Integrated analyses of ionomics, phytohormone profiles, transcriptomics, and metabolomics reveal a pivotal role of carbon-nano sol in promoting the growth of tobacco plants.

BACKGROUND: Carbon nano sol (CNS) can markedly affect the plant growth and development. However, few systematic analyses have been conducted on the underlying regulatory mechanisms in plants, including tobacco (Nicotiana tabacum L.). RESULTS: Integrated analyses of phenome, ionome, transcriptome, and metabolome were performed in this study to elucidate the physiological and molecular mechanisms underlying the CNS-promoting growth of tobacco plants. We found that 0.3% CNS, facilitating the shoot and root growth of tobacco plants, significantly increased shoot potassium concentrations. Antioxidant, metabolite, and phytohormone profiles showed that 0.3% CNS obviously reduced reactive oxygen species production and increased antioxidant enzyme activity and auxin accumulation. Comparative transcriptomics revealed that the GO and KEGG terms involving responses to oxidative stress, DNA binding, and photosynthesis were highly enriched in response to exogenous CNS application. Differential expression profiling showed that NtNPF7.3/NtNRT1.5, potentially involved in potassium/auxin transport, was significantly upregulated under the 0.3% CNS treatment. High-resolution metabolic fingerprints showed that 141 and 163 metabolites, some of which were proposed as growth regulators, were differentially accumulated in the roots and shoots under the 0.3% CNS treatment, respectively. CONCLUSIONS: Taken together, this study revealed the physiological and molecular mechanism underlying CNS-mediated growth promotion in tobacco plants, and these findings provide potential support for improving plant growth through the use of CNS.

PCMDB: a curated and comprehensive resource of plant cell markers.

The advent of single-cell sequencing opened a new era in transcriptomic and genomic research. To understand cell composition using single-cell studies, a variety of cell markers have been widely used to label individual cell types. However, the specific database of cell markers for use by the plant research community remains very limited. To overcome this problem, we developed the Plant Cell Marker DataBase (PCMDB, http://www.tobaccodb.org/pcmdb/), which is based on a uniform annotation pipeline. By manually curating over 130 000 research publications, we collected a total of 81 117 cell marker genes of 263 cell types in 22 tissues across six plant species. Tissue- and cell-specific expression patterns can be visualized using multiple tools: eFP Browser, Bar, and UMAP/TSNE graph. The PCMDB also supports several analysis tools, including SCSA and SingleR, which allows for user annotation of cell types. To provide information about plant species currently unsupported in PCMDB, potential marker genes for other plant species can be searched based on homology with the supported species. PCMDB is a user-friendly hierarchical platform that contains five built-in search engines. We believe PCMDB will constitute a useful resource for researchers working on cell type annotation and the prediction of the biological function of individual cells.

NRAMP6c plays a key role in plant cadmium accumulation and resistance in tobacco (Nicotiana tabacum L.).

Tobacco plants (Nicotiana tabacum L.) exhibit considerable potential for phytoremediation of soil cadmium (Cd) pollutants, owing to their substantial biomass and efficient metal accumulation capabilities. The reduction of Cd accumulation in tobacco holds promise for minimizing Cd intake in individuals exposed to cigar smoking. NRAMP transporters are pivotal in the processes of Cd accumulation and resistance in plants; however, limited research has explored the functions of NRAMPs in tobacco plants. In this investigation, we focused on NtNRAMP6c, one of the three homologs of NRAMP6 in tobacco. We observed a robust induction of NtNRAMP6c expression in response to both Cd toxicity and iron (Fe) deficiency, with the highest expression levels detected in the roots. Subsequent subcellular localization and heterologous expression analyses disclosed that NtNRAMP6c functions as a plasma membrane-localized Cd transporter. Moreover, its overexpression significantly heightened the sensitivity of yeast cells to Cd toxicity. Through CRISPR-Cas9-mediated knockout of NtNRAMP6c, we achieved a reduction in Cd accumulation and an enhancement in Cd resistance in tobacco plants. Comparative transcriptomic analysis unveiled substantial alterations in the transcriptional profiles of genes associated with metal ion transport, photosynthesis, and macromolecule catabolism upon NtNRAMP6c knockout. Furthermore, our study employed plant metabolomics and rhizosphere metagenomics to demonstrate that NtNRAMP6c knockout led to changes in phytohormone homeostasis, as well as shifts in the composition and abundance of microbial communities. These findings bear significant biological implications for the utilization of tobacco in phytoremediation strategies targeting Cd pollutants in contaminated soils, and concurrently, in mitigating Cd accumulation in tobacco production destined for cigar consumption.

