microRNA Target Sites Research Articles

Simplified Lipid Nanoparticles for Tissue- And Cell-Targeted mRNA Delivery Facilitate Precision Tumor Therapy in a Lung Metastasis Mouse Model.

mRNA-based applications have achieved remarkable success in the development of next-generation vaccines and the treatment of diverse liver diseases. Overcoming the challenge of delivering mRNA to extrahepatic tissues, especially specific cells within tissues, is crucial for precision therapy. In this study, a platform is developed for selective mRNA delivery to desired cells within tissues by combining lipid nanoparticle (LNP)-based targeted delivery with mRNA sequence-controlled expression. Through systematic optimization, a three-component LNP platform is developed, enabling targeted mRNA delivery to the lung, liver, and spleen. The incorporation of unique microRNA target sites into the mRNA scaffold further enhances control over protein translation in specific cells within the target tissue. This combined strategy, named SELECT (Simplified LNP with Engineered mRNA for Cell-type Targeting), demonstrates its efficacy in distinguishing mRNA expression between tumor and normal cells based on intracellular microRNA abundance. SELECT encapsulating mRNA encoding a tumor-specific cytotoxic protein, human ELANE, exhibits selective mRNA delivery to tumor lesions and significant inhibition of tumor growth in a mouse model of melanoma lung metastasis. Overall, SELECT has great potential as a new precision tumor treatment approach and also offers promising prospects for other mRNA therapies targeting specific cell types.

Advancing microRNA target site prediction with transformer and base-pairing patterns.

MicroRNAs (miRNAs) are short non-coding RNAs involved in various cellular processes, playing a crucial role in gene regulation. Identifying miRNA targets remains a central challenge and is pivotal for elucidating the complex gene regulatory networks. Traditional computational approaches have predominantly focused on identifying miRNA targets through perfect Watson-Crick base pairings within the seed region, referred to as canonical sites. However, emerging evidence suggests that perfect seed matches are not a prerequisite for miRNA-mediated regulation, underscoring the importance of also recognizing imperfect, or non-canonical, sites. To address this challenge, we propose Mimosa, a new computational approach that employs the Transformer framework to enhance the prediction of miRNA targets. Mimosa distinguishes itself by integrating contextual, positional and base-pairing information to capture in-depth attributes, thereby improving its predictive capabilities. Its unique ability to identify non-canonical base-pairing patterns makes Mimosa a standout model, reducing the reliance on pre-selecting candidate targets. Mimosa achieves superior performance in gene-level predictions and also shows impressive performance in site-level predictions across various non-human species through extensive benchmarking tests. To facilitate research efforts in miRNA targeting, we have developed an easy-to-use web server for comprehensive end-to-end predictions, which is publicly available at http://monash.bioweb.cloud.edu.au/Mimosa.

Uncovering periodontitis-associated markers through the aggregation of transcriptomics information from diverse sources.

Periodontitis, a common chronic inflammatory disease, significantly impacted oral health. To provide novel biological indicators for the diagnosis and treatment of periodontitis, we analyzed public microarray datasets to identify biomarkers associated with periodontitis. The Gene Expression Omnibus (GEO) datasets GSE16134 and GSE106090 were downloaded. We performed differential analysis and robust rank aggregation (RRA) to obtain a list of differential genes. To obtain the core modules and core genes related to periodontitis, we evaluated differential genes through enrichment analysis, correlation analysis, protein-protein interaction (PPI) network and competing endogenous RNA (ceRNA) network analysis. Potential biomarkers for periodontitis were identified through comparative analysis of dual networks (PPI network and ceRNA network). PPI network analysis was performed in STRING. The ceRNA network consisted of RRA differentially expressed messenger RNAs (RRA_DEmRNAs) and RRA differentially expressed long non-coding RNAs (RRA_DElncRNAs), which regulated each other's expression by sharing microRNA (miRNA) target sites. RRA_DEmRNAs were significantly enriched in inflammation-related biological processes, osteoblast differentiation, inflammatory response pathways and immunomodulatory pathways. Comparing the core ceRNA module and the core PPI module, C1QA, CENPK, CENPU and BST2 were found to be the common genes of the two core modules, and C1QA was highly correlated with inflammatory functionality. C1QA and BST2 were significantly enriched in immune-regulatory pathways. Meanwhile, LINC01133 played a significant role in regulating the expression of the core genes during the pathogenesis of periodontitis. The identified biomarkers C1QA, CENPK, CENPU, BST2 and LINC01133 provided valuable insight into periodontitis pathology.

