Screening out Biomarkers of Tetrastigma hemsleyanum for Anti-Cancer and Anti-Inflammatory Based on Spectrum-Effect Relationship Coupled with UPLC-Q-TOF-MS.

Tetrastigma hemsleyanum Diels et Gilg. (T. hemsleyanum) is an economically and medicinally valuable species within the genus Tetrastigma. However, the material basis of its pharmacological action and the biomarkers associated with its anti-cancer and anti-inflammatory effects are still unclear. Additionally, the T. hemsleyanum industry cannot grow because there is a lack of a scientific, universal, and measurable quality control system. This study aimed to explore the chemical basis quality markers related to the anti-cancer and anti-inflammatory effects of T. hemsleyanum to establish an effective quality evaluation method. UPLC-Q-TOF-MSE fingerprint profiles of T. hemsleyanum from different origins were established. Pharmacodynamic studies used HepG2 and HuH-7 cells and LPS-induced RAW264.7 to evaluate the anti-tumor and anti-inflammatory effects of the active ingredients. The spectrum-effect relationships between UPLC fingerprints and anti-cancer and anti-inflammatory activities were evaluated using PCA and PLSR statistical methods. Moreover, docking analysis was performed to identify specific active biomarkers with molecular targets associated with cancer and inflammation. Chlorogenic acid, quinic acid, catechin, kaempferol 3-rutinoside, apigenin-8-C-glucoside, and linolenic acid were associated with anticancer activity, while chlorogenic acid, quercetin, quinic acid, kaempferol 3-rutinoside, rutinum, apigenin-8-C-glucoside, and linolenic acid were associated with anti-inflammatory activity. The spectrum-effect relationship of T. hemsleyanum was successfully established, and the biomarkers for anti-cancer and anti-inflammatory effects were preliminary confirmed. These findings provide a theoretical basis for the elucidation of the substance basis of T. hemsleyanum and lay the foundation for its rapid identification, quality control, industrial research, and utilization.

Phase 1 Trial of an RNA Interference Therapy for Acute Intermittent Porphyria.

BACKGROUND: Induction of delta aminolevulinic acid synthase 1 ( ALAS1) gene expression and accumulation of neurotoxic intermediates result in neurovisceral attacks and disease manifestations in patients with acute intermittent porphyria, a rare inherited disease of heme biosynthesis. Givosiran is an investigational RNA interference therapeutic agent that inhibits hepatic ALAS1 synthesis. METHODS: We conducted a phase 1 trial of givosiran in patients with acute intermittent porphyria. In part A of the trial, patients without recent porphyria attacks (i.e., no attacks in the 6 months before baseline) were randomly assigned to receive a single subcutaneous injection of one of five ascending doses of givosiran (0.035, 0.10, 0.35, 1.0, or 2.5 mg per kilogram of body weight) or placebo. In part B, patients without recent attacks were randomly assigned to receive once-monthly injections of one of two doses of givosiran (0.35 or 1.0 mg per kilogram) or placebo (total of two injections 28 days apart). In part C, patients who had recurrent attacks were randomly assigned to receive injections of one of two doses of givosiran (2.5 or 5.0 mg per kilogram) or placebo once monthly (total of four injections) or once quarterly (total of two injections) during a 12-week period, starting on day 0. Safety, pharmacokinetic, pharmacodynamic, and exploratory efficacy outcomes were evaluated. RESULTS: A total of 23 patients in parts A and B and 17 patients in part C underwent randomization. Common adverse events included nasopharyngitis, abdominal pain, and diarrhea. Serious adverse events occurred in 6 patients who received givosiran in parts A through C combined. In part C, all 6 patients who were assigned to receive once-monthly injections of givosiran had sustained reductions in ALAS1 messenger RNA (mRNA), delta aminolevulinic acid, and porphobilinogen levels to near normal. These reductions were associated with a 79% lower mean annualized attack rate than that observed with placebo (exploratory efficacy end point). CONCLUSIONS: Once-monthly injections of givosiran in patients who had recurrent porphyria attacks resulted in mainly low-grade adverse events, reductions in induced ALAS1 mRNA levels, nearly normalized levels of the neurotoxic intermediates delta aminolevulinic acid and porphobilinogen, and a lower attack rate than that observed with placebo. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02452372 .).

Characterization and Comparative Analysis of Chloroplast Genomes in Five Uncaria Species Endemic to China.

Uncaria, a perennial vine from the Rubiaceae family, is a typical Chinese traditional medicine. Currently, uncertainty exists over the Uncaria genus' evolutionary relationships and germplasm identification. The complete chloroplast genomes of four Uncaria species mentioned in the Chinese Pharmacopoeia and Uncaria scandens (an easily confused counterfeit) were sequenced and annotated. The findings demonstrated that the whole chloroplast genome of Uncaria genus is 153,780-155,138 bp in full length, encoding a total of 128-131 genes, containing 83-86 protein-coding genes, eight rRNAs and 37 tRNAs. These regions, which include eleven highly variable loci and 31-49 SSRs, can be used to create significant molecular markers for the Uncaria genus. The phylogenetic tree was constructed according to protein-coding genes and the whole chloroplast genome sequences of five Uncaria species using four methods. The topology of the two phylogenetic trees showed no difference. The sequences of U. rhynchophylla and U. scandens are clustered in one group, while the U. hirsuta and U. macrophylla are clustered in another group. U. sessilifructus is clustered together with the above two small clades. New insights on the relationship were revealed via phylogenetic research in five Uncaria species. This study will provide a theoretical basis for identifying U. rhynchophylla and its counterfeits, as well as the species of the Uncaria genus. This research provides the initial chloroplast genome report of Uncaria, contributes to elucidating the chloroplast genome evolution of Uncaria in China.

Melatonin enhances cotton immunity to Verticillium wilt via manipulating lignin and gossypol biosynthesis.

Plants endure challenging environments in which they are constantly threatened by diverse pathogens. The soil-borne fungus Verticillium dahliae is a devastating pathogen affecting many plant species including cotton, in which it significantly reduces crop yield and fiber quality. Melatonin involvement in plant immunity to pathogens has been reported, but the mechanisms of melatonin-induced plant resistance are unclear. In this study, the role of melatonin in enhancing cotton resistance to V. dahliae was investigated. At the transcriptome level, exogenous melatonin increased the expression of genes in phenylpropanoid, mevalonate (MVA), and gossypol pathways after V. dahliae inoculation. As a result, lignin and gossypol, the products of these metabolic pathways, significantly increased. Silencing the serotonin N-acetyltransferase 1 (GhSNAT1) and caffeic acid O-methyltransferase (GhCOMT) melatonin biosynthesis genes compromised cotton resistance, with reduced lignin and gossypol levels after V. dahliae inoculation. Exogenous melatonin pre-treatment prior to V. dahliae inoculation restored the level of cotton resistance reduced by the above gene silencing effects. Melatonin levels were higher in resistant cotton cultivars than in susceptible cultivars after V. dahliae inoculation. The findings indicate that melatonin affects lignin and gossypol synthesis genes in phenylpropanoid, MVA, and gossypol pathways, thereby enhancing cotton resistance to V. dahliae.

Identification and profiling of upland cotton microRNAs at fiber initiation stage under exogenous IAA application.

BACKGROUND: Cotton is the most essential textile crop worldwide, and phytohormones are critical for cotton fiber development. One example is the role of auxin in fiber initiation, but we know little molecular basis. MicroRNAs (miRNAs) have a significant function in cotton development; nevertheless their role in fiber initiation remains unclear. Here, exogenous IAA was applied to cotton plant before anthesis. Utilizing small RNA sequencing, the mechanism underlying miRNA-mediated regulation of fiber initiation under exogenous IAA treatment was investigated. RESULTS: With exogenous IAA application, the endogenous IAA and GA contents of IAA treated (IT) ovules were higher than control (CK) ovules at the fiber initiation stage, while endogenous ABA content was lower in IT than CK. Using scanning electron microscopy, we found the fiber number and size were significantly promoted in IT at 0 DPA. Fiber quality analysis showed that fiber length, uniformity, strength, elongation, and micronaire of IT were higher than CK, though not statistically significant, while lint percent was significantly higher in IT. We generated six small RNA libraries using - 3, 0, and 3 DPA ovules of IT and CK, and identified 58 known miRNAs and 83 novel miRNAs together with the target genes. The differential expressed miRNAs number between IT and CK at - 3, 0, 3 DPA was 34, 16 and 24, respectively. Gene ontology and KEGG pathway enrichment analyses for the target genes of the miRNAs expressed in a differential manner showed that they were significantly enriched in 30 terms and 8 pathways. QRT-PCR for those identified miRNAs and the target genes related to phytohormones and fiber development was performed, and results suggested a potential role of these miRNAs in fiber initiation. CONCLUSIONS: The exogenous IAA application affected the relative phytohormone contents in ovule and promoted fiber initiation in cotton. Identification and profiling of miRNAs and their targets at the fiber initiation stage provided insights for miRNAs' regulation function of fiber initiation. These findings not only shed light on the regulatory network of fiber growth but also offer clues for cotton fiber amelioration strategies in cotton.

[Correlation analysis of main agronomic traits and contents of diterpene lactones of Andrographis paniculata].

In order to study the correlation between the traits of Andrographis paniculata. The main agronomic traits and the content of four kinds of diterpene lactons were measured by the seedlings and the unmutagenized seeds carried by the spacecraft,and multiple comparisons,correlations and principal component analysis were carried out. The results showed that the agronomic traits of A. paniculata have different degrees of difference after being carried by space. The contents of diterpene lactones were quite different. The variation coefficients of deoxyandrographolide content,fresh weight,leaf dry weight,dehydrated andrographolide content,dry weight,neoandrographolide content and andrographolide content were all over 35%. There was a significant correlation between multiple traits,and the leaf weight ratio was significantly positively correlated with the number of primary tillers,leaf dry weight and dry weight,and was significantly negatively correlated with the content of deoxyandrographolide. Andrographolide content was a significantly negatively correlated with the number of leaves and the number of primary tillers,and positively correlated with the other three lactones. Five principal components were extracted from principal component analysis,and the cumulative contribution rate was 83. 127%,which were yield factor,plant type factor,leaf type factor,component factor and seed weight factor. Among the traits affecting the quality of A. paniculata,the yield factor for reliability of the selection of A. paniculata is higher than other factors. There are abundant variations among the traits of A. paniculata,carried in space which increase the genetic diversity. The principal component analysis method can be used to select the principal component factors according to the breeding requirements,which provides a theoretical basis for the breeding of high-yield and high-quality A. paniculata and the high-yield and stable cultivation of A. paniculata.

Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling.

To study the multistep process of cervical cancer development, we analyzed 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CIN1- CIN3), and cervical cancer (CxCa) from multiple perspectives, revealing a cascade of progressive changes. Compared with normal tissue, expression of many DNA replication/repair and cell proliferation genes was increased in CIN1/CIN2 lesions and further sustained in CIN3, consistent with high-risk human papillomavirus (HPV)-induced tumor suppressor inactivation. The CIN3-to-CxCa transition showed metabolic shifts, including decreased expression of mitochondrial electron transport complex components and ribosomal protein genes. Significantly, despite clinical, epidemiological, and animal model results linking estrogen and estrogen receptor alpha (ERα) to CxCa, ERα expression declined >15-fold from normalcy to cancer, showing the strongest inverse correlation of any gene with the increasing expression of p16, a marker for HPV-linked cancers. This drop in ERα in CIN and tumor cells was confirmed at the protein level. However, ERα expression in stromal cells continued throughout CxCa development. Our further studies localized stromal ERα to FSP1+, CD34+, SMA- precursor fibrocytes adjacent to normal and precancerous CIN epithelium, and FSP1-, CD34-, SMA+ activated fibroblasts in CxCas. Moreover, rank correlations with ERα mRNA identified IL-8, CXCL12, CXCL14, their receptors, and other angiogenesis and immune cell infiltration and inflammatory factors as candidates for ERα-induced stroma-tumor signaling pathways. The results indicate that estrogen signaling in cervical cancer has dramatic differences from ERα+ breast cancers, and imply that estrogen signaling increasingly proceeds indirectly through ERα in tumor-associated stromal fibroblasts.

QTL mapping with different genetic systems for nine non-essential amino acids of cottonseeds.

Amino acid is an important nutrient resource for both human and animals. Using a set of 188 RILs population derived from an elite hybrid cross of upland cotton cultivars 'HS46' × 'MARCABUCAG8US-1-88' and their immortal F2 (IF2) with reciprocal backcrosses BC1F1 and BC2F1 (BC) populations in two environments, the QTLs located on the embryo genome and maternal plant genome for nine amino acids of cottonseed were studied across environments. The QTL Network-CL-2.0-seed software was used to analyze the QTLs and their genetic effects for nine amino acids. A total of 56 QTLs for nine amino acids were detected in both populations, with many having over 5% of phenotypic variation. Ten of the total QTLs could be simultaneously found in the IF2 and BC populations. For most QTLs, the genetic effects from embryo genome were more important than those from maternal plant genome for the performance of nine amino acids. Significant embryo additive main effects and maternal additive main effect with their environment interaction effects from many QTLs were also found in present experiment. Some QTLs with larger phenotypic variation were important for improving the amino-acid contents in cottonseeds.

Short-term subcutaneous grass pollen immunotherapy under the umbrella of anti-IL-4: A randomized controlled trial.

BACKGROUND: Allergen immunotherapy is currently the only disease-modifying treatment available for allergic rhinitis and allergic asthma. OBJECTIVES: We sought to evaluate the induction of sustained tolerance to allergen when anti-IL-4 was combined with a suboptimal course of grass pollen subcutaneous immunotherapy (SCIT) using the allergen-induced skin late-phase response (LPR) and exploratory immune monitoring as surrogate markers of therapeutic response. METHODS: In this randomized, double-blind, 3-group parallel design trial, 37 participants with seasonal allergic rhinitis received suboptimal SCIT (30,000 standardized quality units) in combination with anti-IL-4 (VAK694) and suboptimal SCIT (30,000 standardized quality units) plus placebo antibody or double placebo (placebo SCIT and placebo antibody) restricted to 13 weeks before the grass pollen season. The primary end point was the size of the LPR at 12 months. Exploratory end points included measures of the immunomodulatory activity of treatment by using IL-4 and IL-10 FluoroSpot assays, flow cytometry of T cells, and measurement of IgE, IgG4, and facilitated antigen binding. RESULTS: Both active treatment arms led to a substantial and sustained reduction of the LPR with no additional suppression with addition of anti-IL-4. Treatment with anti-IL-4 and SCIT compared with SCIT alone led to a sustained reduction in allergen-specific IL-4-producing cell counts (P < .01). Both active treatment arms led to induction of dual IL-4/IL-10-producing cells during the pollen season. CONCLUSION: The combination of anti-IL-4 with SCIT provided no additional benefit over SCIT alone in suppressing the allergen-induced skin LPR. A larger trial is needed to assess whether the observed ex vivo downregulation of TH2 responses might translate into clinical benefit.

Selection and validation of reliable reference genes in Gossypium raimondii.

OBJECTIVE: To identify reliable reference genes for gene expression analysis in Gossypium raimondii. RESULTS: Five different software tools, geNorm, NormFinder, BestKeeper, ReFinder and ∆Ct method were employed to analyze the qRT-PCR data systematically of 12 housekeeping genes. SAD and TUA11 showed relatively stable expression levels in all tissues (i.e. leaves, shoots, buds, and sepals). We then limited our analysis to each plant part and identified tissue-specific reference genes. Our results showed TUA11, TUB6 and EF1a, EF1a, MZA and GAPC2, MZA, GAPC2, SAD and TUA11, and UBQ and MZA were reliable reference genes in leaves, shoots, buds, and sepals, respectively. CONCLUSION: Some genes were commonly identified as candidate reference genes in more than two tissue, while others were tissue-specific. Thus, our study allows choosing an appropriate control gene based on sampling for gene expression analysis.

MicroRNA-target gene responses to lead-induced stress in cotton (Gossypium hirsutum L.).

MicroRNAs (miRNAs) play key roles in plant responses to various metal stresses. To investigate the miRNA-mediated plant response to heavy metals, cotton (Gossypium hirsutum L.), the most important fiber crop in the world, was exposed to different concentrations (0, 25, 50, 100, and 200 µM) of lead (Pb) and then the toxicological effects were investigated. The expression patterns of 16 stress-responsive miRNAs and 10 target genes were monitored in cotton leaves and roots by quantitative real-time PCR (qRT-PCR); of these selected genes, several miRNAs and their target genes are involved in root development. The results show a reciprocal regulation of cotton response to lead stress by miRNAs. The characterization of the miRNAs and the associated target genes in response to lead exposure would help in defining the potential roles of miRNAs in plant adaptation to heavy metal stress and further understanding miRNA regulation in response to abiotic stress.

High-throughput deep sequencing shows that microRNAs play important roles in switchgrass responses to drought and salinity stress.

MicroRNAs (miRNAs) are an important class of small regulatory RNAs. The goal of this study was to analyse stress-responsive miRNAs in switchgrass (Panicum virgatum), the emerging biofuel crop, to facilitate choosing gene targets for improving biomass and biofuel yield. After sequencing three small RNA libraries constructed from control, salt- and drought-treated switchgrass using Illumina sequencing technology, we identified 670 known miRNA families from a total of more than 50 million short reads. A total of 273 miRNAs were identified with precursors: 126 conserved miRNAs and 147 novel miRNAs. Of them, 265 miRNAs were found to have their opposite sequences (miRNA*) with 2-nt overhang on the 3' end. Of them, 194 were detected in switchgrass transcriptome sequences generated from 31 high-throughput RNA sequencing (RNA-Seq) data sets in NCBI. Many miRNAs were differentially or uniquely expressed during salinity or drought stress treatment. We also discovered 11 miRNA clusters containing 29 miRNAs. These identified miRNAs potentially targeted 28549 genes with a various function, including transcription factors, stress-response proteins and cellulose biosynthesis-related proteins. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the identified miRNAs and their targets were classified to 3779 GO terms including 1534 molecular functions, 1851 biological processes and 394 cellular components and were enriched to 147 KEGG pathways. Interestingly, 195 miRNA families and 450 targets were involved in the biosynthesis pathways of carbon, glucose, starch, fatty acid and lignin and in xylem formation, which could aid in designing next-generation switchgrass for biomass and biofuel.

Limited agreement of independent RNAi screens for virus-required host genes owes more to false-negative than false-positive factors.

Systematic, genome-wide RNA interference (RNAi) analysis is a powerful approach to identify gene functions that support or modulate selected biological processes. An emerging challenge shared with some other genome-wide approaches is that independent RNAi studies often show limited agreement in their lists of implicated genes. To better understand this, we analyzed four genome-wide RNAi studies that identified host genes involved in influenza virus replication. These studies collectively identified and validated the roles of 614 cell genes, but pair-wise overlap among the four gene lists was only 3% to 15% (average 6.7%). However, a number of functional categories were overrepresented in multiple studies. The pair-wise overlap of these enriched-category lists was high, ∼19%, implying more agreement among studies than apparent at the gene level. Probing this further, we found that the gene lists implicated by independent studies were highly connected in interacting networks by independent functional measures such as protein-protein interactions, at rates significantly higher than predicted by chance. We also developed a general, model-based approach to gauge the effects of false-positive and false-negative factors and to estimate, from a limited number of studies, the total number of genes involved in a process. For influenza virus replication, this novel statistical approach estimates the total number of cell genes involved to be ∼2,800. This and multiple other aspects of our experimental and computational results imply that, when following good quality control practices, the low overlap between studies is primarily due to false negatives rather than false-positive gene identifications. These results and methods have implications for and applications to multiple forms of genome-wide analysis.

CD70-Targeted Allogeneic CAR T-Cell Therapy for Advanced Clear Cell Renal Cell Carcinoma.

Therapeutic approaches for clear cell renal cell carcinoma (ccRCC) remain limited; however, chimeric antigen receptor (CAR) T-cell therapies may offer novel treatment options. CTX130, an allogeneic CD70-targeting CAR T-cell product, was developed for the treatment of advanced or refractory ccRCC. We report that CTX130 showed favorable preclinical proliferation and cytotoxicity profiles and completely regressed RCC xenograft tumors. We also report results from 16 patients with relapsed/refractory ccRCC who received CTX130 in a phase I, multicenter, first-in-human clinical trial. No patients encountered dose-limiting toxicity, and disease control was achieved in 81.3% of patients. One patient remains in a durable complete response at 3 years. Finally, we report on a next-generation CAR T construct, CTX131, in which synergistic potency edits to CTX130 confer improved expansion and efficacy in preclinical studies. These data represent a proof of concept for the treatment of ccRCC and other CD70+ malignancies with CD70- targeted allogeneic CAR T cells. Significance: Although the role of CAR T cells is well established in hematologic malignancies, the clinical experience in solid tumors has been disappointing. This clinical trial demonstrates the first complete response in a patient with RCC, reinforcing the potential benefit of CAR T cells in the treatment of solid tumors.

Mapping genes for plant structure, development and evolution: functional mapping meets ontology.

One of the fundamental tasks in biology is the identification of genes that control the structure and developmental pattern of complex traits and their responses to the environment during trait development. Functional mapping provides a statistical means for detecting quantitative trait loci (QTLs) that underlie developmental traits, such as growth trajectories, and for testing the interplay between gene action and development. Here we describe how functional mapping and studies of plant ontology can be integrated so as to elucidate the expression mechanisms of QTLs that control plant growth, morphology, development, and adaptation to changing environments. This approach can also be used to construct an evo-devo framework for inferring the evolution of developmental traits.

A model-based analysis to infer the functional content of a gene list.

An important challenge in statistical genomics concerns integrating experimental data with exogenous information about gene function. A number of statistical methods are available to address this challenge, but most do not accommodate complexities in the functional record. To infer activity of a functional category (e.g., a gene ontology term), most methods use gene-level data on that category, but do not use other functional properties of the same genes. Not doing so creates undue errors in inference. Recent developments in model-based category analysis aim to overcome this difficulty, but in attempting to do so they are faced with serious computational problems. This paper investigates statistical properties and the structure of posterior computation in one such model for the analysis of functional category data. We examine the graphical structures underlying posterior computation in the original parameterization and in a new parameterization aimed at leveraging elements of the model. We characterize identifiability of the underlying activation states, describe a new prior distribution, and introduce approximations that aim to support numerical methods for posterior inference.

Assessment of Genetic Diversity in Bupleurum spp. Basing Agronomic Traits, Medicinal Components and ISSR Markers.

Radix bupleuri is one of the bulk medicinal materials in China and it is widely adopted in clinical applications and drug discovery. The investigation of agronomic traits, active component content and genetic diversity in diverse Radix bupleuri germplasms may provide evidence to promote the selection of better strains. In this research, 13 germplasms from various sources were used to investigate the variations between different Radix bupleuri germplasms. Nine biological characteristics were noted in the field, and the levels of the two primary active ingredients were determined using high performance liquid chromatography (HPLC). Moreover, the molecular marker technique of inter-simple sequence repeat (ISSR) and the unweighted pair group method with arithmetic means (UPGMA) were employed to evaluate the molecular genetic diversity. The findings showed that there was a wide range of variation among the many varieties of Radix bupleuri, with coefficients of variation for agronomic traits and active component content ranging from 7.62% to 41.54% and 36.47% to 53.70%, respectively. Moreover, there are different degrees of relationship between the two. Since there was a significant correlation between root weight and saikosaponin content, it was possible to classify a plant based on its weight and anticipate its saikosaponin content. The 13 species were divided into four groups based on their germplasm by genetic markers-based cluster analysis. This indicated the possibility that the component content would not necessarily be related to germplasm and might easily be influenced by environmental factors. The use of ISSR marker technology made it possible to precisely identify the various Radix bupleuri provenances and its counterfeit products. There may be a way to prevent the misunderstandings caused by the appearance and composition of Chinese medicinal substances. In our study, the germplasm of Radix bupleuri that was widely circulated in the market was comprehensively evaluated in terms of agronomic traits, active components and molecular level, and identified by simple means, to provide a theoretical basis for the evaluation and screening of fine germplasms of Radix bupleuri.

mRNA-Seq and miRNA-Seq Analyses Provide Insights into the Mechanism of Pinellia ternata Bulbil Initiation Induced by Phytohormones.

Pinellia ternata (Thunb.) Breit (abbreviated as P. ternata) is a plant with an important medicinal value whose yield is restricted by many factors, such as low reproductive efficiency and continuous cropping obstacles. As an essential breeding material for P. ternata growth and production, the bulbils have significant advantages such as a high survival rate and short breeding cycles. However, the location effect, influencing factors, and molecular mechanism of bulbil occurrence and formation have not been fully explored. In this study, exogenously applied phytohormones were used to induce in vitro petiole of P. ternata to produce bulbil structure. Transcriptome sequencing of mRNA and miRNA were performed in the induced petiole (TCp) and the induced bulbil (TCb). Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for the identification of key genes and pathways involved in bulbil development. A total of 58,019 differentially expressed genes (DEGs) were identified. The GO and KEGG analysis indicated that DEGs were mainly enriched in plant hormone signal transduction and the starch and sucrose metabolism pathway. The expression profiles of miR167a, miR171a, and miR156a during bulbil induction were verified by qRT-PCR, indicating that these three miRNAs and their target genes may be involved in the process of bulbil induction and play an important role. However, further molecular biological experiments are required to confirm the functions of the identified bulbil development-related miRNAs and targets.

Systematic Identification and Validation of Suitable Reference Genes for the Normalization of Gene Expression in Prunella vulgaris under Different Organs and Spike Development Stages.

The quantitative real-time PCR (qRT-PCR) is an efficient and sensitive method for determining gene expression levels, but the accuracy of the results substantially depends on the stability of the reference gene (RG). Therefore, choosing an appropriate reference gene is a critical step in normalizing qRT-PCR data. Prunella vulgaris L. is a traditional Chinese medicine herb widely used in China. Its main medicinal part is the fruiting spike which is termed Spica Prunellae. However, thus far, few studies have been conducted on the mechanism of Spica Prunellae development. Meanwhile, no reliable RGs have been reported in P. vulgaris. The expression levels of 14 candidate RGs were analyzed in this study in various organs and at different stages of Spica Prunellae development. Four statistical algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) were utilized to identify the RGs' stability, and an integrated stability rating was generated via the RefFinder website online. The final ranking results revealed that eIF-2 was the most stable RG, whereas VAB2 was the least suitable as an RG. Furthermore, eIF-2 + Histon3.3 was identified as the best RG combination in different periods and the total samples. Finally, the expressions of the PvTAT and Pv4CL2 genes related to the regulation of rosmarinic acid synthesis in different organs were used to verify the stable and unstable RGs. The stable RGs in P. vulgaris were originally identified and verified in this work. This achievement provides strong support for obtaining a reliable qPCR analysis and lays the foundation for in-depth research on the developmental mechanism of Spica Prunellae.

Selection of suitable candidate genes for mRNA expression normalization in bulbil development of Pinellia ternata.

Pinellia ternata (Thunb.) Breit. (Abbreviated as P. ternata). It is a commonly prescribed Chinese traditional medicinal herb for the treatment of phlegm, cough, and morning sick. Bulbil reproduction is one of the main reproductive methods of P. ternata. The accurate quantification of gene expression patterns associated with bulbil development might be helpful to explore the molecular mechanism involved in P. ternata reproduction. Quantitative real-time PCR was the most preferred method for expression profile and function analysis of mRNA. However, the reference genes in different tissues of P. ternata in different periods of bulbil development have not been studied in detail. In present study, the expression stability of eight candidate reference genes were determined with programs: geNorm, NormFinder, BestKeeper, and refFinder. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the top- rated reference gene in all samples of P. ternata, while different combinations of reference gene proved to be the most stable depending on development stage and tissue type. Furthermore, the reliability of GAPDH expression was verified by six P. ternata related genes in hormone and nutrient biosynthesis pathways, and the expression profiles of these genes were agreed with the results of RNA-seq digital gene expression analysis. These results can contribute to studies of gene expression patterns and functional analysis of P. ternata involved in bulbil development.