Urinary exosomal prostate-specific antigen is a noninvasive biomarker to detect prostate cancer: Not only old wine in new bottles.

The study aimed at evaluating the performance of urinary exosomal prostate-specific antigen (UE-PSA) to predict the results of initial prostate biopsies and discriminate clinically significant prostate cancer (Gleason score ≥ 7, csPCa) from nonsignificant PCa (Gleason score < 7, nsPCa) plus benign patients. Two hundred seventy-two consecutive participants were admitted who underwent a prostate biopsy. The UE-PSA expression was detected by enzyme-linked immunosorbent assay (ELISA). The predictive power and clinical value of UE-PSA was assessed by receiver operating characteristic (ROC), decision curve analysis (DCA) and waterfall plots. UE-PSA was upregulated in PCa compared to benign patients (P < .001) and csPCa compared to nsPCa plus benign patients (P < .001). UE-PSA achieved an AUC of 0.953 (0.905-0.989) in distinguishing PCa from benign patients and an AUC of 0.879 (0.808-0.941) in predicting csPCa from nsPCa plus benign patients. These results were validated in an additional multicenter cohort. In addition, DCA showed that UE-PSA achieved the highest net benefit at almost any threshold probability compared to tPSA and %fPSA. As the waterfall plot showed, the UE-PSA assay could avoid 57.6% (155 cases) and 34.6% (93 cases) unnecessary biopsies while only missing 2.6% (7 cases) and 1.5% (4 cases) of the cases of csPCa at the cutoff value of 90% and 95% sensitivity, respectively. We validated that UE-PSA presented great diagnostic power and clinical utility to diagnose PCa and csPCa. UE-PSA could be a promising noninvasive biomarker to improve PCa detection.

Artificial intelligence for the diagnosis of clinically significant prostate cancer based on multimodal data: a multicenter study.

BACKGROUND: The introduction of multiparameter MRI and novel biomarkers has greatly improved the prediction of clinically significant prostate cancer (csPCa). However, decision-making regarding prostate biopsy and prebiopsy examinations is still difficult. We aimed to establish a quick and economic tool to improve the detection of csPCa based on routinely performed clinical examinations through an automated machine learning platform (AutoML). METHODS: This study included a multicenter retrospective cohort and two prospective cohorts with 4747 cases from 9 hospitals across China. The multimodal data, including demographics, clinical characteristics, laboratory tests, and ultrasound reports, of consecutive participants were retrieved using extract-transform-load tools. AutoML was applied to explore potential data processing patterns and the most suitable algorithm to build the Prostate Cancer Artificial Intelligence Diagnostic System (PCAIDS). The diagnostic performance was determined by the receiver operating characteristic curve (ROC) for discriminating csPCa from insignificant prostate cancer (PCa) and benign disease. The clinical utility was evaluated by decision curve analysis (DCA) and waterfall plots. RESULTS: The random forest algorithm was applied in the feature selection, and the AutoML algorithm was applied for model establishment. The area under the curve (AUC) value in identifying csPCa was 0.853 in the training cohort, 0.820 in the validation cohort, 0.807 in the Changhai prospective cohort, and 0.850 in the Zhongda prospective cohort. DCA showed that the PCAIDS was superior to PSA or fPSA/tPSA for diagnosing csPCa with a higher net benefit for all threshold probabilities in all cohorts. Setting a fixed sensitivity of 95%, a total of 32.2%, 17.6%, and 26.3% of unnecessary biopsies could be avoided with less than 5% of csPCa missed in the validation cohort, Changhai and Zhongda prospective cohorts, respectively. CONCLUSIONS: The PCAIDS was an effective tool to inform decision-making regarding the need for prostate biopsy and prebiopsy examinations such as mpMRI. Further prospective and international studies are warranted to validate the findings of this study. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100048428. Registered on 06 July 2021.