Genome-Wide Identification and Expression Analysis of the Broad-Complex, Tramtrack, and Bric-à-Brac Domain-Containing Protein Gene Family in Potato

The BTB (broad-complex, tramtrack, and bric-à-brac) domain, also known as the POZ (POX virus and zinc finger) domain, is a conserved protein–protein interaction domain present in various organisms. In this study, we conducted a genome-wide search to identify and characterize BTB genes in Solanum tuberosum. A total of 57 StBTBs were identified and analyzed for their physicochemical properties, chromosomal distribution, gene structure, conserved motifs, phylogenetic relationships, tissue-specific expression patterns, and responses to hormonal and stress treatments. We found that StBTBs were unevenly distributed across potato chromosomes and exhibited diverse gene structures and conserved motifs. Tissue-specific expression analysis revealed differential expression patterns across various potato tissues, implying their roles in plant growth and development. Furthermore, differential expression analysis under hormonal and stress treatments indicated the involvement of StBTBs in abiotic and biotic stress responses and hormone signaling pathways. Protein–protein interaction analysis identified potential interactions with ribosomal proteins, suggesting roles in translational regulation. Additionally, microRNA target site analysis revealed regulatory relationships between StBTBs and miRNAs. Our study provides a comprehensive understanding of the StBTB gene family in potato, laying the groundwork for further functional characterization and manipulation of these genes to improve stress tolerance and agricultural productivity in potato and related plant species.

Characterization of the Fatty Acyl-CoA Reductase (FAR) Gene Family and Its Response to Abiotic Stress in Rice (Oryza sativa L.).

Fatty acyl-CoA reductase (FAR) is an important NADPH-dependent enzyme that can produce primary alcohol from fatty acyl-CoA or fatty acyl-carrier proteins as substrates. It plays a pivotal role in plant growth, development, and stress resistance. Herein, we performed genome-wide identification and expression analysis of FAR members in rice using bioinformatics methods. A total of eight OsFAR genes were identified, and the OsFARs were comprehensively analyzed in terms of phylogenetic relationships, duplication events, protein motifs, etc. The cis-elements of the OsFARs were predicted to respond to growth and development, light, hormones, and abiotic stresses. Gene ontology annotation analysis revealed that OsFAR proteins participate in biological processes as fatty acyl-CoA reductase during lipid metabolism. Numerous microRNA target sites were present in OsFARs mRNAs. The expression analysis showed that OsFARs were expressed at different levels during different developmental periods and in various tissues. Furthermore, the expression levels of OsFARs were altered under abiotic stresses, suggesting that FARs may be involved in abiotic stress tolerance in rice. The findings presented here serve as a solid basis for further exploring the functions of OsFARs.

Characterization of the DUF868 gene family in Nicotiana and functional analysis of NtDUF868-E5 involved in pigment metabolism

