Analysis of herbivore-responsive long noncoding ribonucleic acids reveals a subset of small peptide-coding transcripts in Nicotiana tabacum.

Long non-coding RNAs (lncRNAs) regulate many biological processes in plants, including defense against pathogens and herbivores. Recently, many small ORFs embedded in lncRNAs have been identified to encode biologically functional peptides (small ORF-encoded peptides [SEPs]) in many species. However, it is unknown whether lncRNAs mediate defense against herbivore attack and whether there are novel functional SEPs for these lncRNAs. By sequencing Spodoptera litura-treated leaves at six time-points in Nicotiana tabacum, 22,436 lncRNAs were identified, of which 787 were differentially expressed. Using a comprehensive mass spectrometry (MS) pipeline, 302 novel SEPs derived from 115 tobacco lncRNAs were identified. Moreover, 61 SEPs showed differential expression after S. litura attack. Importantly, several of these peptides were characterized through 3D structure prediction, subcellular localization validation by laser confocal microscopy, and western blotting. Subsequent bioinformatic analysis revealed some specific chemical and physical properties of these novel SEPs, which probably represent the largest number of SEPs identified in plants to date. Our study not only identifies potential lncRNA regulators of plant response to herbivore attack but also serves as a valuable resource for the functional characterization of SEP-encoding lncRNAs.

Haploid induction in allotetraploid tobacco using DMPs mutation.

MAIN CONCLUSION: dmp1dmp2dmp3 mutants created by CRISPR/Cas9 could trigger maternal haploids in the allotetraploid model plant Nicotiana tabacum L. Double haploid (DH) technology is becoming increasingly important because it can significantly accelerate the breeding process. Haploid induction plays a fundamental role in the production of DH lines. Haploid induction has been realized and applied in diploid plants using DMP genes. However, it has yet to be elucidated whether haploid induction could be established in polyploid plants. In the current study, three homologues of the DMP genes (NtDMP1, 2, and 3) were identified in the allotetraploid plant Nicotiana tabacum, and the encoded proteins localized in the endoplasmic reticulum. Loss-of-function mutations in all three genes triggered maternal haploids with an induction rate of 1.52-1.75%. Compared with wild-type tobacco, the created haploid inducer exhibited differences in pollen vigor and seed germination rate. Furthermore, to rapidly and easily screen haploids, a visible haploid identification system was established based on a powdery mildew resistance phenotype. Findings from this study lay the foundation for the potential application of haploid inducers in allotetraploid plants such as tobacco.

Types of Interferons and Their Expression in Plant Systems.

Interferons (IFNs) are divided into 3 types (type I, type II, and type III) on the basis of sequence homology and functional properties. Recombinant IFNs have been approved by regulatory agencies in many countries for clinical treatment of hepatitis B, hepatitis C, and other diseases; these IFNs are mainly produced in microorganisms and mammalian cell systems. However, there are serious obstacles to the production of recombinant IFNs in microorganism systems; for example, the recombinant IFN may have different glycosylation patterns from the native protein, be present in insoluble inclusion bodies, be contaminated with impurities such as endotoxins and nucleic acids, have a short half-life in human blood, and incur high production costs. Some medicinal proteins have been successfully expressed in plants and used in clinical applications, suggesting that plants may also be a good system for IFN expression. However, there are still many technical problems that need to be addressed before the clinical application of plant-expressed IFNs, such as increasing the amount of recombinant protein expression and ensuring that the IFN is modified with the correct type of glycosylation. In this article, we review the classification of IFNs, their roles in antiviral signal transduction pathways, their clinical applications, and their expression in plant systems.

Molecular Rewiring of the Jasmonate Signaling Pathway to Control Auxin-Responsive Gene Expression.