Metagenomic insights into the changes in the rhizosphere microbial community caused by the root-knot nematode Meloidogyne incognita in tobacco.

Root-knot nematode (RKN) disease is a destructive soil disease that affects crop health and causes huge losses in crop production. To explore the relationships between soil environments, rhizobacterial communities, and plant health, rhizosphere bacterial communities were analyzed using metagenomic sequencing in tobacco samples with different grades of RKN disease. The results showed that the community structure and function of the plant rhizosphere were significantly correlated to the RKN disease. RKN density and urease content were key factors affecting the rhizosphere bacterial community. Urease accelerated the catabolism of urea and led to the production of high concentrations of ammonia, which directly suppressed the development of RKNs or by improving the nutritional and growth status of microorganisms that were antagonistic to RKNs. Further experiments showed that the suppression role of ammonia should be attributed to the direct inhibition of NH3. The bacterial members that were positively correlated with RKN density, contained many plant cell wall degrading enzymes, which might destroy plant cell walls and promote the colonization of RKN in tobacco roots. The analysis of metatranscriptome and metabolism demonstrated the role of these cell wall degrading enzymes. This study offers a comprehensive insight into the relationships between RKNs, bacteria, and soil environmental factors and provides new ideas for the biological control of RKNs.

PLncDB V2.0: a comprehensive encyclopedia of plant long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with little or no protein coding potential. The expanding list of lncRNAs and accumulating evidence of their functions in plants have necessitated the creation of a comprehensive database for lncRNA research. However, currently available plant lncRNA databases have some deficiencies, including the lack of lncRNA data from some model plants, uneven annotation standards, a lack of visualization for expression patterns, and the absence of epigenetic information. To overcome these problems, we upgraded our Plant Long noncoding RNA Database (PLncDB, http://plncdb.tobaccodb.org/), which was based on a uniform annotation pipeline. PLncDB V2.0 currently contains 1 246 372 lncRNAs for 80 plant species based on 13 834 RNA-Seq datasets, integrating lncRNA information from four other resources including EVLncRNAs, RNAcentral and etc. Expression patterns and epigenetic signals can be visualized using multiple tools (JBrowse, eFP Browser and EPexplorer). Targets and regulatory networks for lncRNAs are also provided for function exploration. In addition, PLncDB V2.0 is hierarchical and user-friendly and has five built-in search engines. We believe PLncDB V2.0 is useful for the plant lncRNA community and data mining studies and provides a comprehensive resource for data-driven lncRNA research in plants.

Metagenomic insight into the microbial degradation of organic compounds in fermented plant leaves.

