[Research progress in original species identification in industry chain of Rhei Radix et Rhizoma].

Rhei Radix et Rhizoma is a kind of commonly used Chinese medicinal materials. Due to the overharvesting, the wild resource is endangering. Large market demand caused severely adulterant of commercial Rhei Radix et Rhizoma medicinal materials and decoction pieces. This manuscript reviewed the advances of the original species authentication in the industrial chain of Rhei Radix et Rhizoma during the latest decade, including characteristics and microscopic features, phytochemical analysis on anthraquinones, and molecular authentication based on DNA barcoding. Accordingly, an original species authentication route for the industrial chain of Rhei Radix et Rhizoma was summarized:(1)the identification of seeds and seedlings by DNA barcoding;(2) the selection of high variable sites based on the chloroplast genome;(3)biomonitoring of the Rhei Radix et Rhizoma medicinal materials and decoction pieces by two-dimensional DNA barcode;(4)traceability of Chinese patent medicines by third-generation sequencing. In conclusion, the combination of molecular identification and traditional identification methods provides a new idea for the identification of the original species of Rhei Radix et Rhizoma in the industrial chain and a essential guidance for the research of drug safety and efficacy of Rhei Radix et Rhizoma.

[Identification of antler powder components based on DNA barcoding technology].

In order to authenticate the components of antler powder in the market, DNA barcoding technology coupled with cloning method were used. Cytochrome c oxidase subunit I (COI) sequences were obtained according to the DNA barcoding standard operation procedure (SOP). For antler powder with possible mixed components, the cloning method was used to get each COI sequence. 65 COI sequences were successfully obtained from commercial antler powders via sequencing PCR products. The results indicates that only 38% of these samples were derived from Cervus nippon Temminck or Cervus elaphus Linnaeus which is recorded in the 2010 edition of "Chinese Pharmacopoeia", while 62% of them were derived from other species. Rangifer tarandus Linnaeus was the most frequent species among the adulterants. Further analysis showed that some samples collected from different regions, companies and prices, contained adulterants. Analysis of 36 COI sequences obtained by the cloning method showed that C. elaphus and C. nippon were main components. In addition, some samples were marked clearly as antler powder on the label, however, C. elaphus or R. tarandus were their main components. In summary, DNA barcoding can accurately and efficiently distinguish the exact content in the commercial antler powder, which provides a new technique to ensure clinical safety and improve quality control of Chinese traditional medicine

[Identification of peucedani radix, peucedani decursivi radix and its adulterants using ITS2 sequence].

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.

[Identification of Placenta hominis and its adulterants using COI barcode].

In order to provide a new method for the identification of Placenta hominis, the COI barcode has been employed to identify the P. hominis medicinal materials and its adulterants. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. NJ tree was constructed by MEGA6.0 software. COI sequences can be successfully obtained from all experimental samples. The intra-specific variation and inter-specific divergence were calculated. The average intra-specific K2P distance of P. hominis was 0.001 and the maximum intra-specific distance was 0.008. The cluster dendrogram constructed can be seen that the same genus is together, and distinguished from its adulterants. It is concluded that P. hominis and its adulterants can be correctly identified by DNA barcoding method.

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

[Identification of gentianae macrophyllae radix using the ITS2 barcodes].

DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine. Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine. There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: (1) the extraction methods for total DNA of the rhizomes of the medicinal materials; (2) intra-specific variation among samples from different places of origin; (3) accuracy and stability of this method. In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology. Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants. After improving the DNA extraction method, genomic DNA from all samples was successfully obtained. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed. Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies. Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.

[Stability and accuracy of the identification of Notopterygii Rhizoma et Radix using the ITS/ITS2 barcodes].

In this study, Notopterygii Rhizoma et Radix was used to verify the stability and accuracy of DNA barcodes in identification of Chinese materia medica for the first time. All genomic DNAs from thirty one samples were extracted. The ITS (internal transcribed spacer) regions were amplified and sequenced bi-directionally. Obtained sequences were assembled using the CodonCode Aligner. And the sequences of the ITS regions were aligned through Clustal-W and the genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model. The neighbor-joining (NJ) phylogenetic trees were constructed. The ITS2 regions were obtained by using the hidden Markov model (HMM)-based annotation methods from the ITS sequences. Results indicated that the lengths of ITS regions of Notopterygii Rhizoma et Radix were 603-604 bp, while the lengths of ITS2 regions were 228 bp. The haplotypes of ITS/ITS2 regions of Notopterygii Rhizoma et Radix were the same as those of the original plant leaves. The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2 regions of Notopterygium incisum and N. franchetii. The NJ trees showed that N. incisum, N. franchetii and its adulterants can be easily differentiated according to their monophyly. Therefore, ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Notopterygii Rhizoma et Radix from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.

Mechanical force-promoted osteoclastic differentiation via periodontal ligament stem cell exosomal protein ANXA3.

Exosomes play a critical role in intracellular communication. The biogenesis and function of exosomes are regulated by multiple biochemical factors. In the present study, we find that mechanical force promotes the biogenesis of exosomes derived from periodontal ligament stem cells (PDLSCs) and alters the exosomal proteome profile to induce osteoclastic differentiation. Mechanistically, mechanical force increases the level of exosomal proteins, especially annexin A3 (ANXA3), which facilitates exosome internalization to activate extracellular signal-regulated kinase (ERK), thus inducing osteoclast differentiation. Moreover, the infusion of exosomes derived from PDLSCs into mice promotes mechanical force-induced tooth movement and increases osteoclasts in the periodontal ligament. Collectively, this study demonstrates that mechanical force treatment promotes the biogenesis of exosomes from PDLSCs and increases exosomal protein ANXA3 to facilitate exosome internalization, which activates ERK phosphorylation, thus inducing osteoclast differentiation. Our findings shed light on new mechanisms for how mechanical force regulates the biology of exosomes and bone metabolism.

DNA methylation and demethylation link the properties of mesenchymal stem cells: Regeneration and immunomodulation.

Mesenchymal stem cells (MSCs) are a heterogeneous population that can be isolated from various tissues, including bone marrow, adipose tissue, umbilical cord blood, and craniofacial tissue. MSCs have attracted increasingly more attention over the years due to their regenerative capacity and function in immunomodulation. The foundation of tissue regeneration is the potential of cells to differentiate into multiple cell lineages and give rise to multiple tissue types. In addition,the immunoregulatory function of MSCs has provided insights into therapeutic treatments for immune-mediated diseases. DNA methylation and demethylation are important epigenetic mechanisms that have been shown to modulate embryonic stem cell maintenance, proliferation, differentiation and apoptosis by activating or suppressing a number of genes. In most studies, DNA hypermethylation is associated with gene suppression, while hypomethylation or demethylation is associated with gene activation. The dynamic balance of DNA methylation and demethylation is required for normal mammalian development and inhibits the onset of abnormal phenotypes. However, the exact role of DNA methylation and demethylation in MSC-based tissue regeneration and immunomodulation requires further investigation. In this review, we discuss how DNA methylation and demethylation function in multi-lineage cell differentiation and immunomodulation of MSCs based on previously published work. Furthermore, we discuss the implications of the role of DNA methylation and demethylation in MSCs for the treatment of metabolic or immune-related diseases.

Evaluation of Periodontal Status after Orthodontic Treatment: A Pilot Study on Patients with Stage IV/Grade C periodontitis.

OBJECTIVE: To evaluate whether the periodontal status was affected in patients with stage IV/grade C periodontitis after orthodontic treatment. METHODS: Twenty-four patients with stage IV/grade C periodontitis who received combined periodontal and orthodontic treatment were included in this study. Probing depth (PD), bleeding on probing (BOP) and percentage of relative bone height (RBH%) were measured and calculated. Parameter changes before and after orthodontic treatment, and their differences between teeth adjacent to extraction sites (TAES) and teeth nonadjacent to extraction sites (TNES) were compared. Three-level analysis was performed to test the influential factors of PD and RBH% reduction after orthodontic treatment. RESULTS: No change of PD, BOP% and RBH% was detected after orthodontic treatment. No difference of PD, BOP% and RBH% was detected between TAES and TNES. BOP negative, excessive horizontal overlap, excessive vertical overlap, crowding, PD at T0 (the last periodontal visit before orthodontic treatment) and RBH% at T0 were positively associated with PD reduction after orthodontic treatment. Being female, excessive horizontal overlap, excessive vertical overlap, crowding, PD at T0 and RBH% at T0 were positively associated with a RBH% reduction. Space was negatively associated with a RBH% reduction. CONCLUSION: Periodontal stability can be obtained for patients with stage IV/grade C periodontitis after orthodontic treatment. Orthodontic treatment with extraction was safe for patients with severe periodontitis, however, attention should be given to TAES.

DNA barcoding of Corydalis, the most taxonomically complicated genus of Papaveraceae.

The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya-Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA-trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P-distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia-recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well-supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.

[Study on adherence and interrelated factors of acquired immunedeficiency syndrome patients receiving antiretroviral treatment].

OBJECTIVE: To study on the compliance antiretroviral(ARV) drugs and interrelated factors of HIV/AIDS patients undertaking highly active antiretroviral therapy(HAART) to improve clinical treatment. METHODS: 3 counties in Henan province were selected including 2 counties from China Cares Program points and one county where HIV/AIDS was serious. All cases studied had already received antiretroviral therapy (ART) for 2-12 months. Several indicators through questionnaire were studied including drug adherence, side effect, symptoms status before and after treatment and ART measures etc. At the same time, blood was collected to analyze CD4+ T-lymphocyte, the virus loads of HIV and drug genotype resistance by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In the therapy group, most patients had taken ARV drugs for 4-8 months, which accounted for 78.24% of the total number, with the adherence rate above 90% as 67.51%. The main reason for the patients not listening to their doctor's advice was due to ART drug's side effects (66.95%), including queasiness, vomiting and tetter. In the therapy group, 82.57% of patients' symptoms were obvious. Adherence had a great impact on the improvement (P < 0.05). After therapy, the total count of patients' CD4+ T-lymphocyte's kept stable or improved with slow speed. At the time of 3 months and 6 months after ART, the rates of improvement were 55.1% and 50.8%. However CD4+ count did not show much difference between the twe group. The prevalence rate of HIV drug resistance strain roise from 13.9% in non-remedial group to 45.4% at the time of 3 months after therapy and 62.7% at the time of 6 months after therapy. The resistance against non-nucleoside reverse transcriptase inhibitors (NNRTI) had improved obviously. CONCLUSION: Patients with HIV/AIDS receiving HAART, compliance seemed directly affect the curative effect and the implementation of therapy schedule and should be improved to avoid drug resistance.