The Mechanism of Cannabichromene and Cannabidiol Alone Versus in Combination in the Alleviation of Arthritis-Related Inflammation.

BACKGROUND: Patients suffering from arthritis have limited treatment options for nonoperative management. In search of pain relief, patients have been taking over-the-counter cannabinoids. Cannabidiol (CBD) and cannabichromene (CBC) are minor cannabinoids with reported analgesic and anti-inflammatory properties and have been implicated as potential therapeutics for arthritis-related pain. To this end, we utilized a murine model to investigate the effectiveness of and mechanism by which CBC alone, CBD alone, or CBD and CBC in combination may provide a reduction in arthritis-associated inflammation. METHODS: Forty-eight mice were included in the study, which were separated into 4 groups: control group (n = 12), treatment with CBD alone (n = 12), treatment with CBC alone (n = 12), and treatment with CBD + CBC (n = 12). We induced inflammation in each mouse utilizing the collagen-induced arthritis model. At scheduled timepoints, mice were clinically assessed for weight gain, swelling, and arthritis severity. In addition, inflammation-associated serum cytokine levels were analyzed for each animal. RESULTS: Thirty-five of 48 mice survived the duration of the study resulting in the following group numbers: control group (n = 8), treatment with CBD alone (n = 9), treatment with CBC alone (n = 9), and treatment with CBD + CBC (n = 9). Animals treated with CBC and CBD + CBC showed significant weight gain between 3 and 5 weeks. Irrespective of treatment, regression analysis comparing all cytokine measurement and physical outcomes found a significant positive correlation between levels of 5 individual cytokines and both arthritis scores and swelling. Animals treated with CBD + CBC showed a significant decrease in swelling between 3 and 5 weeks compared with the control group. Cannabinoid treatment selectively affected the gene expression of eotaxin and lipopolysaccharide-induced CXC chemokine with combined treatment of CBC + CBD. CONCLUSION: Treatment with cannabinoids resulted in decreased clinical markers of inflammation. Further, the anti-inflammatory effect of CBC and CBD in conjunction was associated with a greater anti-inflammatory effect than either minor cannabinoid alone. Future work will elucidate the possibility of synergistic or entourage effects of minor cannabinoids used in combination for the treatment of arthritis-related pain and inflammation.

Proteomic and single-cell landscape reveals novel pathogenic mechanisms of HBV-infected intrahepatic cholangiocarcinoma.

Despite the epidemiological association between intrahepatic cholangiocarcinoma (ICC) and hepatitis B virus (HBV) infection, little is known about the relevant oncogenic effects. A cohort of 32 HBV-infected ICC and 89 non-HBV-ICC patients were characterized using whole-exome sequencing, proteomic analysis, and single-cell RNA sequencing. Proteomic analysis revealed decreased cell-cell junction levels in HBV-ICC patients. The cell-cell junction level had an inverse relationship with the epithelial-mesenchymal transition (EMT) program in ICC patients. Analysis of the immune landscape found that more CD8 T cells and Th2 cells were present in HBV-ICC patients. Single-cell analysis indicated that transforming growth factor beta signaling-related EMT program changes increased in tumor cells of HBV-ICC patients. Moreover, ICAM1+ tumor-associated macrophages are correlated with a poor prognosis and contributed to the EMT in HBV-ICC patients. Our findings provide new insights into the behavior of HBV-infected ICC driven by various pathogenic mechanisms involving decreased cell junction levels and increased progression of the EMT program.

EMLI-ICC: an ensemble machine learning-based integration algorithm for metastasis prediction and risk stratification in intrahepatic cholangiocarcinoma.