The plant hormone jasmonic acid (JA) has an important role in many aspects of plant defense response and developmental process. JA triggers interaction between the F-box protein COI1 and the transcriptional repressors of the JAZ family that leads the later to proteasomal degradation. The Jas-motif of JAZs is critical for mediating the COI1 and JAZs interaction in the presence of JA. Here, by using the protoplast transient gene expression system we reported that the Jas-motif of JAZ1 was necessary and sufficient to target a foreign reporter protein for COI1-facilitated degradation. We fused the Jas-motif to the SHY2 transcriptional repressor of auxin signaling pathway to create a chimeric protein JaSHY. Interestingly, JaSHY retained the transcriptional repressor function while become degradable by the JA coreceptor COI1 in a JA-dependent fashion. Moreover, the JA-induced and COI1-facilitated degradation of JaSHY led to activation of a synthetic auxin-responsive promoter activity. These results showed that the modular components of JA signal transduction pathway can be artificially redirected to regulate auxin signaling pathway and control auxin-responsive gene expression. Our work provides a general strategy for using synthetic biology approaches to explore and design cell signaling networks to generate new cellular functions in plant systems.

Highly efficient production of diverse rare ginsenosides using combinatorial biotechnology.

The rare ginsenosides are recognized as the functionalized molecules after the oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by methyl jasmonate-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six ß-glycosidases and their combination with yields ranging from 5.54 to 32.66 mg L-1 . The yield of Rh2 was furthermore increased by 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature conditions, with the highest yield reaching 51.17 mg L-1 (17.06% of protopanaxadiol-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.

miR­338­3p inhibits A549 lung cancer cell proliferation and invasion by targeting AKT and ß­catenin signaling pathways.

The aim of the present study was to explore the underlying mechanism of microRNA­338­3p (miR­338­3p) in lung cancer cell (A549) invasion and proliferation. A microarray assay showed that miR­338­3p upregulated 216 and downregulated 147 genes in A549 cells, and the differentially expressed genes were enriched for several signaling pathways, including AKT and ß­catenin signaling pathways. Western blotting results showed that phosphorylated (p)­AKT at Ser473 and at Thr308 and p­ß­catenin at Ser552 were downregulated. Inhibiting AKT signaling pathway decreased ß­catenin signaling pathway. Overexpression of ß­catenin promoted the invasion of A549 cells. In addition, miR­338­3p showed inhibitory effect on the primary tumor developed from KrasG12D mice. Together, the data of the present study support that miR­338­3p inhibits lung cancer cell invasion and proliferation by downregulating AKT/ß­catenin signaling pathway. The present findings supported the potential use of miR­338­3p in the treatment of lung cancer.

[Development and application of monoclonal antibodies against human free prostate specific antigen(f-PSA)].

Objective Construction of monoclonal antibody cell lines against human free prostate specific antigen (f-PSA), and establish double antibody sandwich CLIA for human f-PSA. Methods Two hybridoma cell lines secreting anti-f-PSA mAb were obtained by hybridoma technique. The cell lines were expanded by spinner bottles and purified by affinity purification, from which the antibodies were tested for antibody titers, specificity, epitopes by ELISA and affinity by surface plasmon resonance. Establishment, analysis and performance evaluation of a double mAbs sandwich CLIA system using standard curve quantitative method. In total 426 (130 gray area) clinical specimens were used for system comparison between our assay and Roche f-PSA assay. Results anti- f-PSA(f-10-1, f-14-1) mAbs were obtained. The CLIA system with detection range of 0.1-30 ng/mL, sensitivity of 0.05 ng/mL, relative detection deviation of ±5% and not cross-react with the tumor marker AFP, CEA and c-PSA. The correlation coefficient of our reagent with Roche's was 0.99. The positive coincidence rate, negative coincidence rate and total coincidence rate of gray area specimens were all higher than 90%. Conclusion mAbs against human f-PSA were successfully prepared, and the double mAbs sandwich CLIA for specific quantitative detection of human f-PSA was established.

MiR-338-3p inhibits TNF-α-induced lipogenesis in human sebocytes.

