Systematic Analysis and Identification of Dysregulated Panel lncRNAs Contributing to Poor Prognosis in Head-Neck Cancer

Head and neck cancer (HNC) is one of the most prevalent cancers worldwide, accounting for approximately 5% of all cancers. While the underlying molecules and their pathogenetic mechanisms in HNC have yet to be well elucidated, recent studies have shown that dysregulation of lncRNAs may disrupt the homeostasis of various biological pathways. However, the understanding of lncRNAs in HNC is still limited by the lack of expression profiling. In the present study, we employed a systematic strategy to identify a panel of lncRNA associated with HNC. A cancer-related lncRNA profile PCR array was screened to explore potential molecules specific for HNC. A total of 55 lncRNAs were found to be dysregulated in HNC cells when compared to normal keratinocytes. Further analysis of the prognostic significance using The Cancer Genome Atlas (TCGA) database revealed 15 lncRNAs highly correlated with overall survival in HNC patients. Additionally, clinical sample expression analysis of the TCGA-HNSC cohort revealed 16 highly dysregulated lncRNAs in HNC, resulting in a combined 31-lncRNA signature panel that could predict prognosis. Validation of these molecules confirmed the considerable level of altered expressions in HNC cells, with XIST, HOXA11-AS, TSIX, MALAT1, WT1-AS, and IPW being the most prominently dysregulated. We further selected a molecule from our panel (XIST) to confirm the validity of these lncRNAs in the regulation of cancer aggressiveness. Gene ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses demonstrated that XIST participated in various cancer-related functions, including cell proliferation and metastasis. XIST silencing with the RNAi technique substantially reduced invasion and migration in several HNC cell lines. Thus, our study defined a 31-lncRNA panel as prognostic signatures in HNC. These perspective results provide a knowledge foundation for further application of these molecules in precision medicine.

New Insights Into the Biological Interaction Between Unripe Citrus Fruits and the Tephritid Fly Bactrocera Minax Based on Omics

The adaptation of phytophagous insects to host defence is an important aspect of plant-insect interactions. The reciprocal adaptability between specialist insects and their hosts have been adequately explored; however, the mechanisms underlying the adaptation of tephritid fruit fly specialists, a group of notorious pests worldwide, to unripen host fruits remain elusive. Here, plant metabolomes and insect transcriptomes were analysed for the first time to explore the interaction between unripe citrus fruits and the Chinese citrus fly Bactrocera minax. Seventeen citrus secondary metabolites, mainly flavones, alkaloids and phenylpropanoids, were identified in the unripe citrus fruit metabolome and they accumulated during larval feeding. Three detoxification genes (1 P450 gene, 2 ABCs genes) were highly expressed in B. minax larvae collected from unripe citrus fruits compared with the ones fed on artificial diets and ripe citrus fruits. Based on omics data, a novel ABC gene was screened through plant allelopathy tests and the gene was significantly upregulated in B. minax larvae treated with defensive secondary metabolites; additionally, the mortality rate of the larvae reached 51% after silencing the ABC gene by RNAi technique. Overall, these results shed light on the mechanisms underlying the biological interactions between tephritid fruit fly specialists and host fruits.

Multiple cytochrome P450 genes: conferring high levels of permethrin resistance in mosquitoes, Culex quinquefasciatus

Insecticides, especially pyrethroids, are the most important in the insect pest control and preventing insect vector-borne human diseases. However, insect pests, including mosquitoes, have developed resistance in the insecticides that used against them. Cytochrome P450s are associated with insecticide resistance through overexpression and detoxification mechanisms in insect species. In this study, we utilized a powerful tool, the RNAi technique, to determine the roles of key P450 genes overexpressed in permethrin resistant mosquitoes that confer insecticide resistance to unravel the molecular basis of resistance mechanisms in the mosquito Culex quinquefasciatus. The results showed that knockdown of 8 key P450 genes using RNAi techniques significantly decreased resistance to permethrin in resistant mosquitoes. In silico modeling and docking analysis further revealed the potential metabolic function of overexpressed P450 genes in the development of insecticide resistance in mosquitoes. These findings not only highlighted the functional importance of these P450 genes in insecticide resistance, but also revealed that overexpression of multiple P450 genes was responsible for the high levels of insecticide resistance in a mosquito population of Culex quinquefasciatus.

The CONJUDOR pipeline for multiplexed knockdown of gene pairs identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans

Multiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in Caenorhabditis elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique ‘CONJUDOR’. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONJUDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONJUDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput.