Domains of unknown function (DUF) proteins represent a large group of uncharacterized protein families. The DUF868 gene family in Nicotiana has not yet been described. In the present study, we identified 12, 11, and 25 DUF868 family members in the genome of Nicotiana sylvestris, N. tomentosiformis, and N. tabacum, respectively. Based on phylogenetic analysis, these were categorized into five groups (A-E). Within each group, the gene structures, motifs, and tertiary structures showed high similarity. NtDUF868 family expansion during evolution was mainly driven by segmental duplication events. MicroRNA (miRNA) target site prediction identified 12 miRNA members that target 16 NtDUF868 family genes. The promoters of these genes contain cis-regulatory elements responsive to light, phytohormones, and abiotic stresses. Expression profiling revealed their tissue- and stage-specific expression patterns. RNA-sequencing and quantitative reverse transcription PCR revealed that the NtDUF868 family genes are potentially involved in the response to abiotic and biotic stresses, particularly drought and hormone stresses, and in the resistance to black shank and bacterial wilt. We generated transformed plants using NtDUF868-E5 overexpression and gene-editing vectors. NtDUF868-E5 overexpression resulted in enhanced tobacco plant growth and development, leading to increased leaf photosynthetic capacity and higher chlorophyll and carotenoid contents. This study provided a comprehensive genome-wide analysis of the DUF868 gene family, shedding light on their potential roles in plant growth and stress responses.

The microRNA target site profile is a novel biomarker in the immunotherapy response.

MicroRNAs (miRNAs) bind on the 3' untranslated region (3'UTR) of messenger RNAs (mRNAs) and regulate mRNA expression in physiological and pathological conditions, including cancer. Thus, studies have identified miRNAs as potential biomarkers by correlating the miRNA expression with the expression of important mRNAs and/or clinical outcomes in cancers. However, tumors undergo pervasive 3'UTR shortening/lengthening events through alternative polyadenylation (APA), which varies the number of miRNA target sites in mRNA, raising the number of miRNA target sites (numTS) as another important regulatory axis of the miRNA binding effects. In this study, we developed the first statistical method, BIOMATA-APA, to identify predictive miRNAs based on numTS features. Running BIOMATA-APA on The Cancer Genome Atlas (TCGA) and independent cohort data both with immunotherapy and no immunotherapy, we demonstrated for the first time that the numTS feature 1) distinguishes different cancer types, 2) predicts tumor proliferation and immune infiltration status, 3) explains more variation in the proportion of tumor-infiltrating immune cells, 4) predicts response to immune checkpoint blockade (ICB) therapy, and 5) adds prognostic power beyond clinical and miRNA expression. To the best of our knowledge, this is the first pan-cancer study to systematically demonstrate numTS as a novel type of biomarker representing the miRNA binding effects underlying tumorigenesis and pave the way to incorporate miRNA target sites for miRNA biomarker identification. Another advantage of examining the miRNA binding effect using numTS is that it requires only RNA-Seq data, not miRNAs, thus resulting in high power in the miRNA biomarker identification.

Circular RNAs Associated with Idiopathic Parkinson’s Disease: Case/Control Study Results on PPMI Dataset

AbstractBackgroundPD is the second most common neurodegenerative disease after AD, for which biomarkers do not exist. We aimed identify circular RNAs associated with Parkinson’s Disease (PD) in a longitudinal dataset and test their potential as Bioamrkers.MethodWe included 2507 European ancestry blood samples from PPMI participants, with transcriptomic data available and distributed across five time‐points. We aligned reads to the reference genome (GRCh38) using STAR in chimeric alignment mode. Annotation and quantification of circRNAs were performed using DCC, followed by a cross‐sectional differential expression analyses for each visit using DESeq2. Significance was assessed and corrected by FDR at the last visit, with nominal p‐value for the remaining visits. We used Circular RNA Interactome web tool to predict microRNA (miRNA) target sites within significantly dysregulated circRNAs. Subsequently, we performed pathway analyses using DIANA tools.ResultTwo circRNAs were significantly dysregulated in PD across all visits: circRHBDD1 (p = 3.34´10−4, Log2FC = ‐0.13) and circDHRSX (p = 1.03´10−3, Log2FC = 0.4). We identified twenty miRNAs that putatively interact with circRHBDD1. Pathway analyses showed that fourteen of them are involved in the Hippo signaling pathway, fifteen in oxytocin signaling pathway, and sixteen in the ErbB1 signaling pathway. We also identified one hundred and twenty‐eight miRNAs interacting with circDHRSX that are also part of the Hippo signaling pathway.ConclusionWe identified circRHBDD1 and circDHRSX to be dysregulated in PD across time. Both of them are enriched in binding sites for miRNAs that target the Hippo and ErbB1 signaling pathways. ErbB1 has already been associated with PD in brain studies, whereas the Hippo pathway is associated to neuronal cell death. We are conducting additional analyses in independent datasets and using mixed models to replicate the results and obtain the best predictive models.