Microbial degradation of organic compounds is an environmentally benign and energy efficient part in product processing. Fermentation of plant leaves involves enzymatic actions of many microorganisms. However, microbes and enzymes discovered from natural degradation communities were still limited by cultural methods. In this study, we used a metagenomics sequence-guided strategy to identify the microbes and enzymes involved in compound degradation and explore the potential synergy among community members in fermented tobacco leaves. The results showed that contents of protein, starch, pectin, lignin, and cellulose varied in fermented leaves from different growing sites. The different compound contents were closely related to taxonomic composition and functional profiles of foliar microbial communities. Microbial communities showed significant correlations with protein, lignin, and cellulose. Vital species for degradations of protein (Bacillus cereus and Terribacillus aidingensis), lignin (Klebsiella pneumoniae and Pantoea ananatis) and cellulose (Pseudomonas putida and Sphingomonas sp. Leaf20) were identified and relating hydrolytic enzymes were annotated. Further, twenty-two metagenome-assembled genomes (MAGs) were assembled from metagenomes and six potential cellulolytic genomes were used to reconstruct the cellulose-degrading process, revealing the potential metabolic cooperation related to cellulose degradation. Our work should deepen the understanding of microbial roles in plant fermentation and provide a new viewpoint for applying microbial consortia to convert plant organic components to small molecules.

At The Forefront of Penile Surgical Reconstruction: A Bibliometric Study of the 100 Most-Cited Articles.

PURPOSE: The citation count of a scientific article is considered as the recognition it received from this field. The purpose of this bibliometric analysis was to identify the top 100 most-cited scientific articles in penile surgical reconstruction. METHODS: The Web of Science database was used to extract the top 100 most-cited articles. Individual articles were reviewed to identify the authorship, published journal, journal impact factor (IF), primary disease, article type, institution and country of origin, and year of publication. RESULTS: The top 100 most-cited articles were published between 1947 and 2013. The number of citations ranged from 23 to 233. Journal of Urology contributed the most articles (n = 36). Articles with a high level of evidence like prospective analysis (n = 5), systematic review and meta-analysis (n = 2), and guideline (n = 1) were all published after 2000. The average citation per year of articles published in high-IF journals was significantly higher than that of other articles (p = 0.0129). There was a positive linear correlation between citation count per year and publication year (r2 = 0.26, p < 0.001). Among the top 100 articles, 74 articles were interlinked via citation of each other. The major topic of co-citation network was the application of flaps in penile reconstruction. CONCLUSIONS: The analysis of top 100 most-cited articles facilitates the comprehensive recognition of current focus in the field of penile surgical reconstruction, which is the exploration of flaps and development of new techniques in penile reconstruction. In the future, more attention should be paid to evidence-based medicine to provide high-level evidence for research. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Circulating exosomal mRNA profiling identifies novel signatures for the detection of prostate cancer.

The landscape and characteristics of circulating exosomal messenger RNAs (emRNAs) are poorly understood, which hampered the accurate detection of circulating emRNAs. Through comparing RNA sequencing data of circulating exosomes with the corresponding data in tissues, we illustrated the different characteristics of emRNAs compared to tissue mRNAs. We then developed an improved strategy for emRNA detection based on the features of circulating emRNAs. Using the optimized detection strategy, we further validated prostate cancer (PCa) associated emRNAs discovered by emRNA-seq in a large cohort of patients and identified emRNA signatures for PCa screening and diagnosis using logistic regression analysis. The receiver operating characteristic curve (ROC) analysis showed that the circulating emRNA-based screening signature yielded an area under the ROC curve (AUC) of 0.948 in distinguishing PCa patients from healthy controls. The circulating emRNA-based diagnostic signature also showed a great performance in predicting prostate biopsy results (AUC: 0.851). In conclusion, our study developed an optimized emRNA detection strategy and identified novel emRNA signatures for the detection of PCa.

Performance of SOFA, qSOFA and SIRS to predict septic shock after percutaneous nephrolithotomy.