The CONDOR pipeline for simultaneous knockdown of multiple genes identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans

ABSTRACTMultiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in C. elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique ‘CONDOR’. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput.

The Basic Leucine Zipper Transcription Factor PlBZP32 Associated with the Oxidative Stress Response Is Critical for Pathogenicity of the Lychee Downy Blight Oomycete Peronophythora litchii

ABSTRACT Basic leucine zipper (bZIP) transcription factors are widespread in eukaryotes, including plants, animals, fungi, and oomycetes. However, the functions of bZIPs in oomycetes are rarely known. In this study, we identified a bZIP protein possessing a special bZIP-PAS structure in Peronophythora litchii, named PlBZP32. We found that PlBZP32 is upregulated in zoospores, in cysts, and during invasive hyphal growth. We studied the functions of PlBZP32 using the RNAi technique to suppress the expression of this gene. PlBZP32-silenced mutants were more sensitive to oxidative stress, showed a lower cyst germination rate, and produced more sporangia than the wild-type strain SHS3. The PlBZP32-silenced mutants were also less invasive on the host plant. Furthermore, we analyzed the activities of extracellular peroxidases and laccases and found that silencing PlBZP32 decreased the activities of P. litchii peroxidase and laccase. To our knowledge, this is the first report that the functions of a bZIP-PAS protein are associated with oxidative stress, asexual development, and pathogenicity in oomycetes. IMPORTANCE In this study, we utilized the RNAi technique to investigate the functions of PlBZP32, which possesses a basic leucine zipper (bZIP)-PAS structure, and provided insights into the contributions of bZIP transcription factors to oxidative stress, the production of sporangia, the germination of cysts, and the pathogenicity of Peronophythora litchii. This study also revealed the role of PlBZP32 in regulating the enzymatic activities of extracellular peroxidases and laccases in the plant-pathogenic oomycete.

Exploring the Patent Landscape of RNAi-based Innovation for Plant Breeding

Background: RNA interference (RNAi) is an innovative technique for plant improvement based on naturally occurring mechanisms which show great potential because of their high specificity and possibility to be applied through innovative methods of topical application in plants. This specific innovation sector is worth analysing from the economic perspective given the great potentiality of RNAi-based plants and products to support modern agriculture in reaching the goals for the improvement of agri-food chains global sustainability. This paper aims to evaluate the global landscape of RNAi innovation by analysing patent data as indicators of innovation output. Methods: We revised all patents relating to RNAi in plants based on a dataset of roughly seven thousand patent families. The analysis classified inventions according to a set of variables able to characterise the dynamics of innovation (i.e. public/private ownership, type of plants involved, main traits) while the use of concentration indices provided insights into the evolution of this sector. Results: Results revealed that RNAi is a technique with promising future applications, able to provide solutions to a great variety of agricultural issues and principally developed by the US and Chinese applicants, whereas European innovation capacity in this field appears to be limited. Conclusion: The innovation landscape of plant breeding is rapidly evolving and RNAi technique is probably going to play a major role in this field.

Characterization of an Intradiol Ring-Cleavage Dioxygenase Gene Associated With Abamectin Resistance in Tetranychus cinnabarinus (Acari: Tetranychidae)

Tetranychus cinnabarinus (Boisduval), i.e., carmine spider mite, is a worldwide pest that can cause serious damage to plants. Problems of resistance have arisen since abamectin usage in the control of T. cinnabarinus. Unfortunately, there are only limited data on the extent of this problem. To understand the development of abamectin resistance in the carmine spider mite, we prokaryotically expressed an intradiol ring-cleavage dioxygenase (ID-RCD) gene sequence, TcID-RCD1, which had a significant upregulated expression of over 7.7 times in an abamectin-resistant strain (AbR) when compared with that of a susceptible strain (SS). The crude enzyme activity also indicated that the AbR had a higher activity than that exhibited in SS. When susceptible individuals were treated with abamectin, TcID-RCD1 was also overexpressed. Furthermore, using the RNA interference (RNAi) technique, TcID-RCD1 was successfully knocked down, with the expression level decreasing significantly to approximately 39% in the SS strain compared with the control. And the mortality of mites feeding on dsTcID-RCD1 increased significantly when treated with abamectin. These results strongly suggest that TcID-RCD1 is involved in abamectin resistance in T. cinnabarinus.