Genome-wide analysis of the heat shock transcription factor family reveals saline-alkali stress responses in Xanthoceras sorbifolium.

The heat shock transcription factor (HSF) family is involved in regulating growth, development, and abiotic stress. The characteristics and biological functions of HSF family member in X. sorbifolium, an important oil and ornamental plant, have never been reported. In this study, 21 XsHSF genes were identified from the genome of X. sorbifolium and named XsHSF1-XsHSF21 based on their chromosomal positions. Those genes were divided into three groups, A, B, and C, containing 12, one, and eight genes, respectively. Among them, 20 XsHSF genes are located on 11 chromosomes. Protein structure analysis suggested that XsHSF proteins were conserved, displaying typical DNA binding domains (DBD) and oligomerization domains (OD). Moreover, HSF proteins within the same group contain specific motifs, such as motif 5 in the HSFC group. All XsHSF genes have one intron in the CDS region, except XsHSF1 which has two introns. Promoter analysis revealed that in addition to defense and stress responsiveness elements, some promoters also contained a MYB binding site and elements involved in multiple hormones responsiveness and anaerobic induction. Duplication analysis revealed that XsHSF1 and XsHSF4 genes were segmentally duplicated while XsHSF2, XsHSF9, and XsHSF13 genes might have arisen from transposition. Expression pattern analysis of leaves and roots following salt-alkali treatment using qRT-PCR indicated that five XsHSF genes were upregulated and one XsHSF gene was downregulated in leaves upon NaCl treatment suggesting these genes may play important roles in salt response. Additionally, the expression levels of most XsHSFs were decreased in leaves and roots following alkali-induced stress, indicating that those XsHSFs may function as negative regulators in alkali tolerance. MicroRNA target site prediction indicated that 16 of the XsHSF genes may be regulated by multiple microRNAs, for example XsHSF2 might be regulated by miR156, miR394, miR395, miR408, miR7129, and miR854. And miR164 may effect the mRNA levels of XsHSF3 and XsHSF17, XsHSF9 gene may be regulated by miR172. The expression trends of miR172 and miR164 in leaves and roots on salt treatments were opposite to the expression trend of XsHSF9 and XsHSF3 genes, respectively. Promoter analysis showed that XsHSFs might be involved in light and hormone responses, plant development, as well as abiotic stress responses. Our results thus provide an overview of the HSF family in X. sorbifolium and lay a foundation for future functional studies to reveal its roles in saline-alkali response.

A novel computational method enables RNA editome profiling during human hematopoiesis from scRNA-seq data

RNA editing is a post-transcriptional modification with a cell-specific manner and important biological implications. Although single-cell RNA-seq (scRNA-seq) is an effective method for studying cellular heterogeneity, it is difficult to detect and study RNA editing events from scRNA-seq data because of the low sequencing coverage. To overcome this, we develop a computational method to systematically identify RNA editing sites of cell types from scRNA-seq data. To demonstrate its effectiveness, we apply it to scRNA-seq data of human hematopoietic stem/progenitor cells (HSPCs) with an annotated lineage differentiation relationship according to previous research and study the impacts of RNA editing on hematopoiesis. The dynamic editing patterns reveal the relevance of RNA editing on different HSPCs. For example, four microRNA (miRNA) target sites on 3ʹ UTR of EIF2AK2 are edited across all HSPC populations, which may abolish the miRNA-mediated inhibition of EIF2AK2. Elevated EIF2AK2 may thus activate the integrated stress response (ISR) pathway to initiate global translational attenuation as a protective mechanism to maintain cellular homeostasis during HSPCs’ differentiation. Besides, our findings also indicate that RNA editing plays an essential role in the coordination of lineage commitment and self-renewal of hematopoietic stem cells (HSCs). Taken together, we demonstrate the capacity of scRNA-seq data to exploit RNA editing events of cell types, and find that RNA editing may exert multiple modules of regulation in hematopoietic processes.

