RNA Interference Assays Research Articles

The orchestration of coding and non-coding genes at the CDH13 locus in coronary artery disease

Abstract Background The CDH13 locus was associated with coronary artery disease (CAD) by genome-wide association studies (GWAS). However, the causal gene(s) and underlying disease mechanisms at this locus remain undetermined. Purpose We intended to pinpoint and functionally validate the causal coding and non-coding genes at the CDH13 locus and delineate the underlying mechanisms for CAD. Methods and results We conducted the transcriptome-wide association analysis (TWAS) using nine types of CAD-relevant tissues from Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) and Genotyped Tissue Expression (GTEx) projects and identified CDH13 (T-cadherin) and four long non-coding RNA (lncRNA) genes as candidate causal genes at The CDH13 locus. One of the antisense(AS) lncRNAs, CDH13-AS2, was predicted to bind on the 3’ untranslated region (UTR) of CDH13 using LncRRIsearch and RNAup servers. The binding interaction was validated by dCas13-medicated RNA immunoprecipitation. Weighted correlation network analysis (WGCNA) using transcriptomic data from CAD-relevant tissues of ~600 human subjects suggested a positive expression correlation between CDH13 and CDH13-AS2 in artery tissues. CRISPR/Cas9-based knockout (KO) of CDH13 or CDH13-AS2 in human umbilical vein endothelial cells (HUVECs) indicated synergistically atherogenic phenotypes, including reduced proliferation and migration, and increased apoptosis and monocyte adhesion. The data matched with the downregulation of CDH13 in artery samples of atherosclerosis patients. The cellular phenotypes were also in line with our in vivo data of Cdh13 and Apoe double-KO (Cdh13-/-/Apoe-/-) mice, which showed increased aortic lesion areas when fed on a high-fat diet compared to Apoe-/- mice. Furthermore, prediction using TargetScan, miRWalk, and scanMiRApp databases identified the potential binding of several EC-expression microRNAs (mir) on 3’UTR of CDH13. Several disease-associated variants were mapped to the predicted binding sites of a few microRNAs, including mir-let7, mir-30, and mir-125. The dual luciferase-based RNA interference (RNAi) assay confirmed the binding of mir-let7 on 3’UTR of CDH13. Transcriptional activation of CDH13-AS2 by the enzymatically inactivated Cas9 (dCas9) system diminished the binding of mir-let7 and increased CDH13 expression. Conclusions At the CDH13 locus, CDH13 and CDH13-AS2 are two causal genes, whose downregulation likely contributes to CAD. CDH13-AS2 bound and stabilized CDH13 partially by preventing mir-let7 mediated degradation of CDH13 mRNA, suggesting therapeutic potentials.

12-OPDA Reductase and OPC-8:0 CoA Ligase Mediate Jasmonic Acid Synthesis in Lasiodiplodia iranensis.

Jasmonic acid (JA), a phytohormone with a distinct flavor, is an important fragrance ingredient. The filamentous fungi Lasiodiplodia sp. can produce JA via fermentation. Based on comparative transcriptome analysis of L. iranensis M2017288 in static and shaking culture, 13 differentially expressed genes potentially related to JA synthesis were screened, including g4442 and g8524, annotated as 12-OPDA reductase (LiOPR) and OPC-8:0 CoA ligase (LiOPCL), respectively. Quantitative real-time PCR and RNA interference assays confirmed that g4442 and g8524 expression levels were positively correlated with the JA synthesis. Recombinant LiOPR protein utilized (15Z)-12-oxophyto-10,15-dienoic acid (12-OPDA) to generate (9S,13S,15Z)-12-oxo-10,11-dihydrophyto-15-enoate (OPC8:0). JA was detected in cascade reactions of both recombinant LiOPR and LiOPCL coupled with Saccharomyces cerevisiae. Phylogenetic analyses revealed that LiOPR and LiOPCL belong to a different evolutionary branch from plants, suggesting that they are essential enzymes for fungal JA synthesis, which is worthy of further investigation.

Two Structural Analogs of Kairomones are Detected by an Odorant Receptor HvarOR28 in the Coccinellid Hippodamia variegata.

