Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Integrated analysis of metabolome and transcriptome reveals key candidate genes involved in flavonoid biosynthesis in Pinellia ternata under heat stress.

Pinellia ternata (Thunb.) Breit. is an important traditional Chinese medicinal herb and very sensitive to high temperatures. To gain a better understanding of flavonoid biosynthesis under heat stress in P. ternata, we performed integrated analyses of metabolome and transcriptome data. P. ternata plants were subjected to a temperature of 38 °C, and samples were collected after 10 d of treatment. A total of 502 differential accumulated metabolites and 5040 different expressed transcripts were identified, with flavonoid biosynthesis predominantly enriched. Integrated metabolomics and transcriptome analysis showed that high temperature treatment upregulated the expression of CYP73A and downregulated the expression of other genes (such as HCT, CCoAOMT, DFR1, DFR2), which might inhibit the biosynthesis of the downstream metabolome, including such metabolites as chlorogenic acid, pelargonidin, cyanidin, and (-)-epigallocatechin in the flavonoid biosynthesis pathway. The transcription expression levels of these genes were validated by real-time PCR. Our results provide valuable insights into flavonoid composition and accumulation patterns and the candidate genes participating in the flavonoid biosynthesis pathways under heat stress in P. ternata.

[Short needling for knee osteoarthritis with blood stasis obstruction and its effect on serum inflammatory factors].

OBJECTIVE: To compare the clinical efficacy and its effect on serum levels of tumor necrosis factor α(TNF-α), interleukin 1ß(IL-1ß), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) between short needling (close-to-bone needling) and conventional acupuncture for knee osteoarthritis (KOA) with blood stasis obstruction. METHODS: A total of 68 KOA patients with blood stasis obstruction were randomized into a short needling group (34 cases, 3 cases dropped off) and a conventional acupuncture group (34 cases, 3 cases dropped off). The same acupoints (Dubi [ST 35], Neixiyan [EX-LE 4], Binzhong [Extra], Liangqiu [ST 34], etc. on the affected side) were selected in the two groups. In the short needling group, short needling technique was adopted, the needles were slowly inserted and the needle bodies were shaken, thus gradually penetrated to the bone. In the conventional acupuncture group, conventional acupuncture was adopted, the needles were penetrated to the muscle. After qi-arrival, Dubi (ST 35) and Neixiyan (EX-LE 4), Zusanli (ST 36) and Liangqiu (ST 34) were connected with CMNS6-1 electronic acupuncture instrument, with disperse-dense wave, 2 Hz/10 Hz in frequency, the current intensity was based on patients' feeling, the needles were retained for 30 min, at the same time, the knee joint was irradiated for 30 min with a special electromagnetic wave apparatus in the two groups. Once every other day, 3 times a week for 4 weeks. Before and after treatment, the Western Ontario and McMaster Universities osteoarthritis index (WOMAC) score, knee joint pain visual analogue scale (VAS) score, inflammatory response related indexes (serum TNF-a, IL-1ß, IL-6 and PGE2) and knee joint ultrasound were observed，and the clinical effect was evaluated in the two groups. RESULTS: After treatment，the pain, stiffness, function scores and total scores of WOMAC were decreased as compared with those before treatment in the two groups (P<0.05), except for the pain score, the changes of above scores in the short needling group were greater than the conventional acupuncture group (P<0.05). After treatment, the VAS scores, serum levels of TNF-a, IL-1ß, IL-6, PGE2 and knee joint synovium thickness, intra-articular effusion were decreased as compared with those before treatment in the two groups (P<0.05), the levels of TNF-a, IL-1ß, IL-6 in the short needling group were lower than the conventional acupuncture group (P<0.05). The total effective rate in the short needling group was 87.1% (27/31), which was superior to 83.9% (26/31) in the conventional acupuncture group (P<0.05). CONCLUSION: Short needling could improve the knee joint function, relieve the pain and inflammatory response, improve the knee joint synovium inflammatory response, reduce the knee joint intra-articular effusion for KOA patients, its effect is better than conventional acupuncture.

Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI.

The medicinal use of traditional Chinese medicine is mainly due to its secondary metabolites. Visualization of the distribution of these metabolites has become a crucial topic in plant science. Mass spectrometry imaging can extract huge volumes of data and provide spatial distribution information about these by analyzing tissue slices. With the advantage of high throughput and higher accuracy, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is often used in biological research and in the study of traditional Chinese medicine. However, the procedures used in this research are complicated and not affordable. In this study, we optimized sectioning and DESI imaging procedures and developed a more cost-effective method to identify the distribution of metabolites and categorize these compounds in plant tissues, with a special focus on traditional Chinese medicines. The study will promote the utilization of DESI in metabolite analysis and standardization of traditional Chinese medicine/ethnic medicine for research-related technologies.

Metabolomics integrated with transcriptomics reveals the distribution of iridoid and crocin metabolic flux in Gardenia jasminoides Ellis.

Gardenia jasminoides Ellis (G. jasminoides) fruits are used as a resource for obtaining natural colorants and in traditional Chinese herbal medicine. However, G. jasminoides presents a relatively long flowering period and different ripening periods, so there are significant differences in the accumulation of metabolites in fruits of different colors. In addition, the complete metabolic pathways of iridoidsand crocins, which are used as medicinal composition of G. jasminoides, are poorly understood at present. In this research, we comprehensively compared the transcriptome and metabolites profiles of the developmental stages and locations of iridoid and crocin biosynthesis. A large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were detected in four groups of samples, and clear variation in the pattern of metabolite abundance and gene expression were observed among different fruit colors and parts. Geniposide and gardenoside mainly accumulated in the sarcocarp of green fruit (GFS) and the sarcocarp of red fruit (FS), respectively. Crocin mainly accumulated in the peel and sarcocarp of red fruits. In the iridoid pathway, we hypothesized that there was a transport mechanism from the sarcocarp to the peel of G. jasminoides because of the inconsistent expression of G8O, 10-HGO and IS associated with differences in fruit ripening. UGTs play an important role in the biosynthesis of the active components of G. jasminoides. Combined transcriptome and metabonomics analysis showed a negative correlation between the biosynthesis of geniposide and crocin. The redirection of the metabolic flux and the regulation of key enzymes may be the main reasons for the changes in the biosynthesis of iridoid and crocin in G. jasminoides fruit. Our study expended valuable information for functional genomic library and provided new insights for metabolic engineering of secondary metabolite in G. Jasminoides.

[Different development phase of transcription proteomics and metabolomics of flower of Lonicera macranthoides].

In order to study the regulation mechanism of secondary metabolites biosynthesis in Lonicera macranthoides, the key genes involved in the regulation of biosynthesis and the mechanism of differential metabolites were explored. In this study, high-throughput sequencing technology was used for transcriptome sequencing of L. macranthoides at different development stages. By using Liquid chromatography-tandem mass spectrometry(LC-MS/MS) technology, the laws of qualitative, quantitative and synthetic accumulation of its metabolites were studied, and the key enzyme genes for the biosynthesis of phenolic acid and flavonoids were screened out according to the differentially expressed genes. A total of 111 differentially accumulate metabolites(DAM) and 6 653 differentially expressed genes(DGE) were obtained by metabonomics and transcriptomics analysis. The metabolites and key enzyme genes in the Erqing(KE) were significantly different from those in the Dabai(KD) and Yinhua(KY) stages. In the phenylalanine biosynthesis pathway, the ion abundance of chlorogenic acid, naringin, quercetin, rutin, coniferol and other metabolites decreased with the development of flowers, while the ion abundance of ferulic acid, coumarin and syringoside increased with the development of flowers. Key enzyme genes such as CHS, HCT, CCR, FLS and COMT positively regulate the downstream metabolites, while PAL, C4H and 4CL negatively regulate the downstream metabolites. This study provides candidate genes and theoretical basis for the further exploration of key enzymes in the biosynthesis of secondary metabolites and for the regulation of the accumulation of secondary metabolites in L. macranthoides by molecular biological methods.

The chromosome-level reference genome of Coptis chinensis provides insights into genomic evolution and berberine biosynthesis.

Coptis chinensis Franch, a perennial herb, is mainly distributed in southeastern China. The rhizome of C. chinensis has been used as a traditional medicine for more than 2000 years in China and many other Asian countries. The pharmacological activities of C. chinensis have been validated by research. Here, we present a de novo high-quality genome of C. chinensis with a chromosome-level genome of ~958.20 Mb, a contig N50 of 1.58 Mb, and a scaffold N50 of 4.53 Mb. We found that the relatively large genome size of C. chinensis was caused by the amplification of long terminal repeat (LTR) retrotransposons. In addition, a whole-genome duplication event in ancestral Ranunculales was discovered. Comparative genomic analysis revealed that the tyrosine decarboxylase (TYDC) and (S)-norcoclaurine synthase (NCS) genes were expanded and that the aspartate aminotransferase gene (ASP5) was positively selected in the berberine metabolic pathway. Expression level and HPLC analyses showed that the berberine content was highest in the roots of C. chinensis in the third and fourth years. The chromosome-level reference genome of C. chinensis provides important genomic data for molecular-assisted breeding and active ingredient biosynthesis.

Transcriptomic and metabolomic analyses provide insights into the biosynthesis of chlorogenic acids in Lonicera macranthoides Hand.-Mazz.

Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in South China. The developmental stage and corolla dehiscence of the flower are the important factors affecting the quality of medicinal ingredients. However, neither the regulatory mechanism controlling chlorogenic acids biosynthesis in L. macranthoides nor the molecular basis of effect of corolla dehiscence on the quality of medicinal materials is fully understood. In this study, metabolomics and transcriptomics were used to analyze the metabolic and transcriptional differences of two different cultivars closed bud type (Bt), and flowering type (Ft), as well as the effect of jasmonic acid methyl ester (MeJA) on chlorogenic acids (CGAs) biosynthesis. In total, large number of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were filtered among three lines of samples. Gene metabolite correlation analyses revealed a 'core set' of 30 genes and 54 genes that were strongly correlated with CGAs biosynthesis and regulating the flowering, respectively. Quantitative real-time polymerase chain reaction results proved the alterations in the expression levels of genes encoding the pathways involved in CGAs biosynthesis. The ion abundances of CGAs were most significantly increased, while some of the CGAs derived and Caffeoyl-CoA-derived substances showed the most largely reduced abundances in the closed bud type (Bt) compared to the flowering type (Ft). MeJA may leads to the activation of downstream genes in CGAs biosynthesis pathway. Overall, there were significant differences in the transcriptional and metabolic levels of CGAs biosynthesis pathway in flower buds of different flowering cultivars. The redirection of metabolic flux may contribute to increased accumulation of CGAs. However, whether MeJA and flowering have direct effects on the accumulation of CGAs needs further studied. These researches effectively expanded the functional genomic library and provide new insights into CGAs biosynthesis in L. macranthoides.

[Transcriptome analysis of Platycodon grandiflorum at different growth years and discovery of key genes for platycodin biosynthesis].

Platycodon grandiflorum is a medicinal and edible medicinal material. Our study is aimed to explore the differences in the gene expression of P. grandiflorum in different growth years, and the expression rules of key genes in the biosynthesis of the main active substances of P. grandiflorum. Illumina Hiseq 4000 sequencing platform was used to sequence the transcriptome of P. grandiflorum in different years. Then, 59 654 unigenes were obtained through filtering, assembly, splicing and bioinformatics analysis of the sequencing data, of which 1 671 unigenes were differentially expressed between at least two samples. The results of cluster analysis showed that there was a great difference in the gene expression of P. grandiflorum from one-year-old to two/three-year-old. There were 1 128 different genes between one-and three-year old P. grandiflorum, and only 57 different genes between two-and three-year-old P. grandiflorum. KEGG enrichment results showed that the differential genes of P. grandiflorum in different years were mainly concentra-ted in the biosynthesis of sesquiterpenes and triterpenes, and the biosynthesis of terpenoid skeletons. In the triterpenoid biosynthesis-related pathways, a total of 15 unigenes were identified, involving 5 enzymes. The expression levels of ACAT, HMGR, FDFT1, SQLE decreased with the increase of the growth year of P. grandiflorum. The expression of HMGS was the highest in the one-year-old P. grandiflorum, followed by the three-year-old sample. This study provides useful data for the development of P. grandiflorum, and also provides a basis for the study of related genes in the biosynthetic pathway of platycodin.

[Variation in physicochemical properties and bacterial community structure in rhizosphere soil of Coptis chinensis tow cropping modes].

The natural forest and artificial shed are the main cropping modes of Coptis chinensis. This study is aimed to reveal the rhizosphere soil bacterial community structure difference between under tow C. chinensis cropping modes-natural forest and artificial shed, and to assist us to completely understand soil quality condition,and provide theoretical guidance for soil improvement and C. chinensis planting. The rhizosphere soil samples of 1-5-year-old C. chinensis under tow cropping modes-natural forest and artificial shed were collected. Illumina high-throughput sequencing technology was used to analyze the alpha diversity, community composition, community structure of soil bacteria under the tow cropping modes,and the effects of soil nutriment indices on soil bacterial community structure. Through the analysis of species number, Shannon, Chao1 index and ACE index of bacterial community, it was found that the bacterial diversity of 1-year-old C. chinensis soil under natural forest cropping mode was significantly lower than that under artificial shed cropping mode, and the diversity of bacterial communities in soil of 2-5-years old C. chinensis were not significant different between two cropping modes. A total of 53 phyla,60 classes,140 orders and 266 families were detected in the rhizosphere soil of C. chinensis under the cropping modes of natural forest, respectively. The rhizosphere soil of C. chinensis under the cropping modes of artificial shed included 54 phyla,65 classes,140 orders and 264 families, respectively. Under the two cropping modes, the top 10 dominant species of bacterial community abundance are the same, they are Proteobacteria, Acidobacteria, Actinobacteria,Bacteroidetes, Planctomycetes, Chloroflexi, Verrucomicrobia, Gemmatimonadetes, Firmicutes and Cyanobacteria, but there are differences in the abundance sequence. The top 10 dominant species of bacterial community abundance accounted for 74.36% to 74.30% of the total bacteria, and 3.15% to 3.92% of the bacteria are unclassified. The results of Metastat analysis showed that the abundance of Gemmatimonadetes in the rhizosphere soil of C. chinensis under the cropping modes the artificial shed was significantly higher than that under the natural forest cropping mode(P<0.05). MRPP analysis of community structure differences showed that under tow cropping modes, there were significant differences in the bacterial community structure of 1-4-year-old soil bacteria, among which the difference between 1-year-old soil samples was the largest. With the increase of cropping years, the difference gradually decreases, and there is no significant difference in the bacterial community structure between 5-year-old soil samples. RDA analysis and correlation analysis of bacterial community structure and soil physical and chemical properties showed that the order of environmental factors on the rhizosphere soil bacteria of Coptis chinensis was: pH>available P> total P> total K>bulk density>total N>available N>organic matter. The results are helpful to understand the soil health of C. chinensis and provide scientific basis and theoretical guidance for soil improvement and C. chinensis planting.

Investigation of Invigorating Qi and Activating Blood Circulation Prescriptions in Treating Qi Deficiency and Blood Stasis Syndrome of Ischemic Stroke Patients: Study Protocol for a Randomized Controlled Trial.

Ischemic stroke (IS) is characterized by high morbidity and high mortality. The integration of Traditional Chinese medicine (TCM) and western medicine has shown promising benefits in relieving symptoms, promoting neurological recovery, and improving the quality of life of patients with IS. In TCM, Qi-deficiency along with blood-stasis (QDBS) syndrome is one of the common types of IS that is treated by invigorating Qi and activating blood circulation. In TCM theory, improving the corresponding degree of prescription-syndrome correlation (PSC) is helpful to improve clinical efficacy. In this study, we intend to use similar prescriptions that invigorate Qi and activate blood circulation: Buyang Huanwu granules (BHG), Naoxintong capsules (NXTC), and Yangyin Tongnao granules (YTG). The goal is to evaluate their level of PSC inpatients with IS with QDBS syndrome and find relevant biomarkers to provide an objective basis for precise treatment of TCM and improve the clinical therapeutic effects. A multicenter, randomized, double-blinded, and placebo-controlled intervention trial will be conducted in IS patients with QDBS syndrome, followed by an add-on of Chinese patent medicine. A total of 160 subjects will be randomly assigned to the BHG, NXTC, YTG, and placebo groups in a 1:2:1:1 allocation ratio. All subjects will undergo 28 days of treatment and then followed for another 180 days. The primary outcome is the changes in the National Institutes of Health Stroke Scale score after 28 days of medication. The secondary outcomes include the modified Rankin scale score, activity of daily living scale score, and TCM symptom score. Data will be analyzed in accordance with a predefined statistical analysis plan. Ethical approval of this trial has been granted by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang Chinese Medical University (ID: 2017-Y-004-02). Written informed consent of patients will be required. This trial is registered in the Chinese Clinical Trial Registry (ChiCTR1800015189), and the results will be disseminated to the public through peer-reviewed journals and academic conferences.

[Effect of acupoint stimulation on the quality of recovery in patients with radical thyroidectomy under the concept of enhanced recovery after surgery: a randomized controlled trial].

OBJECTIVE: To observe the effect of acupoint stimulation on the quality of recovery in patients with radical thyroidectomy under the concept of enhanced recovery after surgery (ERAS). METHODS: A total of 62 patients with radical thyroidectomy were randomized into an observation group and a control group, 31 cases in each one. In both of the two groups, general anesthesia with tracheal intubation was applied, the same anesthesia induction and maintenance medication were given. In the observation group, auricular point pressing with magnetic beads was adopted at bilateral shenmen (TF4) and transcutaneous electrical acupoint stimulation (dilatational wave, 2 Hz/100 Hz in frequency, 6 to 12 mA) was performed at bilateral Hegu (LI 4) and Neiguan (PC 6) from 30 min before anesthesia induction to the end of the anesthesia. In the control group, medical adhesive plaster was pasted at bilateral shenmen (TF4) and the electrodes were plastered at bilateral Hegu (LI 4) and Neiguan (PC 6) with no corresponding stimulation. In both of the two groups, visual analogue scale for anxiety (VAS-A) score was observed to evaluate the anxiety severity before anesthesia induction; the total intraoperative dosages of sufentanil, remifentanil and propofol were recorded; the numerical rating scale (NRS) score was used to assess the pain severity of instant time (T0) and 30 min (T1) of entering post-anesthesia recovery room (PACU), motor and static mode at 2 h (T2), 6 h (T3), 12 h (T4), 24 h (T5) after surgery; time of first anal exhaust, time of getting out of bed after surgery, total hospitalization time and the incidences of postoperative nausea and vomiting were observed; the quality of recovery was assessed by the 40-item quality of recovery score (QoR-40). RESULTS: The VAS-A score and the total intraoperative dosage of remifentanil in the observation group were reduced compared with the control group (P<0.05). The NRS scores at T0-T4 in the observation group were lower than those in the control group (P<0.01, P<0.05), while the difference between the two groups in NRS score at T5 was not significant (P>0.05). The time of first anal exhaust and getting out of bed after surgery in the observation group were advanced than those in the control group (P<0.05), there was no significant difference between the two groups in total hospitalization time and incidences of postoperative nausea and vomiting (P>0.05). Compared with the control group, the QoR-40 score was increased in the observation group (P<0.05). CONCLUSION: Acupoint stimulation can improve the preoperative anxiety in patients with radical thyroidectomy, reduce the intraoperative anesthetic dosage and postoperative pain, advance the time of anal exhaust and getting out of bed, improve the quality of postoperative recovery and enhance the recovery process.

[Effect of transcutaneous electrical acupoint stimulation on pulmonary function and oxidative stress response in lower extremity surgery patients using tourniquet].

OBJECTIVE: To investigate the effect of transcutaneous electrical acupoint stimulation (TEAS) on pulmonary function, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content in patients using tourniquet after lower extremity surgery. METHODS: A total of 40 patients who underwent lower extremity surgery were equally randomized into control group and TEAS group by using a random number table. All patients underwent lumbar epidural anesthesia combined with block anesthesia. The patients in the TEAS group were given TEAS at Zusanli (ST36) and Sanyinjiao (SP6) beginning from 30 min before surgery to the end of surgery, and those in the control group received TEAS at the same acupoints with minimum current intensity. Mean arterial pressure (MAP) and heart rate (HR) were recorded at each time point (T0: pre-surgery /TEAS, T1: 5 min after anesthesia, T2: 1 min before tourniquet-loosening, T3: 1 min after tourniquet-loosening, T4: 5 min after tourniquet-loosening, and T5: 6 h after tourniquet-loosening). Blood samples (4 mL) was collected from the radial artery before TEAS and 6 h after loosening tourniquet for analyzing blood gas parameters as partial pressure of caron dioxide(PCO2), arterial partial pressure of oxygen (PaO2), alveolar partial pressure of oxygen (PAO2), alveolar-arterial oxygen pressure difference (PA-aDO2) and respiratory index (RI) by using a blood gas analyzer, and plasma SOD activity and MDA content were assayed by using xanthine oxidase method and thiobarbituric acid colorimetry method, respectively. RESULTS: Intra-group comparison showed that compared with T0, a significant increase was found in PA-aDO2 and RI at T5 and a significant reduction in PaO2 and PaO2/ PAO2 (a/A) ratio in the control group (P<0.05), and the same changes in the TEAS group (P<0.05) except a/A ratio. Comparison between two groups showed that at T5, both PaO2 and a/A levels were significantly higher in the TEAS group than in the control group (P<0.05), and both PA-aDO2 and RI levels were obviously lower in the TEAS group than in the control group (P<0.05), suggesting an improvement of the pulmonary function after TEAS. At T5, plasma SOD activity was significantly decreased and plasma MDA content was remarkably increased in the control group relevant to T0 (P<0.05), SOD activity was significantly higher in the TEAS group than in the control group (P<0.05), and MDA content was evidently lower in the TEAS group than in the control group (P<0.05), suggesting a reduction of oxidative stress response after TEAS. CONCLUSION: TEAS at ST36 and SP6 can improve pulmonary function and attenuate oxidative stress response in patients using tourniquet after lower extremity surgery.

Methionine Supply During Late-Gestation Triggers Offspring Sex-Specific Divergent Changes in Metabolic and Epigenetic Signatures in Bovine Placenta.

Background: Nonruminant male and female offspring respond differently to gestational nutrition, with placenta contributing to the underlying mechanisms. However, similar data are lacking in large ruminants. Objectives: The aim of this study was to investigate the impact of methionine supply during late-gestation on metabolism and DNA methylation in placenta from cows carrying male or female calves. Methods: During the last 28 d of pregnancy, cows were individually fed a control diet (CON) or the control diet plus rumen-protected d,l-methionine (MET; 0.9 g/kg dry matter intake). Placentomes collected at term were classified according to cow dietary treatment and offspring sex as follows: Male CON (n = 7), Male MET (n = 7), Female CON (n = 8), and Female MET (n = 8). Calf growth was measured until 9 wk of age. Targeted metabolomics, RT-PCR, global DNA methylation, and activity of selected enzymes in one-carbon metabolism and transsulfuration pathways were performed. Statistics were conducted via ANOVA using MIXED models. Results: At birth, Male MET calves were heavier than Male CON calves (7.6%, P = 0.02), but body mass was similar at 9 wk of age. In contrast, compared with Female CON, Female MET calves had greater body mass at 9 wk of age (6.3%, P = 0.03). Compared with Male CON, placenta from Male MET calves had greater concentrations of tricarboxylic acid (TCA) cycle and transsulfuration intermediates (23-100%, P < 0.05), along with greater 5-methyltetrahydrofolatehomocysteine methyltransferase activity (67%, P = 0.03). Compared with Female CON, placenta from Female MET calves had greater concentrations of one-carbon metabolism intermediates (13-52%, P < 0.05). DNA methyltransferase 3A (DNMT3A) was upregulated (43%, P < 0.01) in placenta from Female MET compared with Female CON calves. Global DNA methylation was lower in placenta from Female MET compared with Female CON calves (45%, P = 0.06). Conclusions: Methionine supply affects placental metabolism, DNA methylation, and body mass of the calf in a sex-specific manner, underscoring its importance as dietary methyl-donor for pregnant cows.

Stems and leaves of Aconitum carmichaelii Debx. as potential herbal resources for treating rheumatoid arthritis: Chemical analysis, toxicity and activity evaluation.

According to folk usage of Aconitum carmichaelii Debx., the present study was designed to determine the feasibility of the stems and leaves of Aconitum carmichaelii Debx. as a new medicinal resource. Fourteen alkaloids in mother roots, fibrous roots, stems, and leaves of Aconitum carmichaelii Debx. were measured by HPLC-MS/MS. And multivariate analysis methods, such as clustering analysis and principal component analysis, were applied to analyze the difference among various parts. In addition, the acute toxicity, analgesia, and anti-inflammatory tests were carried out. The results suggested that the contents of alkaloids in mother roots and fibrous roots were approximate, but those of leaves and stems were different from mother roots and fibrous roots. The results of the acute toxicity testing demonstrated the toxicity of fibrous root was strongest, and mother roots were slightly less toxic than fibrous roots. The stems and leaves were far less toxic than mother and fibrous roots. In addition, the analgesia and inflammatory tests showed the effects of the various tissues had no difference each other. These results provided a basis for developing new complementary and alternative treatments for rheumatoid arthritis patients. Simultaneously, the approach may also turn wastes into treasure and promote the development of circular economy.

Acupoint Embedding of Polyglactin 910 Sutures in Patients with Chronic Pain due to Cervical Spondylotic Radiculopathy: A Multicenter, Randomized, Controlled Clinical Trial.

OBJECTIVE: We aimed to investigate the effectiveness of acupoint polyglactin 910 (PGLA) embedding in patients with cervical spondylotic radiculopathy (CSR). METHODS: A total of 102 CSR patients with neck and shoulder pain were recruited and assigned randomly into three groups: the sham acupoint embedding (SAE) group, the middle-layer acupoint PGLA embedding (MAPE) group, and the deep-layer acupoint PGLA embedding (DAPE) group. The primary outcomes were Visual Analog Scale (VAS) scores showing the analgesic effects of treatment. Secondary outcomes included clinical symptoms (evaluated by the Yasuhisa Tanaka 20 (YT-20) score and the neck disability index (NDI)) and patient health status (evaluated by the 36-item short-form survey (SF-36)) as reported in the trial. RESULTS: Compared with the SAE group, VAS scores were significantly reduced at 1, 2, 3, 4, and 10 weeks after the first treatment in both the DAPE and MAPE groups (P < 0.001). Moreover, there were statistically significant increases in the weekly YT-20 scores and significant reductions of the weekly NDI scores compared with baseline values in both the DAPE and MAPE groups (P < 0.001). Compared with baseline values, both the physical component summary (PCS) and the mental component summary scores of the SF-36 at 2, 3, 4, and 10 weeks were significantly higher in the DAPE and MAPE groups (P < 0.001). There were significant lower VAS scores (P < 0.01), higher PCS scores (P < 0.05) at 3 weeks, and lower NDI scores (P < 0.05) at 4 weeks in the DAPE group compared with the MAPE group. CONCLUSIONS: Both DAPE and MAPE showed significant and long-lasting effects on alleviating pain and improving clinical symptoms as well as quality of life in CSR patients with neck and shoulder pain. A more intense effect was seen in the DAPE group compared with the MAPE group.

[Transcriptome analysis reveals candidate genes involved in flavonols biosyhthesis in Sophora japonica].

In order to study the pathways of biosynthesis of flavonoids in Sophora japonica, 113 797 unigenes were obtained by Trinity software, with an average length of 803 bp, of which 72 752 unigenes were obtained from the database by high-throughput sequencing, and a total of 38 891 SSR loci were searched. Through the metabolic pathway analysis, we found that there were 135 unigenes involved in the biosynthesis of flavonoids and 959 unigene involved in other secondary metabolic pathways. Further analysis of genes involved in rutin biosynthesis revealed that 24 were associated with CHS, 52 were associated with FLS, and 11 were associated with UFGT. The obtained data of S. japonica transcriptome lays the foundation for studying the pathways of biosynthesis of flavonoids in S. japonica and provides theoretical basis for the formation of the quality of S. japonica.

Screening for the anti-inflammation quality markers of Xiaojin Pills based on HPLC-MS/MS method, COX-2 inhibition test and protein interaction network.

Nowadays, breast disorders seriously affect women's health in an increasing number. In China, Xiaojin Pills are commonly used in the treatment of breast diseases. Doctors have concluded that the combined use of Xiaojin Pills with conventional therapy can significantly improve the efficacy with fewer side effects. However, the prescription of Xiaojin Pills is complicated and their quality control methods cannot completely ensure the quality of Xiaojin Pills. On the basis of its mechanism, our study combined chemical evaluation and biological evaluation to identify the anti-inflammatory markers of Xiaojin Pills. In this manuscript, 13 compounds in Xiaojin Pills were quantified. At the same time, the cyclooxygenase-2 inhibition rates of different Xiaojin Pills were measured and the possible markers were screened by spectrum-effect relationship. Further, anti-inflammatory activities of markers were verified and protein interaction network was analyzed, identifying the components of Protocatechuate, Beta-Boswellic acid and Levistilide A as the anti-inflammatory quality markers of Xiaojin Pills. We hope our studies can provide a scientific theoretical basis for accurately quality control of Xiaojin Pills and reasonable suggestions for pharmaceutical companies and new ideas for the quality control of other medicines.

Composition and diversity of rhizosphere fungal community in Coptis chinensis Franch. continuous cropping fields.

In this study, effects of continuous cropping on soil properties, enzyme activities, and relative abundance, community composition and diversity of fungal taxa were investigated. Rhizosphere soil from field continuously cropped for one-year, three-year and five-year by Coptis chinensis Franch. was collected and analyzed. Illumina high-throughput sequencing analysis showed that continuous cropping of C. chinensis resulted in a significant and continuous decline in the richness and diversity of soil fungal population. Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota were the dominant phyla of fungi detected in rhizosphere soil. Fungal genera such as Phoma, Volutella, Pachycudonia, Heterodermia, Gibberella, Cladosporium, Trichocladium, and Sporothrix, were more dominant in continuously cropped samples for three-year and five-year compared to that for one-year. By contrast, genera, such as Zygosaccharomyces, Pseudotaeniolina, Hydnum, Umbelopsis, Humicola, Crustoderma, Psilocybe, Coralloidiomyces, Mortierella, Polyporus, Pyrenula, and Monographella showed higher relative abundance in one-year samples than that in three-year and five-year samples. Cluster analysis of the fungal communities from three samples of rhizosphere soil from C. chinensis field revealed that the fungal community composition, diversity, and structure were significantly affected by the continuous cropping. Continuous cropping of C. chinensis also led to significant declines in soil pH, urease, and catalase activities. Redundancy analysis showed that the soil pH had the most significant effect on soil fungal population under continuous cropping of C. chinensis.

[Analysis on genetic diversity and genetic relationship of medicinal species in Coptis by SCoT].

The genetic diversity and genetic relationship among four medicinal species of Coptis were detected by the approach of sequence-related amplified polymorphism (SCoT). The associated genetic parameters were calculated by POPGENE1.31. The systematic diagram of genetic relationship were clustered by TREECONW. The results showed that a total of 434 bands were produced by using 28 primers, of which 430 were polymorphic loci. There was a high level of genetic diversity among species (PPB=99.1%,Na=1.990 6,Ne=1.329 3,H=0.212 2,I=0.337 8). However, genetic diversity was lower within species, the average of genetic parameters wasPPB=16.8%,Na=1.168 2, Ne=1.073 0,H=0.043 7,I=0.067 7. The Nei's genetic differentiation coefficient was 0.794 0, that indicated that most of the genetic variation existed among species. By clustering analysis, different individuals gathered in the same group. The results confirmed that SCoT marker can be used as one of the effective methods to reveal the genetic diversity and relationship among medicinal species of Coptis.