MicroRNAs - small RNAs with a big influence on brain excitability.

MicroRNAs are non-coding RNAs, approximately 22nt in length, which serve to negatively regulate gene expression through binding to complementary sequences in the 3' untranslated region (3'UTR) of target mRNA. The microRNA-target interaction does not require perfect complementarity, meaning that an individual microRNA often has a pool of hundreds of gene targets. Equally, one 3'UTR can contain target sites for many different microRNAs. This gives rise to a complex web of molecular interactions. An emerging concept is that microRNAs have a role as 'master' regulators of certain cellular properties, simultaneously mediating the subtle repression of multiple related genes within a pathway or system, thereby achieving a common phenotypic output. One such example is regulation of brain excitability. There are numerous examples of microRNAs which can target ion channels, ion transporters and genes associated with synaptic transmission. Often, the expression of the microRNA itself is regulated in an activity-dependent manner, thereby forming homeostatic loops. Limitations in our understanding arise from the sheer complexity of microRNA-target interactions, which are difficult to capture experimentally and computationally. Further, many microRNA studies rely on animal model systems, but many microRNAs (and mRNA targets) have sequences which are either not conserved or are entirely unique in the human brain. This leaves many exciting and challenging opportunities to further progress the field in an attempt to fully understand the roles of microRNAs in brain function.

Genome-Wide Analysis of DREB Family Genes and Characterization of Cold Stress Responses in the Woody Plant Prunus nana.

Dehydration response element binding factor (DREB) is a family of plant-specific transcription factors, whose members participate in the regulation of plant responses to various abiotic stresses. Prunus nana, also known as the wild almond, is a member of the Rosaceae family that is rare and found to grow in the wild in China. These wild almond trees are found in hilly regions in northern Xinjiang, and exhibit greater drought and cold stress resistance than cultivated almond varieties. However, the response of P. nana DREBs (PnaDREBs) under low temperature stress is still unclear. In this study, 46 DREB genes were identified in the wild almond genome, with this number being slightly lower than that in the sweet almond (Prunus dulcis cultivar 'Nonpareil'). These DREB genes in wild almond were separated into two classes. All PnaDREB genes were located on six chromosomes. PnaDREB proteins that were classified in the same groups contained specific shared motifs, and promoter analyses revealed that PnaDREB genes harbored a range of stress-responsive elements associated with drought, low-temperature stress, light responsivity, and hormone-responsive cis-regulatory elements within their promoter regions. MicroRNA target site prediction analyses also suggested that 79 miRNAs may regulate the expression of 40 of these PnaDREB genes, with PnaDREB2. To examine if these identified PnaDREB genes responded to low temperature stress, 15 of these genes were selected including seven homologous to Arabidopsis C-repeat binding factor (CBFs), and their expression was assessed following incubation for 2 h at 25 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In summary, this analysis provides an overview of the P. nana PnaDREB gene family and provides a foundation for further studies of the ability of different PnaDREB genes to regulate cold stress responses in almond plants.

Genome-Wide Identification and Expression Analysis of RCC1 Gene Family under Abiotic Stresses in Rice (Oryza sativa L.)

