Eukaryotic Translation Elongation Research Articles

Exploring Canine Mammary Cancer through Liquid Biopsy: Proteomic Profiling of Small Extracellular Vesicles.

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.

UCHL3 promotes hepatocellular carcinoma progression by stabilizing EEF1A1 through deubiquitination

BackgroundHepatocellular carcinoma (HCC) ranks as the second leading cause of global cancer-related deaths and is characterized by a poor prognosis. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) have been proved to play important roles in various human cancers, whereas the deubiquitination of EEF1A1 was poorly understood.MethodsThe binding and regulatory relationship between Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) and EEF1A1 was validated using clinical tissue samples, reverse transcription quantitative real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, co-immunoprecipitation, and immunofluorescence, as well as ubiquitin detection and cyclohexamide tracking experiments. Finally, the impact of the UCHL3/EEF1A1 axis on HCC malignant behavior was analyzed through functional experiments and nude mouse models.ResultsUCHL3 was found to have a high expression level in HCC tissues. Tissue samples from 60 HCC patients were used to evaluate the correlation between UCHL3 and EEF1A1. UCHL3 binds to EEF1A1 through the lysine site, which reduces the ubiquitination level of EEF1A1. Functional experiments and nude mouse models have demonstrated that the UCHL3/EEF1A1 axis promotes the migration, stemness, and drug resistance of HCC cells. Reducing the expression of EEF1A1 can reverse the effect of UCHL3 on the malignant behavior of HCC cells.ConclusionOur findings revealed that UCHL3 binds and stabilizes EEF1A1 through deubiquitination. UCHL3 and EEF1A1 formed a functional axis in facilitating the malignant progression of HCC, proving new insights for the anti-tumor targeted therapy for HCC.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation.

Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0), TATA-binding protein (TBP), GAPDH, actin beta (ACTB), tubulin (TUB), aminolevulinic acid synthase 1 (ALAS1), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ), eukaryotic translational elongation factor 1 alpha (EF1a), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA), and beta-2-microglobulin (B2M)] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches. AIM To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR. METHODS We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (RPLP0 ), TATA-binding protein (TBP ), GAPDH , actin beta (ACTB ), tubulin (TUB ), aminolevulinic acid synthase 1 (ALAS1 ), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (YWHAZ ), eukaryotic translational elongation factor 1 alpha (EF1a ), succinate dehydrogenase complex, subunit A, flavoprotein (SDHA ), and beta-2-microglobulin (B2M )] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups. RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms’ two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking. CONCLUSION For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

EEF1D stabilized by SRSF9 promotes colorectal cancer via enhancing the proliferation and metastasis.

Colorectal cancer (CRC) is the third most common cancer and causes high mortality worldwide. Although CRC has been studied widely, the molecular mechanism is not completely known. Eukaryotic translation elongation factor 1 delta (EEF1D) participates in the progression of various tumors, however, the effect of EEF1D on CRC remains unclear. Here, we aimed to identify the potential mechanism of EEF1D in CRC. The expression levels of EEF1D were assessed in CRC samples. Functional analysis of EEF1D in CRC was detected in vitro and in vivo. The regulatory mechanism of EEF1D was identified with RNA immunoprecipitation, RNA pull-down assay, and proteomics analysis. Our findings confirmed that EEF1D was upregulated in human CRC tissues. Functionally, EEF1D overexpression accelerated cell proliferation and metastasis, whereas EEF1D knockdown inhibited cell proliferation and metastasis both in vitro and in vivo CRC models. Furthermore, we showed that EEF1D was upregulated by SRSF9 via binding to 3'UTR of EEF1D mRNA. EEF1D knockdown reversed the malignant phenotype induced by SRSF9 overexpression. These findings demonstrated that EEF1D promotes CRC progression, and EEF1D may be a molecular target against CRC.

Transcriptional Changes and Preservation of Muscle Protein Content in Hibernating Black Bears