In natural environments, general plant volatiles and herbivore-induced plant volatiles (HIPVs) serve as critical clues for predatory natural enemies in the search for prey. The insect olfactory system plays a vital role in perceiving plant volatiles including HIPVs. In this study, we found that HIPV (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) and the plant volatile geranyl acetate (GA), two structurally similar chemicals, displayed electrophysiological activities on the antennae of the ladybird Hippodamia variegata, but were only attractive to adult females in behavior. Moreover, mated female ladybirds laid a significantly higher number of eggs on TMTT-treated and GA-treated cotton leaves compared to controls. Screening of female-biased odorant receptors (ORs) from the antennal transcriptomes, performing Xenopus oocytes expression coupled with two-electrode voltage clamp recordings, suggested that HvarOR28 specifically tuned to TMTT and GA. Molecular docking and site-directed mutagenesis revealed that the amino acid residues Tyr143 and Phe81 of HvarOR28 are the key site for binding with TMTT and GA. Furthermore, RNA interference (RNAi) assay demonstrated that HvarOR28-silenced individuals demonstrated a notable decrease in electrophysiological responses, even female adults almost lost behavioral preference for the two compounds. Thus, it could be concluded that HvarOR28 in H. variegata contributes to facilitating egg laying through the perception of TMTT and GA. These findings may help to develop new olfactory modulators based on the behaviorally active ligands of HvarOR28.

Pericentriolar material 1 promotes intestinal inflammation in ulcerative colitis by activating NLRP3/gasdermin D‐mediated macrophage pyroptosis

AbstractExcessive proinflammatory cytokine release induced by pyroptosis plays a vital role in intestinal mucosal inflammation in ulcerative colitis (UC). Several pyroptosis‐related factors are regulated by the centrosome. Pericentriolar material 1 (PCM1) is a primary component of centriolar satellites that is present as cytoplasmic granules around the centrosome. Our previous study revealed that PCM1 was highly expressed in UC patients, but the role of PCM1 in UC remains unknown. This study aimed to elucidate the role of PCM1 in the development of UC, especially the mechanism in pyroptosis process of UC. Clinical mucosal sample and dextran sulfate sodium (DSS)‐induced colitis mouse were used to reveal the association between PCM1 and intestinal inflammation. Intestinal epithelial cell‐specific PCM1‐knockout mice were constructed to determine the role of PCM1 in colitis. Finally, PCM1 RNA interference and overexpression assays in THP1 cells were employed to study the molecular mechanisms of PCM1 in inflammatory responses and pyroptosis. We found that PCM1 expression was upregulated in the colonic mucosa of UC patients and positively correlated with inflammatory indicators. PCM1 expression was elevated in DSS‐induced colitis mice and was reduced after methylprednisolone treatment. In the DSS colitis model, intestinal‐specific PCM1‐knockout mice exhibited milder intestinal inflammation and lower pyroptosis levels than wild‐type mice. In cell level, PCM1 exerted a proinflammatory effect by activating the NLRP3 inflammasome and triggering subsequent gasdermin D‐mediated pyroptosis to release IL‐1β and IL‐18. In conclusion, PCM1 mediates activation of the NLRP3 inflammasome and gasdermin D‐dependent pyroptosis, ultimately accelerating intestinal inflammation in UC. These findings revealed a previously unknown role of PCM1 in initiating intestinal mucosal inflammation and pyroptosis in UC, and this factor is expected to be a regulator in the complex inflammatory network of UC.

Chlorbenzuron downregulated HcLCP-17 expression by depressing two 20E-responsive transcription factors Br-C and βFTZ-F1 in Hyphantria cunea (Lepidoptera: Erebidae) larvae.

Cuticular proteins (CPs) play essential roles in forming cuticular structures in insects. However, the specific functions and regulatory mechanisms of CPs remain largely unexplored. In this study, the Larval cuticular protein 17 (HcLCP-17) gene was identified from Hyphantria cunea, a highly destructive and polyphagous forest pest. To investigate the role of HcLCP-17 in cuticular function and transcriptional regulation mediated by 20E-responsive transcription factors (ERTFs), we employed RNA interference (RNAi) and yeast one-hybrid assay techniques. Additionally, we examined the molecular mechanism by which chlorbenzuron, a type of benzoylphenylurea (BPU) that functions as a chitin synthesis inhibitor (CSI), affects the 20E signaling pathway and ultimately regulates HcLCP-17 expression. HcLCP-17 encodes a polypeptide consisting of 393 amino acids, which includes a chitin-binding domain. Silencing HcLCP-17 resulted in a disturbance in the structural organization of the larval cuticle and a notable reduction in chitin levels. HcLCP-17 expression was controlled by the interaction between Broad-Complex (Br-C)and beta Fushi Tarazu Factor-1 (βFTZ-F1) with its promoter fragment. Furthermore, the inhibitory effect of chlorbenzuron on HcLCP-17 expression was found to be potentially mediated by Br-C and βFTZ-F1. The study presents a novel mode of action for the 20E signaling pathway in regulating the expression of CPs and reveals the potential mode-of-action of BPUs in insect cuticles. These findings provide a theoretical basis for future utilization of LCP-17 as a pesticide target making a significant contribution to the development of effective pest management strategies. © 2024 Society of Chemical Industry.