OBJECTIVE: The new clinical criteria termed SOFA and qSOFA were demonstrated to be more accurate than SIRS in screening patients at high risk of sepsis. We aim to evaluate the ability of SOFA, qSOFA and SIRS to predict septic shock after PCNL. PATIENTS AND METHODS: Consecutive patients undergoing PCNL were included to assess the performance of SOFA, qSOFA and SIRS in predicting septic shock, the AUC of ROC curve and decision curve analysis were used, and the optimal cutoff values and their achieving time were calculated. RESULTS: Of the 431 included patients, 12 (2.7%) cases developed septic shock. Compared with non-septic shock patients, patients with septic shock were more likely to be female, have positive history of urine culture and higher urine leukocyte count, and show increased postoperative serum creatinine, PCT and decreased leukocyte. The optimal cutoff of SOFA, qSOFA and SIRS was > 2, > 0 and > 1, respectively. All of the 12 patients with verified septic shock met SOFA and SIRS criteria, while only 11 cases met qSOFA criterion. SOFA had the identical highest sensitivity (100%) and greater specificity (87% vs. 81%) than SIRS. qSOFA had higher specificity (92%) than both SOFA and SIRS at the expense of lower sensitivity (92%). The AUC of SOFA (0.973) to predict septic shock was greater than that of qSOFA (0.928) and SIRS (0.935). When combined with SIRS, SOFA outperformed qSOFA for discrimination of septic shock (AUC 0.987 vs. 0.978). Decision curve analysis indicated SOFA was clearly superior to both qSOFA and SIRS with a higher net benefit and net reduction in intervention. The qSOFA achieved the best time-based predictive efficiency, with the shortest median time to meet its cutoff, followed by SOFA and SIRS. CONCLUSION: The performance of SOFA in predicting septic shock after PCNL was slightly greater than qSOFA and SIRS. The comprehensive application of various criteria is recommended to assist early detection of septic shock following PCNL.

Prostate Cancer Diagnosis Using Urine Sediment Analysis-Based α-Methylacyl-CoA Racemase Score: A Single-Center Experience.

To evaluate the diagnostic value of α-methylacyl-CoA racemase (AMACR) score in Han Chinese patients with prostate cancer (PCa) through urine sediment analysis. We collected 292 urine sediment samples after digital rectal examination. Levels of AMACR and prostate-specific antigen (PSA) messenger RNA (mRNAs) were evaluated by quantitative real time-polymerase chain reaction. The diagnostic value of AMACR score was assessed by receiver-operating characteristic analysis (ROC), Mann-Whitney test, logistic regression analysis and decision curve analysis. In all patients (n = 292), the area under the curve (AUC) for serum PSA, AMACR score, and a combinative model of these 2 parameters were 0.745 (95% confidence interval [CI]: 0.691-0.794), 0.753 (95% CI: 0.700-0.802), and 0.784 (95% CI: 0.732-0.830). No statistical difference was found between AMACR score and serum PSA (P = .826), while the combinative model was better than AMACR score (Z = 5.222, P < .001). Among patients with serum PSA level of 4 to 10 ng/mL (n = 121), the AMACR score was significantly higher in patients with PCa (P = 0.0002), while serum PSA showed no difference (P = 0.3023). Alpha-methylacyl-CoA racemase score (AUC = 0.712, 95% CI: 0.623-0.790) and a combinative model (AUC = 0.714, 95% CI: 0.626-0.793) showed a better diagnostic value than serum PSA (AUC = 0.559, 95% CI: 0.466-0.649), (P = .048, P = .042). Decision curve analysis showed a biopsy prediction model including AMACR score have a better net benefit when the threshold probability greater than 20%. The diagnostic model combing serum PSA and AMACR score has a better diagnostic value in patients with abnormal PSA level (including PSA level ranging from 4-10 ng/mL), and could reduce unnecessary prostate biopsy in clinical use.

Detection and characterization of a novel hepacivirus in long-tailed ground squirrels (Spermophilus undulatus) in China.

Rodent populations are known to be reservoirs of viruses with the potential to infect humans. However, a large number of such viruses remain undiscovered. In this study, we investigated the shedding of unknown viruses in long-tailed ground squirrel (Spermophilus undulatus) feces by high-throughput sequencing. A novel and highly divergent virus related to members of the genus Hepacivirus was identified in ground squirrel liver. This virus, tentatively named RHV-GS2015, was found to have a genome organization that is typical of hepaciviruses, including a long open reading frame encoding a polyprotein of 2763 aa. Sequence alignment of RHV-GS2015 with the most closely related hepaciviruses yielded p-distances of the NS3 and NS5B regions of 0.546 and 0.476, respectively, supporting the conclusion that RHV-GS2015 is a member of a new hepacivirus species, which we propose to be named "Hepacivirus P". Phylogenetic analysis of the NS3 and NS5B regions indicated that RHV-GS2015 shares common ancestry with other rodent hepaciviruses (species Hepacivirus E, and species Hepacivirus F), Norway rat hepacivirus 1 (species Hepacivirus G), and Norway rat hepacivirus 2 (species Hepacivirus H). A phylogenetic tree including the seven previously identified rodent hepaciviruses revealed extreme genetic heterogeneity among these viruses. RHV-GS2015 was detected in 7 out of 12 ground squirrel pools and was present in liver, lung, and spleen tissues. Furthermore, livers showed extremely high viral loads of RHV-GS2015, ranging from 2.5 × 106 to 2.0 × 108 copies/g. It is reasonable to assume that this novel virus is hepatotropic, like hepatitis C virus. The discovery of RHV-GS2015 extends our knowledge of the genetic diversity and host range of hepaciviruses, helping to elucidate their origins and evolution.

Integrated transcriptomics and metabolomics analysis to characterize cold stress responses in Nicotiana tabacum.

BACKGROUND: CB-1 and K326 are closely related tobacco cultivars; however, their cold tolerance capacities are different. K326 is much more cold tolerant than CB-1. RESULTS: We studied the transcriptomes and metabolomes of CB-1 and K326 leaf samples treated with cold stress. Totally, we have identified 14,590 differentially expressed genes (DEGs) in CB-1 and 14,605 DEGs in K326; there was also 200 differentially expressed metabolites in CB-1 and 194 in K326. Moreover, there were many overlapping genes (around 50%) that were cold-responsive in both plant cultivars, although there were also many differences in the cold responsive genes between the two cultivars. Importantly, for most of the overlapping cold responsive genes, the extent of the changes in expression were typically much more pronounced in K326 than in CB-1, which may help explain the superior cold tolerance of K326. Similar results were found in the metabolome analysis, particularly with the analysis of primary metabolites, including amino acids, organic acids, and sugars. The large number of specific responsive genes and metabolites highlight the complex regulatory mechanisms associated with cold stress in tobacco. In addition, our work implies that the energy metabolism and hormones may function distinctly between CB-1 and K326. CONCLUSIONS: Differences in gene expression and metabolite levels following cold stress treatment seem likely to have contributed to the observed difference in the cold tolerance phenotype of these two tobacco cultivars.

Analysis of the sucrose synthase gene family in tobacco: structure, phylogeny, and expression patterns.

MAIN CONCLUSION: Provide an evolutionary and an empirical molecular genetic foundation of the Sus gene family in tobacco and will be beneficial for further investigations of Sus gene functions Sucrose synthase (Sus) has been well characterized as the key enzyme participating in sucrose metabolism, and the gene family encoding different Sus isozymes has been cloned and characterized in several plant species. However, scant information about this gene family is available to date in tobacco. Here, we identified 14, 6, and 7 Sus genes in the genomes of Nicotiana tabacum, N. sylvestris and N. tomentosiformis, respectively. These tobacco Sus family members shared high levels of similarity in their nucleotide and amino acid sequences. Phylogenetic analysis revealed distinct evolutionary paths for the tobacco Sus genes. Sus1-4, Sus5, and Sus6-7 originated from three Sus precursors, respectively, which were generated by duplication before the split of monocots and eudicots. There were two additional duplications, before and after the differentiation of the Solanaceae, which separately gave rise to Sus3/4 and Sus1/2. Gene exon/intron structure analysis showed that the tobacco Sus genes contain varying numbers of conserved introns, resulting from intron loss under different selection pressures during the course of evolution. The expression patterns of the NtSus genes differed from each other in various tobacco tissues. Transcripts of Ntab0259170 and Ntab0259180 were detected in leaves at all tested developmental stages, suggesting that these two genes play a predominant role in sucrose metabolism during leaf development. Expression of Ntab0288750 and Ntab0234340 were conspicuously induced by low temperature and virus treatment, indicating that these two isozymes are important in meeting the increased glycolytic demand that occurs during abiotic stress. Our results provide an evolutionary and an empirical molecular genetic foundation of the Sus gene family in tobacco, and will be beneficial for further investigations of Sus gene functions in the processes of tobacco leaf development and tobacco resistance to environmental stresses.

A chromosome-level haplotype-resolved genome assembly of oriental tobacco budworm (Helicoverpa assulta).

Oriental tobacco budworm (Helicoverpa assulta) and cotton bollworm (Helicoverpa armigera) are two closely related species within the genus Helicoverpa. They have similar appearances and consistent damage patterns, often leading to confusion. However, the cotton bollworm is a typical polyphagous insect, while the oriental tobacco budworm belongs to the oligophagous insects. In this study, we used Nanopore, PacBio, and Illumina platforms to sequence the genome of H. assulta and used Hifiasm to create a haplotype-resolved draft genome. The Hi-C technique helped anchor 33 primary contigs to 32 chromosomes, including two sex chromosomes, Z and W. The final primary haploid genome assembly was approximately 415.19 Mb in length. BUSCO analysis revealed a high degree of completeness, with 99.0% gene coverage in this genome assembly. The repeat sequences constituted 38.39% of the genome assembly, and we annotated 17093 protein-coding genes. The high-quality genome assembly of the oriental tobacco budworm serves as a valuable genetic resource that enhances our comprehension of how they select hosts in a complex odour environment. It will also aid in developing an effective control policy.

Investigating nicotine pathway-related long non-coding RNAs in tobacco.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with low or no protein-coding ability, which play essential roles in various biological processes in plants. Tobacco is an ideal model plant for studying nicotine biosynthesis and metabolism, and there is little research on lncRNAs in this field. Therefore, how to take advantage of the mature tobacco system to profoundly investigate the lncRNAs involved in the nicotine pathway is intriguing. By exploiting 549 public RNA-Seq datasets of tobacco, 30,212 lncRNA candidates were identified, including 24,084 large intervening non-coding RNAs (lincRNAs), 5,778 natural antisense transcripts (NATs) and 350 intronic non-coding RNAs (incRNAs). Compared with protein-coding genes, lncRNAs have distinct properties in terms of exon number, sequence length, A/U content, and tissue-specific expression pattern. lincRNAs showed an asymmetric evolutionary pattern, with a higher proportion (68.71%) expressed from the Nicotiana sylvestris (S) subgenome. We predicted the potential cis/trans-regulatory effects on protein-coding genes. One hundred four lncRNAs were detected as precursors of 30 known microRNA (miRNA) family members, and 110 lncRNAs were expected to be the potential endogenous target mimics for 39 miRNAs. By combining the results of weighted gene co-expression network analysis with the differentially expressed gene analysis of topping RNA-seq data, we constructed a sub-network containing eight lncRNAs and 25 nicotine-related coding genes. We confirmed that the expression of seven lncRNAs could be affected by MeJA treatment and may be controlled by the transcription factor NtMYC2 using a quantitative PCR assay and gene editing. The results suggested that lncRNAs are involved in the nicotine pathway. Our findings further deepened the understanding of the features and functions of lncRNAs and provided new candidates for regulating nicotine biosynthesis in tobacco.

Comparing pretreatment strategies to increase the yield and purity of human urinary extracellular vesicles.