Robust strategies to identify patients at high risk for tumor metastasis, such as those frequently observed in intrahepatic cholangiocarcinoma (ICC), remain limited. While gene/protein expression profiling holds great potential as an approach to cancer diagnosis and prognosis, previously developed protocols using multiple diagnostic signatures for expression-based metastasis prediction have not been widely applied successfully because batch effects and different data types greatly decreased the predictive performance of gene/protein expression profile-based signatures in interlaboratory and data type dependent validation. To address this problem and assist in more precise diagnosis, we performed a genome-wide integrative proteome and transcriptome analysis and developed an ensemble machine learning-based integration algorithm for metastasis prediction (EMLI-Metastasis) and risk stratification (EMLI-Prognosis) in ICC. Based on massive proteome (216) and transcriptome (244) data sets, 132 feature (biomarker) genes were selected and used to train the EMLI-Metastasis algorithm. To accurately detect the metastasis of ICC patients, we developed a weighted ensemble machine learning method based on k-Top Scoring Pairs (k-TSP) method. This approach generates a metastasis classifier for each bootstrap aggregating training data set. Ten binary expression rank-based classifiers were generated for detection of metastasis separately. To further improve the accuracy of the method, the 10 binary metastasis classifiers were combined by weighted voting based on the score from the prediction results of each classifier. The prediction accuracy of the EMLI-Metastasis algorithm achieved 97.1% and 85.0% in proteome and transcriptome datasets, respectively. Among the 132 feature genes, 21 gene-pair signatures were developed to establish a metastasis-related prognosis risk-stratification model in ICC (EMLI-Prognosis). Based on EMLI-Prognosis algorithm, patients in the high-risk group had significantly dismal overall survival relative to the low-risk group in the clinical cohort (P-value < 0.05). Taken together, the EMLI-ICC algorithm provides a powerful and robust means for accurate metastasis prediction and risk stratification across proteome and transcriptome data types that is superior to currently used clinicopathological features in patients with ICC. Our developed algorithm could have profound implications not just in improved clinical care in cancer metastasis risk prediction, but also more broadly in machine-learning-based multi-cohort diagnosis method development. To make the EMLI-ICC algorithm easily accessible for clinical application, we established a web-based server for metastasis risk prediction (http://ibi.zju.edu.cn/EMLI/).

A Randomized Controlled Trial of Topical Cannabidiol for the Treatment of Thumb Basal Joint Arthritis.

PURPOSE: Since the passage of the Agricultural Improvement Act of 2018, hand surgeons have increasingly encountered patients seeking counseling on over-the-counter, topical cannabidiol (CBD) for the treatment of pain. To this end, we designed a human clinical trial to investigate the therapeutic potential of CBD for the treatment of pain associated with thumb basal joint arthritis. METHODS: Following Food and Drug Administration and institutional approval, a phase 1 skin test was completed with 10 healthy participants monitored for 1 week after twice-daily application of 1 mL of topical CBD (6.2 mg/mL) with shea butter. After no adverse events were identified, we proceeded with a phase 2, double-blinded, randomized controlled trial. Eighteen participants with symptomatic thumb basal joint arthritis were randomized to 2 weeks of twice-daily treatment with CBD (6.2 mg/mL CBD with shea butter) or shea butter alone, followed by a 1-week washout period and then crossover for 2 weeks with the other treatment. Safety data and physical examination measurements were obtained at baseline and after completion of each treatment arm. RESULTS: Cannabidiol treatment resulted in improvements from baseline among patient-reported outcome measures, including Visual Analog Scale pain; Disabilities of the Arm, Shoulder, and Hand; and Single Assessment Numeric Evaluation scores, compared to the control arm during the study period. There were similar physical parameters identified with range of motion, grip, and pinch strength. CONCLUSIONS: In this single-center, randomized controlled trial, topical CBD treatment demonstrated significant improvements in thumb basal joint arthritis-related pain and disability without adverse events. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic II.

Horizontal transfer and evolution of the biosynthetic gene cluster for benzoxazinoids in plants.

Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites that have been identified in multiple grass species and are encoded by the Bx biosynthetic gene cluster (BGC) in maize. Data mining of 41 high-quality grass genomes identified complete Bx clusters (containing genes Bx1-Bx5 and Bx8) in three genera (Zea, Echinochloa, and Dichanthelium) of Panicoideae and partial clusters in Triticeae. The Bx cluster probably originated from gene duplication and chromosomal translocation of native homologs of Bx genes. An ancient Bx cluster that included additional Bx genes (e.g., Bx6) is presumed to have been present in ancestral Panicoideae. The ancient Bx cluster was putatively gained by the Triticeae ancestor via horizontal transfer (HT) from the ancestral Panicoideae and later separated into multiple segments on different chromosomes. Bx6 appears to have been under less constrained selection compared with the Bx cluster during the evolution of Panicoideae, as evidenced by the fact that it was translocated away from the Bx cluster in Zea mays, moved to other chromosomes in Echinochloa, and even lost in Dichanthelium. Further investigations indicate that purifying selection and polyploidization have shaped the evolutionary trajectory of Bx clusters in the grass family. This study provides the first candidate case of HT of a BGC between plants and sheds new light on the evolution of BGCs.