OBJECTIVE: To suppress TNF-α-induced lipogenesis in sebocytes (associated with acne development) with microRNA-338-3p (miR-338-3p) and to explore the underlying mechanisms. RESULTS: TNF-α increased lipid droplet formation in sebocytes which were used as in vitro model of inflammation-induced acne. Flow cytometry and TLC assays validated that miR-338-3p could suppress TNF-α-induced lipid droplet formation, down-regulate the expression of PREX2a, and inactivate AKT signaling in sebocytes. In addition, suppression of AKT activity by the PI3 K and AKT inhibitors diminished TNF-α-induced lipogenesis. PREX2a siRNA mimics the effects of miR-338-3p on AKT phosphorylation and lipogenesis. PREX2a overexpression consistently restored lipogenesis and AKT phosphorylation attenuated by miR-338-3p. CONCLUSIONS: MiR-338-3p suppresses the TNF-α-induced lipogenesis in sebocytes by targeting PREX2a and down-regulating PI3K/AKT signaling.

[Preparation and characterization of the monoclonal antibodies against human CA19-9 and establishment of a sandwich CLIA system].

OBJECTIVE: To prepare the monoclonal antibodies (mAbs) against human carbohydrate antigen 19-9 (CA19-9) and establish a double-antibody sandwich chemiluminescent immunoassay (CLIA) detection system for CA19-9 in the human serum. METHODS: BALB/c mice were immunized with human CA19-9 antigen. The mAbs were obtained by hybridoma technique. The purity, titer, specificity and pairing of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity and repetitiveness after optimization of coating buffer, coating concentration and pipetting mode, and the serum sample was tested with it. RESULTS: Four mAbs named #1-1, #2-1, #3-1 and #4-1 were obtained against human CA19-9. The titers of the anti-CA19-9 mAbs were above 10(-8). The mAbs had nearly no cross-reaction with CA125, CA15-3 and CA72-4. The double-antibody sandwich CLIA system was established by #3-1 mAb and #2-1 mAb-HRP. After optimization, its property was detection range of 0-1 000 U/mL, limit detection of 2.0 U/mL, linear correlation coefficient of 0.9999. The results which were contrasted with Roche test showed that: Kappa>0.75, r(correlation coefficient)>0.9. CONCLUSION: The mAbs against human CA9-9 have been prepared and a sandwich CLIA system for detecting CA19-9 in the human serum has been established successfully.

[Preparation and characterization of the monoclonal antibodies against human CA15-3 and establishment of a sandwich CLIA system].

OBJECTIVE: To prepare the monoclonal antibodies (mAbs) against human carbohydrate antigen 15-3 (CA15-3) and establish a double-antibody sandwich chemiluminescent immunoassay (CLIA) system for detecting CA15-3 in the human serum. METHODS: BALB/c mice were immunized with human CA15-3 antigen. Spleen cells of the immunized mice were fused with Sp2/0 cells and the positive hybridoma cells were selected and subcloned. The supernatant was taken for purify mAbs using protein A chromatography. The purity, titer, epitope and subtype of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity, repetitiveness and specificity. RESULTS: Five hybridoma cell lines named #3-1-3, #5-2-2, #11-2-2, #12-1-3 and #16-1-3 were obtained respectively, which are of strong positive signal and high secretion. The titers of the mAbs secreted by these hydridomas were above 10(-8); g/mL. All of the mAbs expressed κ light chains, and their heavy chains were as follows: #3-1-3 mAb had IgG2a, #5-2-2 mAb and #12-1-3 mAb had IgG2b, #11-2-2 mAb and #16-1-3 mAb had IgG3. The double-antibody sandwich CLIA system had a good linear relationship between 0.59 U/mL and 300 U/mL. The recovery rate for accuracy was 97.45% and the limit of detection was 0.59 U/mL. The assay was highly linear (correlation coefficient 0.9978) and highly repeatable [coefficient of variation (CV) <10%]. This CLIA system was also highly specific without cross-reactivity to the tumor marker AFP, CEA, CA50, CA19-9 and CA72-4. CONCLUSION: The mAbs against human CA15-3 have been prepared and a sandwich CLIA system for detecting CA15-3 in the human serum has been established successfully, which provides a basis for CA15-3 quantification and clinical application.