Examination of the roles of CgTH in tyrosine metabolism and pigmentation of Pacific oyster (Crassostrea gigas)

The visually appealing shell colours and intricate patterns of shellfish not only attract consumers but also enhance their economic value. Understanding the inheritance pattern of shell colour and the molecular mechanism of pigmentation is highly important for enhancing breeding efficiency. This study involved the identification of a tyrosine hydroxylase gene (CgTH) and the investigation of its function in the metabolism of tyrosine and pigmentation in Pacific oysters (Crassostrea gigas). CgTH contains a conserved Biopterin_H domain that consists of a binding site for iron atoms and a binding site for tetrahydrobiopterin (BH4). Quantitative PCR (qPCR) and immunofluorescence analysis revealed that the expression of CgTH was higher in the mantle of black shell oysters in comparison to the white strain. The in vitro and in vivo experiments confirmed that CgTH is involved in tyrosine metabolism through the catalysis of the conversion of L-tyrosine to L-DOPA. In order to examine its functions in melanin synthesis in oysters, 3-Iodo-L-tyrosine (3-IT) treatment, RNA interference (RNAi), and L-DOPA treatment assays were conducted. The results of our study showed that 3-IT effectively inhibited CgTH activity, leading to a suppression of tyrosine metabolism. Knockdown of CgTH hindered the cAMP-CREM-MITF axis and decreased pigmentation. On the other hand, L-DOPA stimulated melanin production through cAMP signaling and counteracted the inhibitory effects of 3-IT on the metabolism of tyrosine. These data indicated that there may be a feedback loop involving CgTH and the cAMP-CREM-MITF axis that regulates tyrosine metabolism and melanogenesis in Pacific oysters. This study enhances our understanding of the molecular mechanism that governs oyster pigmentation and offers a possible molecular indicator for the targeted breeding of particular strains of Pacific oysters.

Commiphora myrrha n-hexane extract suppressed breast cancer progression through induction of G0/G1 phase arrest and apoptotic cell death by inhibiting the Cyclin D1/CDK4-Rb signaling pathway.

Breast cancer (BC) is one of the most frequently observed malignancies globally, yet drug development for BC has been encountering escalating challenges. Commiphora myrrha is derived from the dried resin of C. myrrha (T. Nees) Engl., and is widely adopted in China for treating BC. However, the anti-BC effect and underlying mechanism of C. myrrha remain largely unclear. MTT assay, EdU assay, and colony formation were used to determine the effect of C. myrrha n-hexane extract (CMHE) on the proliferation of human BC cells. Cell cycle distribution and apoptosis were assessed via flow cytometry analysis. Moreover, metastatic potential was evaluated using wound-scratch assay and matrigel invasion assay. The 4T1 breast cancer-bearing mouse model was established to evaluate the anti-BC efficacy of CMHE in vivo. RNA-sequencing analysis, quantitative real-time PCR, immunoblotting, immunohistochemical analysis, RNA interference assay, and database analysis were conducted to uncover the underlying mechanism of the anti-BC effect of CMHE. We demonstrated the significant inhibition in the proliferative capability of BC cell lines MDA-MB-231 and MCF-7 by CMHE. Moreover, CMHE-induced G0/G1 phase arrest and apoptosis of the above two BC cell lines were also observed. CMHE dramatically repressed the metastatic potential of these two cells in vitro. Additionally, the administration of CMHE remarkably suppressed tumor growth in 4T1 tumor-bearing mice. No obvious toxic or side effects of CMHE administration in mice were noted. Furthermore, immunohistochemical (IHC) analysis demonstrated that CMHE treatment inhibited the proliferative and metastatic abilities of cancer cells, while also promoting apoptosis in the tumor tissues of mice. Based on RNA sequencing analysis, quantitative real-time PCR, immunoblotting, and IHC assay, the administration of CMHE downregulated Cyclin D1/CDK4-Rb signaling pathway in BC. Furthermore, RNA interference assay and database analysis showed that downregulated Cyclin D1/CDK4 signaling cascade participated in the anti-BC activity of CMHE. CMHE treatment resulted in the suppression of BC cell growth through the stimulation of cell cycle arrest at the G0/G1 phase and the induction of apoptotic cell death via the inhibition of the Cyclin D1/CDK4-Rb pathway, thereby enhancing the anti-BC effect of CMHE. CMHE has potential anti-BC effects, particularly in those harboring aberrant activation of Cyclin D1/CDK4-Rb signaling.

A positive loop between relish and cuticle proteins and their roles in regulating AMPs expression during bacterial infection in Eriocheir sinensis

Cuticle proteins (CPs) are the vital components of the cuticle and chitin lining covering the digestive tract of crustaceans. In this study, four new CP genes (designated as EsCP3, EsCP4, EsCP5, and EsCP8) were initially cloned and identified from the Chinese mitten crab Eriocheir sinensis. EsCP3/4/5/8 included 375, 411, 381, and 570 bp open reading frame encoding 124, 136, 126, and 189 amino acid proteins, respectively. Except for EsCP8, EsCP3/4/5 all contained a Chitin_bind_4 domain. EsCP3/4/5/8 were clustered into different groups in the phylogenetic tree. Quantitative real-time PCR results indicated that four EsCP genes have different patterns of tissue distribution. Changes in the expression levels of these four EsCP genes were observed in the intestine of crabs under Vibrio parahaemolyticus challenge. RNA interference assay showed that the knockdown of EsCPs in the intestine could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. In addition, the knockdown of EsRelish in the intestine decreased the expression levels of these four EsCP genes. These results indicated that EsCPs were involved in regulating the expression of AMPs, and EsCPs were regulated by EsRelish.

TPCA-1 compound, inhibiting testis-specific serine/threonine protein kinase 3 for potential male sterile in Bombyx mori.

Protein kinases are a type of transferase enzyme that catalyze the phosphorylation of protein substrates, including receptor proteins. Testis-specific serine/threonine kinases (TSSKs) are a highly conserved group of protein kinases found in various organisms. They play an essential role in male reproduction by influencing sperm development and function. In this study, we report on the characterization of BmTSSK3, a TSSK from the silkworm, Bombyx mori. We found that BmTSSK3 is specifically expressed in the testis and localized to the sperm flagella, particularly in the sperm tail cyst. Furthermore, we developed BmTSSK3 inhibitors through molecular docking and binding assays. Small molecules 5-(4-Fluorophenyl)-2-ureidothiophene-3-carboxamide (TPCA-1)and Imidurea were identified to bind to BmTSSK3. Using site-specific mutation technology, we identified amino acid residues R134 and S184 as crucial binding sites for small molecules. RNA interference assay and Western blot analysis showed that knockdown of BmTSSK3 significantly decreased histone 3 phosphorylation. To confirm the inhibitory effect of these small molecules, we treated silkworm testes with TPCA-1 and observed a strong inhibitory effect. TPCA-1 is an inhibitor of BmTSSK3, which raises its potential as a future candidate for male sterility of the silkworm. Thus, this study may offer a novel strategy for sterile silkworms as well as insects. © 2024 Society of Chemical Industry.

Target screening using RNA interference in the sheep abomasal nematode parasite Haemonchus contortus

RNA interference (RNAi) on parasitic nematodes has been described as a valuable tool for screening putative targets that could be used as novel drug and/or vaccine candidates. This study aimed to set up a pipeline to identify potential targets using RNAi for vaccine/anti-parasite therapy development against Haemonchus contortus, a blood-feeding abomasal nematode parasite. The available H. contortus sequence data was mined for targets, which were tested for essentiality using RNAi electroporation assays. A total of 56 genes were identified and tested for knockdown using electroporation of first-stage larvae (L1) H. contortus with the target double-stranded RNA. Electroporation of L1 proved to be effective overall; 17 targets had a strong phenotype and significant reduction in alive H. contortus, and another 24 had a moderate phenotype with a significant reduction in larvae development. A total of 28 targets showed a significant reduction in the development of H. contortus larvae to the infective stage (L3) following the RNAi assay. Down-regulation of target transcript levels was evaluated in some targets by semi-quantitative PCR. Four out of five genes tested showed complete knockdown of mRNA levels via semi-quantitative PCR, whereas the knockdown was partial for one. In conclusion, the results indicate that the RNAi pathway is confirmed in H. contortus and that several target genes have the potential to be investigated further as possible vaccine candidates.

The cytochrome P450 gene CYP4g1 driven by high temperature confers abamectin tolerance on Liriomyza trifolii through promoting cuticular hydrocarbons biosynthesis

Liriomyza trifolii, an invasive pest, poses a substantial threat to horticultural and vegetable plants. It spreads rapidly, especially in hot weather, leading to large-scale outbreaks with strong thermotolerance and insecticide resistance. In this study, mortality and LtCYP4g1 expression in L. trifolii were evaluated after thermal and insecticides exposure. Furthermore, functional verification of LtCYP4g1 was conducted through RNA interference and bacterial survival assays in Escherichia coli containing recombinant LtCYP4g1 protein. Results indicated that a short time exposure to high temperature incresed insecticide tolerance of L. trifolii, attributed to decreased mortality and induced LtCYP4g1 expression; LtCYP4g1 was involved in stimulating synthesis of cuticular hydrocarbons (CHCs) and elevating epicuticle lipid content and thickness, and E. coli cells overexpressing LtCYP4g1 exhibited significant tolerance to thermal and insecticide stress. In general, P450-mediated tolerance of L. trifolii was enhanced by high temperature, with LtCYP4g1 playing a role in promoting biosynthesis of CHCs for thickening epidermal lipid barrier and reducing cuticular penetration. This study provides a framework for delving into the function of CYP450s in insecticide detoxification and illustrates the role of global warming in driving the evolution of L. trifolii.

P-268 Unique Argonaute isoforms from mammalian oocytes: unveiling their true purpose

Abstract Study question How does the mouse oocyte-specific AGO2 isoform (AGO2ooc) affect RNA interference (RNAi) and microRNA (miRNA) pathways? Summary answer Mouse AGO2ooc presents reduced RNAi-like endonucleolytic cleavage of perfectly complementary targets. Functional analysis of its impact on fertility and oocyte transcriptome control is in progress. What is known already Argonaute proteins play a pivotal role in effector complexes dedicated to miRNA and RNAi pathways, where small RNAs mediate sequence-specific repression of target RNAs. While all mammalian AGO proteins (AGO1 to AGO4) can function in the miRNA pathway, inhibiting target translation, AGO2 uniquely engages in the RNAi pathway, directing the endonucleolytic cleavage of targets perfectly complementary to small RNAs. In mammalian somatic cells, miRNAs are part of a crucial posttranscriptional regulation network, while RNAi is mostly non-functional. Mouse oocytes, however, deviate from this norm: miRNA regulation proves dispensable, while the abrogation of RNAi results in meiotic spindle defects and sterility. Study design, size, duration Experimental study. Participants/materials, setting, methods We pooled 25 fully-grown GV oocytes in triplicates from non-stimulated 10-12 weeks-old C57BI/6NCrl females for RNA isolation and sequencing. To assess RNAi-like and miRNA-like activity, we transfected Ago2ooc/ooc clones and WT cells with NanoLuc luciferase reporters bearing one perfect complementary (1xP, RNAi-like), four bulged (4xB, miRNA-like) or four mutated (4xM, control) sites for Let-7a and miR-30c. A firefly luciferase reporter without any site was co-transfected for normalization purposes. Main results and the role of chance RNA sequencing revealed an alternative exon-1 in mouse oocyte AGO2, producing an isoform (AGO2ooc) with different amino acid composition in the N-terminus. Interestingly, the somatic version of this N-terminus presents amino acid residues known to be post-translationally modified. These residues are absent in AGO2ooc. To functionally analyze the oocyte-specific N-terminus, we genetically engineered NIH3T3 fibroblasts to express the oocyte alternative exon-1 (Ago2ooc/ooc). Notably, AGO2ooc-expressing cells presented reduced RNAi-like (2-5 fold) but indistinguishable miRNA-like activity when compared to WT cells in reporter assays for Let-7a and miR-30c. The lower RNAi-like activity presented by AGO2ooc-expressing cells was further confirmed in an independent RNAi assay employing the expression of dsRNA hairpins. Both isoforms showed similar abundance in fibroblasts and similar cleavage activity in vitro. Next, we wondered whether AGO2ooc also displays reduced RNAi activity in vivo. To answer that, we generated mouse models whose oocytes would express the somatic AGO2 isoform instead of the AGO2ooc, or N-terminal variants with mutations in putative key amino acid residues. These models will be further utilized to assess fertility and oocyte transcriptome. Limitations, reasons for caution We must investigate whether AGO2ooc also displays differential RNAi activity in mouse oocytes and is relevant for oocyte competence, using the developed mouse models. Wider implications of the findings Mouse oocytes are known to exceptionally operate RNAi. Remarkably, AGO2, the effector nuclease associated with RNAi-like target cleavage has a different N-terminus in oocytes, suggesting that this factor could have adapted to this atypical regulation. We hypothesize that AGO2ooc controls RNAi repression, gene expression and oocyte competence. Trial registration number not applicable

The homeodomain of the <i>Raphanus sativus</i> WOX4 binds to the promoter of the <i>LOG3</i> cytokinin biosynthesis gene

BACKGROUND: The WOX4 transcription factor plays a crucial role in maintaining the organisation of cambium meristem during secondary growth, but its direct targets are unknown. AIM: The aim of our work were to study the effect of WOX4 overexpression on the root development and gene expression in radish (Raphanus sativus L.), a root crop related to Arabidopsis thaliana, and to search for direct targets of the WOX4 in radish. MATERIALS AND METHODS: Radish line 19 of the St. Petersburg State University radish genetic collection was used. Plants were grown on Murashige–Skoog medium and then in soil at 23оС and 16 h of daylight. Total DNA was extracted from radish seedlings using the CTAB method. The PCR-amplified full-length RsWOX4-2 gene, gene fragments or homeobox sequence were cloned into the vectors for overexpression (pB7WG2D), RNA interference (pH7GWIWG2) and yeast one-hybrid assay (pDEST22), respectively, using the Gateway system. The vectors for overexpression and RNA interference of RsWOX4-2 were transformed into Escherichia coli DH10B and then into Agrobacteium rhizogenes Arqua chemically competent cells. Radish seedlings were transformed with A. rhizogenes containing vectors for overexpression and RNA interference of RsWOX4-2, and GUS-overexpressing A. rhizogenes was used as a control. Total RNA from transgenic radish roots was extracted with Trizol reagent. RNA reverse transcription was performed using dT-18 primers and RevertAid reverse transcriptase. qPCR was performed using the Eva Green reagent kit on a CFX96 thermocycler with fluorescence detection system. Results were processed using the 2–ΔΔCT method. Yeast transformation was performed using the competent Saccharomyces cerevisiae cells of Y2H Gold strain. For the yeast one-hybrid assay, the obtained yeast colonies transformed with plasmids containing TF homeodomain sequence and promoter regions of genes were grown on DDO and TDO selective media with different concentrations of 3-amino-1,2,4-triazole. Statistical processing based on Student’s t-test and graphing were performed using the ggplot2 package for the R programming language (v.4.0.2). RESULTS: Overexpression of the RsWOX4-2 gene affects the structure of the radish root stele and alters the number of vessels and cambium cells. Overexpression and RNA interference of the RsWOX4-2 causes changes in the expression levels of putative target genes with the WOX family transcription factor conserved binding sites in their promoters. Using the yeast one-hybrid assay, we have shown that the DNA-binding homeodomain of RsWOX4-2 interacts with the TAATCC site in the promoter of the RsLOG3 gene, which encodes the enzyme for cytokinin biosynthesis. CONCLUSIONS: We have demonstrated the effect of RsWOX4-2 overexpression on radish root stele and gene expression and identified the RsLOG3 as the putative direct target of the WOX4 transcription factor in radish.

Nematicidal activity and action mode of a methyl-accepting chemotaxis protein from Pseudomonas syringae against Caenorhabditis elegans

The conventional phytopathogen Pseudomonas syringae reportedly possesses several virulence determinants against Caenorhabditis elegans; however, their action mechanisms remain elusive. This study reports the nematicidal activity and action receptor of a methyl-accepting chemotaxis protein (MCP03) of a wild-type P. syringae MB03 against C. elegans. Purified MCP03 exhibited nematicidal toxicity against C. elegans at a half-lethal concentration of 124.4 μg mL−1, alongside detrimental effects on the growth and brood size of C. elegans. Additionally, MCP03-treated worms exhibited severe pathological destruction of the intestine and depressed wrinkles of the cuticle. Yeast two-hybrid assays identified a subunit of COP9 signalosome, namely CSN-5, which functioned as an MCP03 action receptor. In vitro pull-down verified the binding interaction between MCP03 and CSN-5. RNA interference assays confirmed that MCP03 antagonizes CSN-5, thereby adversely affecting the brood size and cuticle integrity of C. elegans. Following MCP03 infection, the expression of genes related to reproduction, growth, and cuticle formation, such as kgb-1, unc-98, and col-117, was considerably downregulated, indicating pathological changes in MCP03-treated nematodes. Therefore, we proposed that MCP03 antagonizes CSN-5, causing lethality as well as detrimental effects on the fertility, growth, and morphogenesis of C. elegans, which can provide new insights into the signaling pathways and mechanisms underlying the nematicidal action of MCP03 toward C. elegans.

Structurally Diverse Alkaloids with Anti-Renal-Fibrosis Activity from the Centipede Scolopendra subspinipes mutilans.

Twelve new alkaloids, scolopenolines A-L (1-7, 9-11, 13, 14), along with six known analogues, were isolated from Scolopendra subspinipes mutilans, identified by analysis of spectroscopic data and quantum chemical and computational methods. Scolopenoline A (1), a unique guanidyl-containing C14 quinoline alkaloid, features a 6/6/5 ring backbone. Scolopenoline B (2) is a novel sulfonyl-containing heterodimer comprising quinoline and tyramine moieties. Scolopenoline G (7) presents a rare C12 quinoline skeleton with a 6/6/5 ring system. Alkaloids 1, 8, 10, and 15-18 display anti-inflammatory activity, while 10 and 16-18 also exhibit anti-renal-fibrosis activity. Drug affinity responsive target stability and RNA-interference assays show that Lamp2 might be a potentially important target protein of 16 for anti-renal-fibrosis activity.

Oxyberberine sensitizes liver cancer cells to sorafenib via inhibiting NOTCH1-USP7-c-Myc pathway.

Sorafenib is the first-line therapy for patients with advanced-stage HCC, but its clinical cure rate is unsatisfactory due to adverse reactions and drug resistance. Novel alternative strategies to overcome sorafenib resistance are urgently needed. Oxyberberine (OBB), a major metabolite of berberine in vivo, exhibits potential antitumor potency in various human malignancies, including liver cancer. However, it remains unknown whether and how OBB sensitizes liver cancer cells to sorafenib. Cell viability, trypan blue staining and flow cytometry assays were employed to determine the synergistic effect of OBB and sorafenib on killing HCC cells. PCR, western blot, co-immunoprecipitation and RNA interference assays were used to decipher the mechanism by which OBB sensitizes sorafenib. HCC xenograft models and clinical HCC samples were utilized to consolidate our findings. We found for the first time that OBB sensitized liver cancer cells to sorafenib, enhancing its inhibitory effect on cell growth and induction of apoptosis in vitro. Interestingly, we observed that OBB enhanced the sensitivity of HCC cells to sorafenib by reducing ubiquitin-specific peptidase 7 (USP7) expression, a well-known tumor-promoting gene. Mechanistically, OBB inhibited notch homolog 1-mediated USP7 transcription, leading to the downregulation of V-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), which synergized with sorafenib to suppress liver cancer. Furthermore, animal results showed that cotreatment with OBB and sorafenib significantly inhibited the tumor growth of liver cancer xenografts in mice. These results indicate that OBB enhances the sensitivity of liver cancer cells to sorafenib through inhibiting notch homolog 1-USP7-c-Myc signaling pathway, which potentially provides a novel therapeutic strategy for liver cancer to improve the effectiveness of sorafenib.

Identification of Growth-Related Gene BAMBI and Analysis of Gene Structure and Function in the Pacific White Shrimp Litopenaeus vannamei.

As one of the most important aquaculture species in the world, the improvement of growth traits of the Pacific white shrimp (Litopenaeus vannamei), has always been a primary focus. In this study, we conducted SNP-specific locus analysis and identified a growth-related gene, BAMBI, in L. vannamei. We analyzed the structure and function of LvBAMBI using genomic, transcriptomic, metabolomic, and RNA interference (RNAi) assays. The LvBAMBI possessed highly conserved structural domains and widely expressed in various tissues. Knockdown of LvBAMBI significantly inhibited the gain of body length and weight of the shrimp, underscoring its role as a growth-promoting factor. Specifically, knockdown of LvBAMBI resulted in a significant downregulation of genes involved in lipid metabolism, protein synthesis, catabolism and transport, and immunity. Conversely, genes related to glucose metabolism exhibited significant upregulations. Analysis of differential metabolites (DMs) in metabolomics further revealed that LvBAMBI knockdown may primarily affect shrimp growth by regulating biological processes related to lipid and glucose metabolism. These results suggested that LvBAMBI plays a crucial role in regulating lipid metabolism, glucose metabolism, and protein transport in shrimp. This study provides valuable insights for future research and utilization of BAMBI genes in shrimp and crustaceans.

Abstract 4883: NFE2L3 regulates the expression of BHLHE40 in triple-negative breast cancer