In plants, the essential roles played by the regulator of chromosome condensation 1 (RCC1) in diverse biological processes, including UV-B (ultraviolet-B radiation) response, hormonal signal transduction, cold tolerance and phenotypic plasticity, have been identified. No comprehensive study on the evolution and function of RCC1 gene family in rice has been carried out. A genome-wide analysis of this gene family is thus required. In this study, a total of 26 OsRCC1s unevenly distributed across 10 chromosomes were identified in rice. Based on their phylogenetic relationship and sequence composition, the OsRCC1 family could be classified into six groups. Members within the same group share a similar gene structure and protein motif/domain composition. Gene duplication analysis revealed that segmental duplication might be the main contributor to the expansion of the RCC1 gene family in rice. Several cis-regulatory elements (CREs) relevant to light, abscisic acid (ABA) and methyl jasmonate (MeJA) are abundant in the promoters of OsRCC1s. A large number of microRNA (miRNA) target sites were present in OsRCC1 mRNAs. Additionally, we used data from gene microarray and qRT-PCR to analyze the expression of OsRCC1 genes during various developmental stages and under abiotic stress conditions. OsRCC1s were found to be highly expressed in panicles and seeds, and most OsRCC1s were differentially expressed under abiotic stresses. Taken together, our study provides a systematic characterization of OsRCC1s and preliminarily explores their diversity as well as their biological functions. Evidence demonstrates that OsRCC1s may play vital roles in both development and abiotic stress response. The results presented here lay a foundation for further investigating the functions of OsRCC1s.

Spatial transcriptomics for profiling the tropism of viral vectors in tissues.

A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.

Metal tolerance gene family in barley: an in silico comprehensive analysis

Metal-tolerance proteins (MTPs) are divalent cation transporters that play critical roles in metal tolerance and ion homeostasis in plants. However, a comprehensive study of MTPs is still lacking in crop plants. The current study aimed to comprehensively identify and characterize the MTP gene family in barley (Hordeum vulgare, Hv), an important crop. In total, 12 HvMTPs were identified in the barley genome in this study. They were divided into three phylogenetic groups (Zn-cation diffusion facilitator proteins [CDFs], Fe/Zn-CDFs, and Mn-CDFs) and further subdivided into seven groups (G1, G5, G6, G7, G8, G9, and G12). The majority of MTPs were hydrophobic proteins found in the vacuolar membrane. Gene duplication analysis of HvMTPs revealed one pair of segmental-like duplications in the barley genome. Evolutionary analysis suggested that barley MTPs underwent purifying natural selection. Additionally, the HvMTPs were analyzed in the pan-genome sequences of barley (20 accessions), which suggests that HvMTPs are highly conserved in barley evolution. Cis-acting regulatory elements, microRNA target sites, and protein–protein interaction analysis indicated the role of HvMTPs in a variety of biological processes. Expression profiling suggests that HvMTPs play an active role in maintaining barley nutrient homeostasis throughout its life cycle, and their expression levels were not significantly altered by abiotic stresses like cold, drought, or heat. The expression of barley HvMTP genes in the presence of heavy metals such as Zn2+, Cu2+, As3+, and Cd2+ revealed that these MTPs were induced by at least one metal ion, implying their involvement in metal tolerance or transportation. The identification and comprehensive investigation of MTP gene family members will provide important gene resources for the genetic improvement of crops for metal tolerance, bioremediation, or biofortification of staple crops.

Genome-Wide Identification and Expression Analysis of the MADS-Box Family in Ginkgo biloba

As the most significant transformation stage of plants, the flowering process has typically been the focus of research. MADS-box gene plays an important regulatory role in flower development. In this study, 26 MADS-box genes were identified from Ginkgo biloba, including 10 type-I genes and 16 type-II genes, which were distributed on eight chromosomes. There was no collinearity between the GbMADS genes, and the homology with genes from other species was low. All GbMADS proteins contain conserved MADS domains. The gene structures of GbMADS in the same gene family or subfamily differed, but the conserved protein motifs had similar distributions. The microRNA (miRNA) target sites of the GbMADS genes were predicted. It was found that the expression of 16 GbMADS genes may be regulated by miRNA. The results of cis-acting element analysis showed that the 26 GbMADS genes contained a large number of hormones regulated and light-responsive elements as well as stress-response elements. Furthermore, the quantitative real-time PCR (qRT-PCR) experimental results showed that most GbMADS genes were differentially expressed in the male and female flowers at different developmental stages. Among them, the only MIKC * gene GbMADS16 has the highest expression in the metaphase development of the microstrobilus (M2) stage and is almost not expressed in female flowers. Taken together, these findings suggest that the MADS-box genes may play an important role in the development and differentiation of G. biloba flowers.