Cannabinoids and Pain for the Plastic Surgeon: What Is the Evidence?

BACKGROUND: Since the passage of the 2018 Farm Bill, practitioners have encountered more patients self-treating pain with over-the-counter topical cannabidiol (CBD) derived from hemp-Cannabis sativa with less than 0.3% delta-9-tetrahydrocannabinol-with reported improvements in pain control and activities of daily living. Cannabidiol has been touted for its capacity to improve inflammatory, arthritic, and neuropathic pain conditions, and increasing numbers of patients are exploring its use as potential replacement for opioids. However, limited rigorous clinical trials have been performed evaluating the safety and efficacy of cannabinoids for the treatment of pain. METHODS: A systematic search of PubMed was performed using the Medical Subject Headings (MeSH) terms "cannabinoid" or "CBD" or "cannabidiol" or "cannabis" or "medical marijuana" and "pain." It yielded 340 article titles. Twelve full-text primary studies of oral or topical CBD for chronic pain were selected for review, including 6 animal (2 randomized clinical trial and 4 prospective trials) and 6 human (4 randomized clinical trial and 2 prospective trials) studies. RESULTS: With respect to the safety and efficacy of oral and topical CBD for treating pain, animal and human studies have shown early positive results with limited minor side effects. However, all human studies may be underpowered with small sample sizes. CONCLUSIONS: With respect to the safety and efficacy of oral and topical CBD for treating pain, the evidence remains inconclusive in that we have a paucity of data to share with our patients who are considering the use of these products, which may be associated with significant costs.

Genomic insights into the evolution of Echinochloa species as weed and orphan crop.

As one of the great survivors of the plant kingdom, barnyard grasses (Echinochloa spp.) are the most noxious and common weeds in paddy ecosystems. Meanwhile, at least two Echinochloa species have been domesticated and cultivated as millets. In order to better understand the genomic forces driving the evolution of Echinochloa species toward weed and crop characteristics, we assemble genomes of three Echinochloa species (allohexaploid E. crus-galli and E. colona, and allotetraploid E. oryzicola) and re-sequence 737 accessions of barnyard grasses and millets from 16 rice-producing countries. Phylogenomic and comparative genomic analyses reveal the complex and reticulate evolution in the speciation of Echinochloa polyploids and provide evidence of constrained disease-related gene copy numbers in Echinochloa. A population-level investigation uncovers deep population differentiation for local adaptation, multiple target-site herbicide resistance mutations of barnyard grasses, and limited domestication of barnyard millets. Our results provide genomic insights into the dual roles of Echinochloa species as weeds and crops as well as essential resources for studying plant polyploidization, adaptation, precision weed control and millet improvements.

NtMYB305a binds to the jasmonate-responsive GAG region of NtPMT1a promoter to regulate nicotine biosynthesis.

MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, which comprises a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30-bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.

Insights into Gene Regulation of Jasmonate-Induced Whole-Plant Senescence of Tobacco under Non-Starvation Conditions.

Jasmonate (JA)-induced plant senescence has been mainly studied with a dark/starvation-promoted system using detached leaves; yet, the induction of whole-plant senescence by JA remains largely unclear. This work reports the finding of a JA-induced whole-plant senescence of tobacco under light/non-starvation conditions and the investigation of underlying regulations. Methyl jasmonate (MeJA) treatment induces the whole-plant senescence of tobacco in a light-intensity-dependent manner, which is suppressed by silencing of NtCOI1 that encodes the receptor protein of JA-Ile (the bioactive derivative of JA). MeJA treatment could induce the senescence-specific cysteine protease gene SAG12 and another cysteine protease gene SAG-L1 to high expression levels in the detached leaf patches under dark conditions but failed to induce their expression in tobacco whole plants under light conditions. Furthermore, MeJA attenuates the RuBisCo activase (RCA) level in the detached leaves but has no effect on this protein in the whole plant under light conditions. A genome-wide transcriptional assay also supports the presence of a differential regulatory pattern of senescence-related genes during MeJA-induced whole-plant senescence under non-starvation conditions and results in the finding of a chlorophylase activity increase in this process. We also observed that the MeJA-induced senescence of tobacco whole plants is reversible, which is accompanied by a structural change of chloroplasts. This work provides novel insights into JA-induced plant senescence under non-starvation conditions and is helpful to dissect the JA-synchronized process of whole-plant senescence.

Signaling pathway perturbation analysis for assessment of biological impact of cigarette smoke on lung cells.

Exposure to cigarette smoke (CS) results in injury to the epithelial cells of the human respiratory tract and has been implicated as a causative factor in the development of chronic obstructive pulmonary disease and lung cancers. The application of omics-scale methodologies has improved the capacity to understand cellular signaling processes underlying response to CS exposure. We report here the development of an algorithm based on quantitative assessment of transcriptomic profiles and signaling pathway perturbation analysis (SPPA) of human bronchial epithelial cells (HBEC) exposed to the toxic components present in CS. HBEC were exposed to CS of different compositions and for different durations using an ISO3308 smoking regime and the impact of exposure was monitored in 2263 signaling pathways in the cell to generate a total effect score that reflects the quantitative degree of impact of external stimuli on the cells. These findings support the conclusion that the SPPA algorithm provides an objective, systematic, sensitive means to evaluate the biological impact of exposures to CS of different compositions making a powerful comparative tool for commercial product evaluation and potentially for other known or potentially toxic environmental smoke substances.

NtAIDP1, a novel NtJAZ interacting protein, binds to an AT-rich region to activate the transcription of jasmonate-inducible genes in tobacco.

In plants, jasmonate ZIM-domain proteins (JAZs) act as critical regulators, interacting physically with transcription factors (TFs) and other transcriptional regulators to modulate jasmonate (JA)-responsive gene expression and participate in crosstalk with other hormone signalling pathways. Identifying novel JAZ-interacting proteins will provide new insights into JA signalling cascades in plants. Here, we performed yeast two-hybrid screening to identify 70 NtJAZ1-interacting proteins, including an A/T-rich interaction domain containing protein 1 (NtAIDP1) from JA-treated tobacco Bright Yellow-2 (BY-2) cells. NtAIDP1 is localised in the nucleus and interacts with NtJAZ1 via its C-terminal heat shock protein 20 (HSP) domain. Aside from NtJAZ1, NtAIDP1 also interacts with other JA-inducible NtJAZs, including NtJAZ2b, NtJAZ2b.2, NtJAZ5, NtJAZ7, NtJAZ11 and NtJAZ12, but not with NtJAZ3, NtJAZ3b or NtJAZ10, and interacts with NtNINJA, NtDELLA1 and NtDELLA2 in the yeast two-hybrid assay. Furthermore, NtAIDP1 binds to the AT-rich region of the GAG fragment of the putrescine N-methyltransferase 1a (NtPMT1a) promoter and activates the transcriptional activity of the GAG fragment, whereas NtMYC2a interacts with and competitively inhibits the transactivational activity of NtAIDP1 in Arabidopsis mesophyll protoplasts. Overexpression of NtAIDP1 promotes the transcription of NtPDF1.2 and NtJAZ1, but has little effect on the expression of NtPMT1a, quinolinate phosphoribosyltransferase 2 (NtQPT2), and NtMYC2a in tobacco. These results indicate that NtAIDP1 is a new component of the JA signalling pathway and is involved in JA-regulated gene expression.

scDetect: a rank-based ensemble learning algorithm for cell type identification of single-cell RNA sequencing in cancer.

MOTIVATION: Single-cell RNA sequencing (scRNA-seq) has enabled the characterization of different cell types in many tissues and tumor samples. Cell type identification is essential for single-cell RNA profiling, currently transforming the life sciences. Often, this is achieved by searching for combinations of genes that have previously been implicated as being cell-type specific, an approach that is not quantitative and does not explicitly take advantage of other scRNA-seq studies. Batch effects and different data platforms greatly decrease the predictive performance in inter-laboratory and different data type validation. RESULTS: Here, we present a new ensemble learning method named as 'scDetect' that combines gene expression rank-based analysis and a majority vote ensemble machine-learning probability-based prediction method capable of highly accurate classification of cells based on scRNA-seq data by different sequencing platforms. Because of tumor heterogeneity, in order to accurately predict tumor cells in the single-cell RNA-seq data, we have also incorporated cell copy number variation consensus clustering and epithelial score in the classification. We applied scDetect to scRNA-seq data from pancreatic tissue, mononuclear cells and tumor biopsies cells and show that scDetect classified individual cells with high accuracy and better than other publicly available tools. AVAILABILITY AND IMPLEMENTATION: scDetect is an open source software. Source code and test data is freely available from Github (https://github.com/IVDgenomicslab/scDetect/) and Zenodo (https://zenodo.org/record/4764132#.YKCOlrH5AYN). The examples and tutorial page is at https://ivdgenomicslab.github.io/scDetect-Introduction/. And scDetect will be available from Bioconductor. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Synthesis of cembratriene-ol and cembratriene-diol in yeast via the MVA pathway.

BACKGROUND: Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis. RESULTS: We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20. CONCLUSIONS: We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.

TOD-CUP: a gene expression rank-based majority vote algorithm for tissue origin diagnosis of cancers of unknown primary.

Gene expression profiling holds great potential as a new approach to histological diagnosis and precision medicine of cancers of unknown primary (CUP). Batch effects and different data types greatly decrease the predictive performance of biomarker-based algorithms, and few methods have been widely applied to identify tissue origin of CUP up to now. To address this problem and assist in more precise diagnosis, we have developed a gene expression rank-based majority vote algorithm for tissue origin diagnosis of CUP (TOD-CUP) of most common cancer types. Based on massive tissue-specific RNA-seq data sets (10 553) found in The Cancer Genome Atlas (TCGA), 538 feature genes (biomarkers) were selected based on their gene expression ranks and used to predict tissue types. The top scoring pairs (TSPs) classifier of the tumor type was optimized by the TCGA training samples. To test the prediction accuracy of our TOD-CUP algorithm, we analyzed (1) two microarray data sets (1029 Agilent and 2277 Affymetrix/Illumina chips) and found 91% and 94% prediction accuracy, respectively, (2) RNA-seq data from five cancer types derived from 141 public metastatic cancer tumor samples and achieved 94% accuracy and (3) a total of 25 clinical cancer samples (including 14 metastatic cancer samples) were able to classify 24/25 samples correctly (96.0% accuracy). Taken together, the TOD-CUP algorithm provides a powerful and robust means to accurately identify the tissue origin of 24 cancer types across different data platforms. To make the TOD-CUP algorithm easily accessible for clinical application, we established a Web-based server for tumor tissue origin diagnosis (http://ibi. zju.edu.cn/todcup/).

Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants.

Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species of Oryza and Echinochloa crus-galli (barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyte Calohypnum plumiforme (formerly Hypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase gene CpDTC1/HpDTC1, which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated the C. plumiforme genome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, the CpDTC1/HpDTC1 gene and the "dehydrogenase momilactone A synthase" gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution in Nicotiana benthamiana suggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (Oryza and Echinochloa) and C. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster in C. plumiforme is functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.

Genome-Wide Identification of the VQ Protein Gene Family of Tobacco (Nicotiana tabacum L.) and Analysis of Its Expression in Response to Phytohormones and Abiotic and Biotic Stresses.

VQ motif-containing proteins (VQ proteins) are transcriptional regulators that work independently or in combination with other transcription factors (TFs) to control plant growth and development and responses to biotic and abiotic stresses. VQ proteins contain a conserved FxxhVQxhTG amino acid motif that is the main element of its interaction with WRKY TFs. We identified 59 members of the tobacco (Nicotiana tabacum L.) NtVQ gene family by in silico analysis and examined their differential expression in response to phytohormonal treatments and following exposure to biotic and abiotic stressors. NtVQ proteins clustered into eight groups based upon their amino acid sequence and presence of various conserved domains. Groups II, IV, V, VI, and VIII contained the largest proportion of NtVQ gene family members differentially expressed in response to one or more phytohormone, and NtVQ proteins with similar domain structures had similar patterns of response to different phytohormones. NtVQ genes differentially expressed in response to temperature alterations and mechanical wounding were also identified. Over half of the NtVQ genes were significantly induced in response to Ralstonia solanacearum infection. This first comprehensive characterization of the NtVQ genes in tobacco lays the foundation for further studies of the NtVQ-mediated regulatory network in plant growth, developmental, and stress-related processes.