Physical inactivity decreases mechanical load on the skeleton, which, when prolonged, leads to loss of muscle mass and strength in most mammalian species. In contrast, hibernating bears demonstrate limited loss of muscle mass and protein content over the prolonged periods (5-6 months) of inactivity and fasting during winter. This suggests that bears have natural adaptive mechanisms preserving muscle mass and functionality. To identify transcriptional changes that underlie molecular mechanisms attenuating muscle loss, we conducted a large-scale gene expression profiling (14,194 genes) in quadriceps muscle of adult black bear males, comparing hibernating animals (n=5) sampled in March, about 1 month before expected emergence, and summer active animals (n=5) using next generation sequencing (RNA-seq) of the transcriptome. In total, 336 differentially expressed (FDR<0.05) genes were up-regulated and 677 genes were down-regulated in muscle of hibernating bears. Gene set enrichment analysis with Reactome database showed significantly elevated proportion of overexpressed genes involved in eukaryotic translation initiation (FDR<0.0001) and elongation (FDR<0.0001) as well as in the mTORC1 mediated signaling pathway (FDR=0.021). In contrast, down-regulated genes were overrepresented among genes involved in catabolism of branched chain amino acid (BCAA: leucine, isoleucine and valine, FDR<0.0001) that suggests preservation of BCAA. These findings imply induction of protein biosynthesis through the mTORC1 signaling positively activated by availability of leucine in muscle during hibernation. Support for this conclusion comes from significant overexpression of Ras-related GTP-binding protein D (RRAGD, FC=2.12, FDR<0.0001), crucial regulator of the mTORC1 response to leucine availability, and up-regulation of eukaryotic translation initiation factor 4B (EIF4B, FC=1.95, FDR=0.012), downstream target of the mTORC1 mediated signaling. In addition to regulation of protein biosynthesis, activation of the mTOR signaling suppresses autophagy-dependent protein degradation. Consistent with reduction of autophagy, microtubule associated protein 1 light chain (MAP1LC3A, FC=-2.46, FDR<0.0001) and unc-51 like autophagy activating kinase (ULK1, FC=-1.69, FDR=0.052) were down-regulated in hibernating muscle. No expression differences were detected for two muscle atrophy markers, key members of the ubiquitin proteasome degradation, FBXO32 (Atrogin-1, MAFBX, FDR=0.85) and TRIM63 (MURF-1, FDR=0.63). The induction of protein biosynthesis and decrease in protein catabolism through the mTORC1 mediated signaling as response to BCAA availability likely contribute to the maintenance of muscle protein content through prolonged periods of immobility and fasting during hibernation. The follow up studies need to include metabolomic quantification of BCAA, phospho-proteomic assessment of key genes (RPS6KB1, RPS6) involved in the mTORC1 signaling and identification of upstream negative regulators suppressing BCAA catabolism in hibernating muscle. The work was supported by NIH COBRE under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects

Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient’s mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient’s mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification.

CDCA5-EEF1A1 interaction promotes progression of clear cell renal cell carcinoma by regulating mTOR signaling

BackgroundCell division cycle associated 5 (CDCA5) plays ontogenetic role in various human cancers. However, its specific function and regulatory mechanism in ccRCC remain uncertain.MethodsImmunohistochemistry and western blots were performed to investigate the expression of CDCA5 in ccRCC tissues. Genetic knockdown and upregulation of CDCA5 were performed to investigate its functional roles in ccRCC proliferation, migration, apoptosis and sunitinib resistance. Furthermore, Co-IP assay and LC–MS/MS were performed to investigate the underlying mechanisms.ResultsWe found that CDCA5 expression is frequently upregulated in ccRCC tumors and is associated with poor prognosis of ccRCC patients. Functionally, CDCA5 promotes proliferation, migration, and sunitinib resistance, while inhibiting apoptosis in ccRCC cells. In vivo mouse xenograft model confirms that silencing of CDCA5 drastically inhibits the growth of ccRCC. Mechanistically, we discovered that CDCA5 interacts with Eukaryotic Translation Elongation Factor 1 Alpha 1 (EEF1A1) to regulate mTOR signaling pathway, thereby promoting ccRCC progression.ConclusionsTaken together, our results demonstrate the significant role of CDCA5 in ccRCC progression. The findings may provide insights for the development of new treatment strategies targeting CDCA5 for ccRCC patients.

Abstract 5697: MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production

Abstract Despite the progress in endocrine therapy, targeted therapy and immunotherapy, the treatment of triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer, is challenging. This is closely associated with the lack of effective molecular targets in TNBC cells for therapeutic intervention. Here we report that the long noncoding RNA (lncRNA) MILIP drives TNBC cell survival, proliferation and tumorigenicity through complexing with transfer RNAs (tRNAs) to promote protein production, and thus represents a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, yet MILIP silencing diminished TNBC cell viability and retarded TNBC xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its well-established role in repressing p53 in other types of cancers. Mechanistical investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with tRNALeu and tRNASer, type II tRNAs, through their variable loops, thus facilitating the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein production and cell viability, with therapeutic potential revealed where targeting MILIP using Gapmers inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identified MILIP as an RNA translation elongation factor to promote protein production in TNBC cells and demonstrated that experimental therapy with MILIP Gapmers inhibits TNBC growth, indicating MILIP targeting is a potential avenue for developing improved TNBC treatment through blocking protein synthesis. Citation Format: Yuchen Feng, Simin Zheng, Xiaohong Zhao, Yuanyuan Zhang, Ran Xu, Liang Xu, Thomas Preiss, Lei Jin, Jinnan Gao, Xu Dong Zhang. MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production [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 5697.

Abstract 3172: Significance of PELP1 signaling in liver cancer progression

Abstract Background: Hepatocellular carcinoma (HCC) is the fastest-rising cause of cancer-related mortality in the United States. Furthermore, advanced HCC has a very poor prognosis, with a median survival time of only about ten months. Effective new targeted therapies are urgently needed for the treatment of HCC. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) is a protooncogene. PELP1 signaling has been implicated in the development of several cancers, however, its role in the progression of HCC remains unclear. The goal of this project is to investigate the significance and therapeutic potential of targeting PELP1 in HCC. Methods: In this investigation, we have used six HCC cell lines (HUH7, HEP3B, SNU398, SNU475, SNU423, and SNU449). Using immunohistochemistry (IHC), the expression of PELP1 in HCC tissue microarrays was examined. PELP1shRNA lentiviral particles were used to create PELP1 knockdown (KD) cells. Using well-established in vitro tests for cell proliferation, colony formation, and apoptosis, the effects of PELP1 KD or PELP1 inhibitor (SMIP34) were investigated. RNA-seq, RT-qPCR, Western blotting, IHC, reporter gene assays, and signaling pathway analysis were used in mechanistic investigations. Preclinical evaluations were conducted using mice xenograft models. Results: TNM plot analysis showed that HCC samples had increased PELP1 expression relative to normal tissue. Immunohistochemistry analysis of tissue microarray confirmed that HCC specimens overexpressed PELP1 in comparison to normal specimens. PELP1 knockdown with shRNA dramatically decreased HCC cell invasion, clonogenicity, and proliferation. Similarly, PELP1 inhibitor SMIP34 treatment significantly reduced cell viability, invasiveness, colony forming capacity, and induced apoptosis of HCC cells. RNA-seq analyses using PELP1 KD cells confirmed that PELP1 modulates the ribosome and the eukaryotic translation elongation pathways. Mechanistic studies confirmed that SMIP34 treatment contributed to the disruption of the Rix complex, which is crucial for ribosomal biogenesis. Puromycin labelling studies confirmed that PELP1 KD or SMIP34 treatment contributes to the reduction of ribosomal biogenesis and new protein synthesis. Furthermore, PELP1 KD or treatment with SMIP34 dramatically slowed the progression of HCC tumors in xenograft models. IHC analysis revealed reduced levels of the proliferation marker Ki67 in PELP1 KD/SMIP34 treated HCC xenograft tumors compared to controls. Conclusions: Overall, the results of our study point to PELP1 as a potential therapeutic target for HCC intervention. Citation Format: Uday Pratap, Khaled Mohamed Nassar, Xue Yang, Adriana Baker, Rahul Gopalam, William C. Arnold, Timilehin Adeniran, Marian Helena Hernandez Fernandez, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Luzhe Sun, Ratna K. Vadlamudi. Significance of PELP1 signaling in liver cancer progression [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 3172.

Abstract A109: Novel germline genetic variants in pseudogenes associated with overall survival in advanced stage ovarian cancer treated with carboplatin, paclitaxel, and bevacizumab: Results from the ROSiA trial

Abstract A109: Novel germline genetic variants in pseudogenes associated with overall survival in advanced stage ovarian cancer treated with carboplatin, paclitaxel, and bevacizumab: Results from the ROSiA trial

Identification and Validation of Reference Genes for qRT-PCR Analysis of Petal-Color-Related Genes in Rosa praelucens.