Extracellular vesicles (EVs) are membranous vesicles released by various cells, and are involved in intercellular communication and disease progression. EVs that are isolated from urine are good indicators of urinary system diseases and help certain urological studies. During isolation of urine EVs, Dithiothreitol (DTT) is widely used to reduce the contamination of the major contaminant Tamm-Horsefall protein (THP),which is the most abundant protein in the human urine and the most difficult contaminant to remove in the isolation of urine EVs. Unfortunately, DTT can interfere with subsequent analysis due to its strong reducing ability and cannot completely remove THP. To optimize the urine EV isolation strategy, we compared two pretreatment protocols: incubating urine with NaCl and DTT before centrifugation. After a series of analyses by nanoparticle tracking analysis (NTA), western blotting (WB), and transmission electron microscopy (TEM), we found that NaCl removed more THP than DTT in a low-speed centrifugation step and that the residual EVs also had lower THP contamination post NaCl treatment. Remarkably, the yield of EVs obtained via the salting-out method was significantly higher than those obtained by the other methods (P = 0.001). Our study is the first to demonstrate that the salting-out method is better than the traditional DTT method in terms of efficiency in removing THP and EV yields.

Comprehensive Analysis of Long Non-coding RNA Modulates Axillary Bud Development in Tobacco (Nicotiana tabacum L.).

Long non-coding RNAs (lncRNAs) regulate gene expression and are crucial for plant growth and development. However, the mechanisms underlying the effects of activated lncRNAs on axillary bud development remain largely unknown. By lncRNA transcriptomes of axillary buds in topped and untopped tobacco plants, we identified a total of 13,694 lncRNAs. LncRNA analysis indicated that the promoted growth of axillary bud by topping might be partially ascribed to the genes related to hormone signal transduction and glycometabolism, trans-regulated by differentially expressed lncRNAs, such as MSTRG.52498.1, MSTRG.60026.1, MSTRG.17770.1, and MSTRG.32431.1. Metabolite profiling indicated that auxin, abscisic acid and gibberellin were decreased in axillary buds of topped tobacco lines, while cytokinin was increased, consistent with the expression levels of related lncRNAs. MSTRG.52498.1, MSTRG.60026.1, MSTRG.17770.1, and MSTRG.32431.1 were shown to be influenced by hormones and sucrose treatments, and were associated with changes of axillary bud growth in the overexpression of NtCCD8 plants (with reduced axillary buds) and RNA interference of NtTB1 plants (with increased axillary buds). Moreover, MSTRG.28151.1 was identified as the antisense lncRNA of NtTB1. Silencing of MSTRG.28151.1 in tobacco significantly attenuated the expression of NtTB1 and resulted in larger axillary buds, suggesting the vital function of MSTRG.28151.1 axillary bud developmen by NtTB1. Our findings shed light on lncRNA-mRNA interactions and their functional roles in axillary bud growth, which would improve our understanding of lncRNAs as important regulators of axillary bud development and plant architecture.

Identification of a novel norovirus species in fox.

A novel Norovirus (NoV) was identified by viral metagenomic analysis in fox fecal samples from the Xinjiang Uygur Autonomous Region of China. The virus exhibited typical genomic characteristics of NoVs. It was closely related to the canine NoV GVII strains with 86.0-86.2% and 91.9% amino acid identities in the capsid protein VP1 and RNA-dependent RNA polymerase (RdRp), respectively. The fox NoV clustered phylogenetically with the two canine NoV GVII strains, and it was distant from other NoVs. According to the new classification criteria of NoVs, the new fox NoV belongs to the same genotype as GVII, similar to canine GVII NoVs. Moreover, key amino acid residues in the Histo-blood group antigen (HBGA) binding sites and the HBGA binding pattern of the fox NoV differed significantly from those of human and canine GVII NoVs. This study identified a new GVII norovirus from wild foxes in China. These findings enrich our understanding of the diversity of NoVs and provide further evidence regarding the genetic heterogeneity of NoVs in carnivores.