Abstract Introduction: Breast Cancer (BrCA) is one of the most common cancers, causing mortality in women worldwide. Among the subtypes of breast cancer, triple-negative breast cancer (TNBC) is the most aggressive due to the absence of three major receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). Treatment options for TNBC are limited because of the lack of these molecular targets. We hypothesized that the identification and characterization of novel transcriptional regulatory relationships in TNBC will provide etiologic insights, enhance therapeutic outcomes, and improve patient survival. Overexpression of NFE2L3 in BrCA has inhibited cell proliferation and metastasis while overexpression of BHLHE40 has produced an inverse effect. This study aims to characterize the relationship between NFE2L3 and BHLHE40 in TNBC cells. Methods: Patient data from the two groups of our focus from the Cancer Genome Atlas BrCA RNAseq gene expression dataset were examined: triple-positive breast cancer (TPBC) and TNBC. Differentially expressed genes (DEGs) between these groups were identified using DESeq2. Pathways over-represented among the DEGs were determined using enrichR. For their impact on biological interactions, those DEGs were mapped to curate protein-protein interactions - the STRING database. Additionally, DEGs were superposed on two network inference algorithm-generated transcriptional regulatory networks (TRNs). Virtual Inference of protein activity by Enriched Regulon analysis was applied to the generated TRN to identify master regulators driving gene expression differences between TPBC and TNBC. Of the master regulators observed, the activity of NFE2L3 was explored in TNBC cells. The TRNs indicate that NFE2L3 regulates the expression of BHLHE40. This regulatory relationship was explored in TNBC cells by RNA interference, qRT-PCR, immunoblotting, and co-immunoprecipitation assays to investigate NFE2L3 and BHLHE40 gene and protein expression and interaction. Results: Among 12,000 DEGs (5% FDR), over-represented pathways included “mitotic signaling” and “G1/S transition”. Master regulators with higher expression in TNBC included NFE2L3, HMGA1, TP53, FOXC1, and SOX9; those with lower expression included FOXA1, BHLHE40, AR, XBP1, and ESR1. On silencing the cells of NFE2L3 and performing qRTPCR analysis, the expression of BHLHE40 was decreased in the TNBC cells. The protein-protein interaction between NFE2L3 and BHLHE40 was also validated. Conclusion: DEGs up-regulated in TNBC participate in cell cycle signaling. And there is a novel transcriptional regulatory relationship between NFE2L3 and BHLHE40 in TNBC cells. Citation Format: Shail Rakesh Modi, Terrick Andey, George Acquaah-Mensah. NFE2L3 regulates the expression of BHLHE40 in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4883.

The pharyngeal taste organ of a blood-feeding insect functions in food recognition

BackgroundObligate blood-feeding insects obtain the nutrients and water necessary to ensure survival from the vertebrate blood. The internal taste sensilla, situated in the pharynx, evaluate the suitability of the ingested food. Here, through multiple approaches, we characterized the pharyngeal organ (PO) of the hematophagous kissing bug Rhodnius prolixus to determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises eight taste sensilla that become bathed with the incoming blood.ResultsWe showed that these taste sensilla house gustatory receptor neurons projecting their axons through the labral nerves to reach the subesophageal zone in the brain. We found that these neurons are electrically activated by relevant appetitive and aversive gustatory stimuli such as NaCl, ATP, and caffeine. Using RNA-Seq, we examined the expression of sensory-related gene families in the PO. We identified gustatory receptors, ionotropic receptors, transient receptor potential channels, pickpocket channels, opsins, takeouts, neuropeptide precursors, neuropeptide receptors, and biogenic amine receptors. RNA interference assays demonstrated that the salt-related pickpocket channel Rproppk014276 is required during feeding of an appetitive solution of NaCl and ATP.ConclusionsWe provide evidence of the role of the pharyngeal organ in food evaluation. This work shows a comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

Methylation of NRIP3 Is a Synthetic Lethal Marker for Combined PI3K and ATR/ATM Inhibitors in Colorectal Cancer.

The aim of this study was to investigate the epigenetic regulation and underlying mechanism of NRIP3 in colorectal cancer (CRC). Eight cell lines (SW480, SW620, DKO, LOVO, HT29, HCT116, DLD1, and RKO), 187 resected margin samples from colorectal cancer tissue, 146 cases with colorectal adenomatous polyps, and 308 colorectal cancer samples were used. Methylation-specific PCR, Western blotting, RNA interference assay, and a xenograft mouse model were used. NRIP3 exhibited methylation in 2.7% (5/187) of resected margin samples from colorectal cancer tissue, 32.2% (47/146) of colorectal adenomatous polyps, and 50.6% (156/308) of CRC samples, and the expression of NRIP3 was regulated by promoter region methylation. The methylation of NRIP3 was found to be significantly associated with late onset (at age 50 years or older), poor tumor differentiation, lymph node metastasis, and poor 5-year overall survival in CRC (all P < 0.05). In addition, NRIP3 methylation was an independent poor prognostic marker ( P < 0.05). NRIP3 inhibited cell proliferation, colony formation, invasion, and migration, while induced G1/S arrest. NRIP3 suppressed CRC growth by inhibiting PI3K-AKT signaling both in vitro and in vivo . Methylation of NRIP3 sensitized CRC cells to combined PI3K and ATR/ATM inhibitors. NRIP3 was frequently methylated in both colorectal adenomatous polyps and CRC. The methylation of NRIP3 may potentially serve as an early detection, late-onset, and poor prognostic marker in CRC. NRIP3 is a potential tumor suppressor. NRIP3 methylation is a potential synthetic lethal marker for combined PI3K and ATR/ATM inhibitors.