The flower's color is regarded as one of the most outstanding features of the rose. Rosa praelucens Byhouwer, an endemic and critically endangered decaploid wild rose species, is abundant in phenotypic diversity, especially in flower color variation, from white to different degrees of pink. The mechanism underlying this variation, e.g., the level of petal-color-related genes, is worth probing. Seven candidate reference genes for qRT-PCR analysis, including tubulin α chain (TUBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H2B (Histone2A), eukaryotic translation elongation factor 1-α (EEF1A), 60S ribosomal protein (RPL37), eukaryotic translation initiation factor 1-α (EIF1A), and aquaporins (AQP), were detected from the transcriptome datasets of full blooming flowers of white-petaled and pink-petaled individuals, and their expression stabilities were evaluated through qRT-PCR analysis. According to stability rankings analysis, EEF1A showed the highest stability and could be chosen as the most suitable reference gene. Moreover, the reliability of EEF1A was demonstrated via qRT-PCR analysis of six petal-color-related target genes, the expression patterns of which, through EEF1A normalization, were found to be consistent with the findings of transcriptome analysis. The result provides an optimal reference gene for exploring the expression level of petal-color-related genes in R. praelucens, which will accelerate the dissection of petal-color-variation mechanisms in R. praelucens.

Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis

Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.

The Role of Rosavin in the Pathophysiology of Bone Metabolism.

Rosavin, a phenylpropanoid in Rhodiola rosea's rhizome, and an adaptogen, is known for enhancing the body's response to environmental stress. It significantly affects cellular metabolism in health and many diseases, particularly influencing bone tissue metabolism. In vitro, rosavin inhibits osteoclastogenesis, disrupts F-actin ring formation, and reduces the expression of osteoclastogenesis-related genes such as cathepsin K, calcitonin receptor (CTR), tumor necrosis factor receptor-associated factor 6 (TRAF6), tartrate-resistant acid phosphatase (TRAP), and matrix metallopeptidase 9 (MMP-9). It also impedes the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), c-Fos, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways and blocks phosphorylation processes crucial for bone resorption. Moreover, rosavin promotes osteogenesis and osteoblast differentiation and increases mouse runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression. In vivo studies show its effectiveness in enhancing bone mineral density (BMD) in postmenopausal osteoporosis (PMOP) mice, restraining osteoclast maturation, and increasing the active osteoblast percentage in bone tissue. It modulates mRNA expressions by increasing eukaryotic translation elongation factor 2 (EEF2) and decreasing histone deacetylase 1 (HDAC1), thereby activating osteoprotective epigenetic mechanisms, and alters many serum markers, including decreasing cross-linked C-telopeptide of type I collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRACP5b), receptor activator for nuclear factor κ B ligand (RANKL), macrophage-colony-stimulating factor (M-CSF), and TRAP, while increasing alkaline phosphatase (ALP) and OCN. Additionally, when combined with zinc and probiotics, it reduces pro-osteoporotic matrix metallopeptidase 3 (MMP-3), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), and enhances anti-osteoporotic interleukin 10 (IL-10) and tissue inhibitor of metalloproteinase 3 (TIMP3) expressions. This paper aims to systematically review rosavin's impact on bone tissue metabolism, exploring its potential in osteoporosis prevention and treatment, and suggesting future research directions.

Role of Eukaryotic Translation Elongation Factor 1 Family Members in the Tumorigenesis and Progression of Lung Adenocarcinoma

Objective To investigate the role and mechanism of eukaryotic translation elongation factor 1(EEF1) family members (EEF1D,EEF1A1,and EEF1A2) in lung adenocarcinoma (LUAD) based on public databases.Methods We examined EEF1 member expression levels in human LUAD samples via The Cancer Genome Atlas in the UCSC Xena browser and the Clinical Proteomic Tumor Analysis Consortium.We analyzed the mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 and their correlations with pathological variables via the Mann-Whitney U test.The Kaplan-Meier curves were established to assess the prognostic values of EEF1D,EEF1A1,and EEF1A2.The single-sample gene set enrichment analysis algorithm was employed to explore the relationship between the expression levels of EEF1 members and tumor immune cell infiltration.Spearman and Pearson correlation analyses were performed to examine the relationship between the expression levels of EEF1 members and those of the genes in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.The immunohistochemical assay was employed to determine the expression levels of EEF1D,EEF1A1,and EEF1A2 in the LUAD tissue (n=75) and paracancer tissue (n=75) samples.Results The mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 showed significant differences between tumor and paracancer tissues (all P<0.001).The patients with high protein levels of EEF1A1 showed bad prognosis in terms of overall survival (P=0.039),and those with high protein levels of EEF1A2 showed good prognosis in terms of overall survival (P=0.012).The influence of the mRNA level of EEF1D on prognosis was associated with pathological characteristics.The expression levels of EEF1 members were significantly associated with the infiltration of various immune cells and the expression of key molecules in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.Conclusion EEF1D,EEF1A1,and EEF1A2 are associated with the progression of LUAD,serving as the candidate prognostic markers for LUAD.