Genome-Wide Identification and Expressional Profiling of the Metal Tolerance Protein Gene Family in Brassica napus.

The Cation Diffusion Facilitator (CDF) family, also named Metal Tolerance Protein (MTP), is one of the gene families involved in heavy metal transport in plants. However, a comprehensive study of MTPs in Brassica napus has not been reported yet. In the present study, we identified 33 BnMTP genes from the rapeseed genome using bioinformatic analyses. Subsequently, we analyzed the phylogenetic relationship, gene structure, chromosome distribution, conserved domains, and motifs of the BnMTP gene family. The 33 BnMTPs were phylogenetically divided into three major clusters (Zn-CDFs, Fe/Zn-CDFs, and Mn-CDFs) and seven groups (group 1, 5, 6, 7, 8, 9, and 12). The structural characteristics of the BnMTP members were similar in the same group, but different among groups. Evolutionary analysis indicated that the BnMTP gene family mainly expanded through whole-genome duplication (WGD) and segmental duplication events. Moreover, the prediction of cis-acting elements and microRNA target sites suggested that BnMTPs might be involved in plant growth, development, and stress responses. In addition, we found the expression of 24 BnMTPs in rapeseed leaves or roots could respond to heavy metal ion treatments. These results provided an important basis for clarifying the biological functions of BnMTPs, especially in heavy metal detoxification, and will be helpful in the phytoremediation of heavy metal pollution in soil.

Generation of a microRNA-Regulated Oncolytic Coxsackievirus B3.

The members of the picornavirus family include various viruses which, due to their impressive oncolytic activity, have the potential to be used for the treatment of cancer. However, the replication of these oncolytic viruses (OV) is not limited to tumor cells but can also take place in various normal tissues. To increase the safety of these OV, target sites (miR-TS) of microRNAs, which are expressed in normal tissues but are absent or only expressed at low levels in cancer cells, can be inserted into the viral genome. Here we describe how miR-TS can easily be inserted into the complementary DNA (cDNA) of coxsackievirus B3 (CVB3) RNA genome using the In-Fusion cloning technology. Here we provide the step-by-step protocol, how miR-TS containing recombinant CVB3 can be generated from these viral cDNA constructs, how the virus is amplified, purified and concentrated, and how the functionality of the miR-TS within the viral genome can be confirmed.

Highly efficient, genotype-independent transformation and gene editing in watermelon (Citrullus lanatus) using a chimeric ClGRF4-GIF1 gene.

Efficient genetic transformation has the potential to advance research and breeding in watermelon (Citrullus lanatus), but regeneration from tissue culture remains challenging. Previous work showed that expressing a fusion of two interacting transcription factors, GROWTH-REGULATING FACTOR4 (GRF4) and GRF-INTERACTING FACTOR1 (GIF1), improved regeneration in wheat (Triticum aestivum). By overexpressing a chimeric fusion of ClGRF4 and ClGIF1, we achieved highly efficient transformation in watermelon. Mutating the mi396 microRNA target site in ClGRF further boosted the transformation efficiency up to 67.27% in a genotype-independent manner. ClGRF4-GIF1 can also be combined with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing tools to achieve highly efficient gene editing in watermelon, which we used to successfully create diploid seedless watermelon. This research thus puts forward a powerful transformation tool for future watermelon research and breeding.

Application Route and Immune Status of the Host Determine Safety and Oncolytic Activity of Oncolytic Coxsackievirus B3 Variant PD-H.

The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3+ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus.