigen-specificity in T1D, improving our understanding of the respective function of thymic-derived antigen-specific Treg suppression in peripheral tissues associated with autoimmune T1D.

Molecular identification of Aquilaria species with distribution records in China using DNA barcode technology

ABSTRACTS Aquilaria species is one of the main plant resources that produce agarwood, which containing black resin with important economic and medicinal values. There are about 15 species known to the genus around the world, but only two can be found in China, i.e. A. sinensis and A. yunnanensis. In this study, A. sinensis and A. yunnanensis that endemic respectively to Hainan and Yunnan were sampled, on the basis of the investigation and observation of their main morphological features in plantation. Five primers, i.e. ITS2, matK, trnL-trnF1, trnL-trnF2, and trnH-psbA, were eventually selected for DNA barcoding. The results showed that the seed surface of A. sinensis is smooth or sparsely pubescent, and the seed appendages were long. While the seed surface of A. yunnanensis is densely covered with yellow hairs and the seed appendages are short. The trnL-trnF1 sequence fragment has significant intraspecific and interspecific genetic distances. However, the species identification success rate of ITS2+matK combination was finally screened to be the highest, which was verified by the BBA method of TaxonDNA. The phylogenetic trees cluster analysis revealed that the classification of A. sinensis and A. yunnanensis is significant, and there is geographic isolation between the two species. Therefore, on the premise of accurate identification of plant morphological characters, ITS2+matK combination can be used to accurately identify the Aquilaria species in China.

A systematic strategy for uncovering quality marker of Asari Radix et Rhizoma on alleviating inflammation based chemometrics analysis of components

Asari Radix et Rhizoma (Asarum), a traditional Chinese medicine (TCM), has been applied in clinical generally. However, due to the lack of valid methods for Asarum quality control, inhomogenous quality and therapy issues have become severe with each passing day. In this study, we aimed to establish a comprehensive multi-system to explore the quality control markers underlying pharmaceutical effects based on chemometrics analysis on the total ingredients of Asarum. In brief, DNA barcoding technology was used to screen out the unadulterated herbs in the 15 batches Asarum collected from different origins. Then, the chemical profiles of volatile/nonvolatile components of 10 batches Asarum with definite resource were obtained by HPLC Q-TOF/MS and GC/MS. Combination with chemometrics methods, 14 characteristic ingredients and 4 qualitative and quantitative markers were figured out preliminarily. Moreover, correlation analysis between the characteristic ingredients and the cytokines integrating the virtual targets prediction of network pharmacology, 3 potential bioactive substance were ascertained. In conclusion, l-asarinin, 2-Methoxy-4-vinylphenol and safrole were considered as the potent candidates for quality control markers based on the comprehensive understanding for therapeutic effects and the chemical information of Asarum, which provided a novel perspective of the development for the quality control of TCM.

Genetic differentiation of predominant mosquito species in Hainan province and characterization of mosquito midgut microbiota

Objective: To identify the mosquito species collected in the field of 8 cities/counties of Hainan province, China and to analyze the characteristics of genetic differentiation of the predominant mosquito species. The types, contents and composition characteristics of the midgut microbiota of various mosquito species were explored to provide evidence for the control and prevention of mosquito-borne diseases in Hainan province. Methods: Adult mosquitoes were collected in the field using light traps, human lure or cattle lure methods. Morphology and DNA-barcoding technology (COI gene) were applied to identify the mosquito species. The V3-V4 hypervariable region of microbes 16S rRNA was used for high-throughput sequencing of the midgut microbiota, and SPSS 21.0 and R v3.1.1 software were employed to perform statistical analysis of the sequencing results and the Beta diversity analysis. Results: Aedes (Ae.) albopictus, Armigeres (Ar.) subalbatus and Culex (Cx.) pipiens pallpens were the three predominant species of the mosquito samples. Ae. albopictus was closely related to MK736660 (India) and JQ235749 (Yunnan), while MT541282 (DF, Dongfang) was a relatively independent population. The composition and structure of bacterial communities significantly varied among most of the samples (P<0.05). At the genus levels, 16 genera overlapped in the midgut in Ae. albopictus, 20 in Ar. subalbatus and 26 in Ar. subalbatus. Besides, Ae. albopictus, Ar. subalbatus and Cx. pipiens pallpens shared 15 out of the 16 overlapping genera. Conclusions: Ae. albopictus, Ar. subalbatus and Cx. pipiens pallpens were close within the same species in Hainan. The main exchange of mosquito species occurs within Hainan province, but also occurs across provincial or even national boundaries. The gut microbiota of mosquito species collected from the same sites were similar. Most of the core bacteria found in the midgut of Ae. albopictus were also present in Ar. subalbatus and Cx. pipiens pallpens, indicating that the breeding environment of Ae. albopictus may cover the breeding environment of Ar. subalbatus and Cx. pipiens pallpens.

Barcoding Technology for Multiplexed Analysis of Metastatic Ability In Vivo.

DNA barcoding allows the quantitative, biomarker-free tracking of individual cell populations in mixed/heterogeneous cell pools. Here, we describe a multiplexed in vivo screening platform based on DNA barcoding technology to interrogate compound libraries for their effect on metastatic seeding in vivo. We apply next-generation sequencing (NGS) technology to quantitatively analyze high-throughput compound screening in mice. Up to 96 compounds and controls can be screened for their effect on metastatic ability in a single mouse.

Comparative study on diatom morphology and molecular identification in drowning cases

In the field of criminal investigations, in the event that a body is found in water, the ability to differentiate whether the cause of death was drowning or the body was murdered then dumped into water elsewhere is difficult but important for case detection. Detecting diatoms in human organs can be used to effectively identify if the cause of death was drowning. At present, diatom detection methods are roughly divided into morphological and molecular detection methods, but both methods have different limitations. In this study, a total of 79 samples from 23 victims in 19 known drowning deaths were collected. The diatom morphological identification combined with DNA metabarcoding technology was used to compare the reliability of the diatom detection method. Microscopic observations revealed that the positive detection rate of diatoms was 52.6 %, 26.3 % and 58.8 % respectively in the kidney, liver and lung samples. DNA metabarcoding analysis found that the positive detection rate of diatoms was 31.6 %, 31.6 % and 35.3 % respectively in kidney, liver and lung samples. When compared with barcode BacirbcL, barcode 18S605 detected more diatoms, while diatoms in BacirbcL were more consistent with environmental samples. The comparative analysis found that microscopic observations were not highly correlated with the identification results of DNA barcoding technology. There were no obvious differences in the effect of internal organs on diatom enrichment, and different organs should be tested at the same time. At present, the DNA barcode reference sequence is gravely insufficient and has many errors, which leads to restrictions in the application of this technology, resulting in many OTU not being accurately identified. This explains why the success rate of molecular identification is not higher than that of microscopic identification. Construction of a reliable diatom DNA barcode reference sequence database is an urgent task for drowning forensics.

Survey of Species Covered by DNA Barcoding Data in BOLD and GenBank for Integration of Data for Museomics

DNA barcoding technology has become employed widely for biodiversity and molecular biology researchers to identify species and analyze their phylogeny. Recently, DNA metabarcoding and environmental DNA (eDNA) technology have developed by expanding the concept of DNA barcoding. These techniques analyze the diversity and quantity of organisms within an environment by detecting biogenic DNA in water and soil. It is particularly popular for monitoring fish species living in rivers and lakes (Takahara et al. 2012). BOLD Systems (Barcode of Life Database systems, Ratnasingham and Hebert 2007) is a database for DNA barcoding, archiving 8.5 million of barcodes (as of August 2020) along with the voucher specimen, from which the DNA barcode sequence is derived, including taxonomy, collected country, and museum vouchered as metadata (e.g. https://www.boldsystems.org/index.php/Public_RecordView?processid=TRIBS054-16). Also, many barcoding data are submitted to GenBank (Sayers et al. 2020), which is a database for DNA sequences managed by NCBI (National Center for Biotechnology Information, US). The number of the records of DNA barcodes, i.e. COI (cytochrome c oxidase I) gene for animal, has grown significantly (Porter and Hajibabaei 2018). BOLD imports DNA barcoding data from GenBank, and lots of DNA barcoding data in GenBank are also assigned BOLD IDs. However, we have to refer to both BOLD and GenBank data when performing DNA barcoding. I have previously investigated the registration of DNA barcoding data in GenBank, especially the association with BOLD, using insects and flowering plants as examples (Nakazato 2019). Here, I surveyed the number of species covered by BOLD and GenBank. I used fish data as an example because eDNA research is particularly focused on fish. I downloaded all GenBank files for vertebrates from NCBI FTP (File Transfer Protocol) sites (as of November 2019). Of the GenBank fish entries, 86,958 (7.3%) were assigned BOLD identifiers (IDs). The NCBI taxonomy database has registrations for 39,127 species of fish, and 20,987 scientific names at the species level (i.e., excluding names that included sp., cf. or aff.). GenBank entries with BOLD IDs covered 11,784 species (30.1%) and 8,665 species-level names (41.3%). I also obtained whole "specimens and sequences combined data" for fish from BOLD systems (as of November 2019). In the BOLD, there are 273,426 entries that are registered as fish. Of these entries, 211,589 BOLD entries were assigned GenBank IDs, i.e. with values in “genbank_accession” column, and 121,748 entries were imported from GenBank, i.e. with "Mined from GenBank, NCBI" description in "institution_storing" column. The BOLD data covered 18,952 fish species and 15,063 species-level names, but 35,500 entries were assigned no species-level names and 22,123 entries were not even filled with family-level names. At the species level, 8,067 names co-occurred in GenBank and BOLD, with 6,997 BOLD-specific names and 599 GenBank-specific names. GenBank has 425,732 fish entries with voucher IDs, of which 340,386 were not assigned a BOLD ID. Of these 340,386 entries, 43,872 entries are registrations for COI genes, which could be candidates for DNA barcodes. These candidates include 4,201 species that are not included in BOLD, thus adding these data will enable us to identify 19,863 fish to the species level. For researchers, it would be very useful if both BOLD and GenBank DNA barcoding data could be searched in one place. For this purpose, it is necessary to integrate data from the two databases. A lot of biodiversity data are recorded based on the Darwin Core standard while DNA sequencing data are sometimes integrated or cross-linked by RDF (Resource Description Framework). It may not be technically difficult to integrate these data, but the species data referenced differ from the EoL (The Encyclopedia of Life) for BOLD and the NCBI taxonomy for GenBank, and the differences in taxonomic systems make it difficult to match by scientific name description. GenBank has fields for the latitude and longitude of the specimens sampled, and Porter and Hajibabaei 2018 argue that this information should be enhanced. However, this information may be better described in the specimen and occurrence databases. The integration of barcoding data with the specimen and occurrence data will solve these problems. Most importantly, it will save the researcher from having to register the same information in multiple databases. In the field of biodiversity, only DNA barcode sequences may have been focused on and used as gene sequences. The museomics community regards museum-preserved specimens as rich resources for DNA studies because their biodiversity information can accompany the extraction and analysis of their DNA (Nakazato 2018). GenBank is useful for biodiversity studies due to its low rate of mislabelling (Leray et al. 2019). In the future, we will be working with a variety of DNA, including genomes from museum specimens as well as DNA barcoding. This will require more integrated use of biodiversity information and DNA sequence data. This integration is also of interest to molecular biologists and bioinformaticians.

Authentication, characterization and contamination detection of cell lines, xenografts and organoids by barcode deep NGS sequencing.

Misidentification and contamination of biobank samples (e.g. cell lines) have plagued biomedical research. Short tandem repeat (STR) and single-nucleotide polymorphism assays are widely used to authenticate biosamples and detect contamination, but with insufficient sensitivity at 5–10% and 3–5%, respectively. Here, we describe a deep NGS-based method with significantly higher sensitivity (≤1%). It can be used to authenticate human and mouse cell lines, xenografts and organoids. It can also reliably identify and quantify contamination of human cell line samples, contaminated with only small amount of other cell samples; detect and quantify species-specific components in human–mouse mixed samples (e.g. xenografts) with 0.1% sensitivity; detect mycoplasma contamination; and infer population structure and gender of human samples. By adopting DNA barcoding technology, we are able to profile 100–200 samples in a single run at per-sample cost comparable to conventional STR assays, providing a truly high-throughput and low-cost assay for building and maintaining high-quality biobanks.

DNA Barcode Technology and Its Application Prospects in Forensic Medicine.

Traditional species identification has gone through five stages -- morphology, cytology, biochemistry, immunology and molecular biology. At present, the use of DNA technology for species identification has become a research hotspot. In the use of DNA for species identification, the presentation and application of DNA barcode is of epoch-making significance. With the successful application of new technology in species identification, forensic species identification has also made corresponding development, and is expected to play an important role in forensic related fields. This paper briefly describes the general situation and principles of DNA barcode technology as well as its advantages and limitations when applied to biological classification, and discusses the future significance and feasibility of DNA barcode technology in forensic applications, in order to provide new ideas for future forensic identification.

Hidden in our pockets: building of a DNA barcode library unveils the first record of Myotis alcathoe for Portugal.

BackgroundThe advent and boom of DNA barcoding technologies have provided a powerful tool for the fields of ecology and systematics. Here, we present the InBIO Barcoding Initiative Database: Portuguese Bats (Chiroptera) dataset containing DNA sequences of 63 specimens representing the 25 bat species currently known for continental Portugal. For that, we sequenced tissues samples obtained in a vast array of projects spanning the last two decades.New informationWe added four new Barcoding Index Numbers (BINs) to existing Chiroptera barcodes on BOLD, two belonging to Myotis escalerai, one to Plecotus auritus and the other to Rhinolophus hipposideros. Surprisingly, one of the samples initially identified in the field as Myotis mystacinus turned out to be Myotis alcathoe, which represents the first record of this species for Portugal. The presence of Nyctalus noctula in Portugal was also genetically confirmed for the first time. This case study shows the power and value of DNA barcoding initiatives to unravel new data that may be hidden on biological collections.

Wildlife Collisions to Aircraft in India a Comparative Analysis of Hazardous Species Involved in Different Time Periods

Wildlife strikes (mainly birds, but also includes bats and other mammals on the ground) with aircraft isa serious economic and safety concern in the aviation industry. The solution to the problem can be evolved byidentifying the species involved in the incidents/ accidents. In the Indian context, such an attempt was started in1980. In the recent past, the Indian Air Force adopted the DNA Bar-coding technology to identify the species involved. The extent of the problems faced by the country and involvement of different species in various time blocks has been compared with the objective of analyzing changes over different periods to gauge the changes and assess the future requirements. The data indicates that over the years, the number of strikes has increased manifold in the civil aviation sector. The number of species involved in strikes has almost doubled. The serious strikes due to Vultures have nearly disappeared and their place has been mainly taken over by Black Kites. In the recent past, Black Kites are the cause of the highest damages and also have the highest probability of causing damages (61.17%) when struck. Adoption of DNA Barcoding technology has helped to identify the species in incidents where minimal bird remnants were found. Although the number of accidents has decreased, the economical losses continue to rise due to the high cost of modern aircraft.

Application of DNA barcoding in fish identification of supermarkets in Henan province, China: More and longer COI gene sequences were obtained by designing new primers

In recent years, DNA barcode technology has been widely used in food identification, especially in the identification of fish. In China, there are few studies on the authenticity of fish products in Henan province of China. In this study, 179 fish samples were collected from supermarkets in Zhengzhou city and Xinxiang city in Henan province, China. COI gene sequences were obtained with PCR technology by designing specific primers and universal primers. COI gene sequences of all samples were obtained to identify species, which is used to investigate species substitution and mislabeling of the fish sold in the two regional markets. The molecular identification results showed that 28.49% (51/179) fish samples were not consistent with the labels. Substitution of high-price fish by low-price fish was prevalent. For example, halibut (Pleuronectiformes) and cod (Gadus) are replaced by striped catfish (Pangasianodon hypophthalmus), and some merchants label bighead carp (Hypophthalmichthys nobilis) as cod (Gadus), there are also accidental labeling errors (such as labels for greenfin horse-faced filefish (Thamnaconus septentrionalis) have been identified as grass carp (Ctenopharyngodon idella) and Wuchang bream (Megalobrama amblycephala) etc. Most of the samples labeled correctly are the fish of low economic value and the fresh fish. This study shows that almost all the commercial fish can be identified by COI DNA barcoding by newly designed primers. Finally, this study also gives a reference of real species of fish fillet in Henan province in China.

BARKOD DNA PADA TANAMAN GAMBIR (Uncaria gambir(Hunter) Roxb.) BERDASARKAN GEN matK DAN rbcL

Gambir ( Uncaria gambir (Hunter) Roxb.) is a superior commodity of West Sumatra that has many benefits and has been used as a drug. Plant identification carried out morphologically has many weaknesses, with the development of electronic and genetic technology now a new method of species identification has been developed. plants and animals, namely DNA barcoding technology that uses standard short pieces of DNA Species identification methods have been agreed to using standard DNA barcodes are rbcL genes and matK genes. The purpose of this study is to find out DNA Barkoding candidates between matK and rbcL which can identify gambier ( Uncaria gambir (Hunter) Roxb.) Which is good. Polymerase Chain Reaction (PCR) technique is used to amplify rbcL and matK gene sequences through universal primers. The DNA sequences of the MATC sequence in the GR sample (Riau Gambir) produced a similarity rate of 98.79% with Nauclea diderrichii , for the rbcL sequence GC (Cubadak Gambir) samples produced a similarity rate of 99.81% with Uncaria macrophylla , and GR (Gambir Riau) samples produced the similarity rate was 96.84% with Uncaria macrophylla based on analysis with BOLD Systems. This similarity indicates the low variation in intraspecific genetics for the identification or confirmation of gambir species

The Role of Aire in the selection of Regulatory T cells in Diabetes

Abstract In autoimmune diseases like T1D, it is proposed that failure in central tolerance mechanisms towards pancreatic islet self-antigens leads to the escape of pathogenic clones and loss of critical immunosuppressive antigen-specific Tregs. The importance of Aire in maintaining central tolerance against self-reactive antigens is evidenced by the multiorgan autoimmune disease seen in individuals with mutation the Aire gene. When Aire-expression is specifically ablated in mouse mTECs, there is a dramatic increase in the number of self-reactive T cells that escape negative selection, in addition, a dramatic loss of Tregs. Utilizing novel high-throughput platforms and single-cell DNA barcoding technology has allowed us to assess several thousand CD4+ T-cell clones. The use of FoxP3-GFP reporter mice permits us to identify individual Tregs that have undergone Aire-dependent selection in the thymus and compare the TCR repertoire to mice that have had Aire specifically ablated from the mTECs. Identification of unique clones and gene signatures within our large datasets has allowed us to start to qualitatively assess the functional role of the identified TCRs within the model of T1D. We have begun comparisons of our extensive datasets to insulin-specific TCRs and TCRs that we have previously identified from diabetic animals. In addition, the peptide specificities of the identified TCRs are being evaluated using Baculovirus MHC-II peptide libraries. Our proposed study aims to expand our knowledge on TCR repertoire of Aire-dependent Tregs and the antigen-specificity in T1D, improving our understanding of the respective function of thymic-derived antigen-specific Treg suppression in peripheral tissues associated with autoimmune T1D.