Shoot Regeneration Is Not a Single Cell Event.

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4-5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

A transcriptomic profile of topping responsive non-coding RNAs in tobacco roots (Nicotiana tabacum).

BACKGROUND: Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs), accomplish remarkable variety of biological functions. However, the composition of ncRNAs and their interactions with coding RNAs in modulating and controlling of cellular process in plants is largely unknown. Using a diverse group of high-throughput sequencing strategies, the mRNA, miRNA, lncRNA and circRNA compositions of tobacco (Nicotiana tabacum) roots determined and their alteration and potential biological functions in response to topping treatment analyzed. RESULTS: A total of 688 miRNAs, 7423 non-redundant lncRNAs and 12,414 circRNAs were identified, among which, some selected differentially expressed RNAs were verified by quantitative real-time PCR. Using the differentially expressed RNAs, a co-expression network was established that included all four types of RNAs. The number of circRNAs identified were higher than that of miRNAs and lncRNAs, but only two circRNAs were present in the co-expression network. LncRNAs appear to be the most active ncRNAs based on their numbers presented in the co-expression network, but none of them seems to be an eTM (endogenous Target Mimicry) of miRNAs. Integrated with analyses of sequence interaction, several mRNA-circRNA-miRNA interaction networks with a potential role in the regulation of nicotine biosynthesis were uncovered, including a QS-circQS-miR6024 interaction network. In this network miR6024 was significantly down-regulated, while the expression levels of its two targets, circQS and its host gene QS, were sharply increased following the topping treatment. CONCLUSIONS: These results illustrated the transcriptomic profiles of tobacco roots, the organ responsible for nicotine biosynthesis. mRNAs always play the most important roles, while ncRNAs are also expressed extensively for topping treatment response, especially circRNAs are the most activated in the ncRNA pool. These studies also provided insights on the coordinated regulation module of coding and non-coding RNAs in a single plant biological sample. The findings reported here indicate that ncRNAs appear to form interaction complex for the regulation of stress response forming regulation networks with transcripts involved in nicotine biosynthesis in tobacco.

Increased Leaf Nicotine Content by Targeting Transcription Factor Gene Expression in Commercial Flue-Cured Tobacco (Nicotiana tabacum L.).

Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.

Evidence for the Role of the Cecal Microbiome in Maintenance of Immune Regulation and Homeostasis.

OBJECTIVE (S): Our objective was to investigate alterations in the cecal microbial composition during the development of type 1 diabetes (T1D) with or without IgM therapy, and correlate these alterations with the corresponding immune profile. METHODS: (1) Female nonobese diabetic (NOD) mice treated with IgM or saline (n = 20/group) were divided into 5-week-old nondiabetic; 9 to 12-week-old prehyperglycemic stage-1; ≥13-week-old prehyperglycemic stage-2; and diabetic groups. 16S rRNA libraries were prepared from bacterial DNA and deep-sequenced. (2) New-onset diabetic mice were treated with IgM (200 µg on Days 1, 3, and 5) and their blood glucose monitored for 2 months. RESULTS: Significant dysbiosis was observed in the cecal microbiome with the progression of T1D development. The alteration in microbiome composition was characterized by an increase in the bacteroidetes:firmicutes ratio. In contrast, IgM conserved normal bacteroidetes:firmicutes ratio and this effect was long-lasting. Furthermore, oral gavage using cecal content from IgM-treated mice significantly diminished the incidence of diabetes compared with controls, indicating that IgM specifically affected mucosa-associated microbes, and that the affect was causal and not an epiphenomenon. Also, regulatory immune cell populations (myeloid-derived suppressor cells and regulatory T cells) were expanded and insulin autoantibody production diminished in the IgM-treated mice. In addition, IgM therapy reversed hyperglycemia in 70% of new-onset diabetic mice (n = 10) and the mice remained normoglycemic for the entire post-treatment observation period. CONCLUSIONS: The cecal microbiome appears to be important in maintaining immune homeostasis and normal immune responses.