Phosphorylated Adapter RNA Export Protein Is Methylated at Lys 381 by an Methyltransferase-like 21C (METTL21C).

Methyltransferase-like 21C (METTL21C) is a member of the non-histone methyltransferase superfamily, which mainly mediates the methylation of lysine (Lys) residues. The main types of modification are Lys dimethylation and trimethylation. However, at present, most of the studies on METTL21C are focused on humans and mice, and there are few reports on poultry. Therefore, chicken embryo fibroblasts (DF-1) were selected as the object of study. To explore the function of chicken METTL21C (chMETTL21C) in the proliferation of DF-1 cells, flow cytometry and qPCR were used to detect the function of chicken METTL21C in the proliferation of DF-1 cells. The results showed that overexpression of METTL21C blocked the cell cycle in the G1max S phase, thus inhibiting cell proliferation. In addition, based on proteomic analysis, stable overexpression of METTL21C may inhibit the proliferation of DF-1 cells by mediating lysine trimethylation of proliferation-related proteins phosphorylated adapter RNA export protein (PHAX), nucleoside diphosphate kinases (NDPKs), eukaryotic transcription extension factor (eukaryotic translation elongation factor 1A,e EF1A), and inversin (Invs). Through immunoprecipitation (co-IP) and liquid chromatography-mass spectrometry (LC-MS/MS) analysis, METTL21C-mediated PHAX Lys-381 methylation was confirmed to be involved in the regulation of DF-1 cell proliferation. The results of this study provide a reference for analyzing the methylation function of METTL21C and the mechanism of regulating the growth and development of chicken cells.

Knockout of the tomato translational elongation factor using CRISPR-Cas9 technology

Pathogenic viruses cause severe tomato losses around the world despite the development of both classical breeding and biotechnological methods. Since replication of phytoviruses involves the interaction between viral components and host plant factors, therefore loss-of-function mutations in the latters can confer viral resistance in plants. There are evidences that eukaryotic translation elongation factor 1 (eEF1) proteins are involved in the replication of some plant viruses. However, the involvement of individual subunits of the eEF1B in the viral cycle is still poorly understood.&#x0D; This work is devoted to the study of the role of the eEF1B factor in the development of tomato virus infection. The contribution of each of the α, β and γ subunits of the eEF1B factor to tomato viral resistance will be determined by CRISPR-Cas9-induced targeted mutagenesis of corresponding gene sequences. As an applied aspect, we expect to find ways to create tomato plants with increased resistance to certain viral diseases. A series of binary vectors contained sequences encoded different RNAs targeting the eEF1B subunit genes was constructed. As a result of Agrobacterium-mediated transformation of tomato, more than 300 independent transgenic lines were obtained. The presence of expression cassettes with functional genes (Cas9 and sgRNAs) was confirmed by PCR. The presence of mutations in target sequences was detected using T7E1 analysis and sequencing. It turned out that the majority of transgenic lines carrying mutations have a chimeric genotype, and mutations of the target genes in the homozygous state were not detected. The propagation of self-pollinated transgenic plants under greenhouse condition and following analyses of target genes to segregate the insertion of foreign DNA and obtain homozygous mutations in eEF1B subunit sequences are in progress.

Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins.

The Chinese hamster ovary (CHO) cell line is the main host for the high-titer production of therapeutic and diagnostic proteins in the biopharmaceutical industry. In most cases, plasmids for efficient protein expression in CHO cells are based on the cytomegalovirus (CMV) promoter. The autologous Chinese hamster eukaryotic translation elongation factor 1α (EEF1A1) promoter is a viable alternative to the CMV promoter in industrial applications. The EEF1A1 promoter and its surrounding DNA regions proved to be effective at maintaining high-level and stable expression of recombinant proteins in CHO cells. EEF1A1-based plasmids' large size can lead to low transfection efficiency and hamper target gene amplification. We hypothesized that an efficient EEF1A1-based expression vector with a long terminal repeat fragment from the Epstein-Barr virus (EBVTR) could be truncated without affecting promoter strength or the long-term stability of target gene expression. We made a series of deletions in the downstream flanking region of the EEF1A1 gene, and then in its upstream flanking region. The resulting plasmids, which coded for the enhanced green fluorescent protein (eGFP), were tested for the level of eGFP expression in the populations of stably transfected CHO DG44 cells and the stability of eGFP expression in the long-term culture in the absence of selection agents. It was shown that in the presence of the EBVTR fragment, the entire downstream flanking region of the EEF1A1 gene could be excluded from the plasmid vector. Shortening of the upstream flanking region of the EEF1A1 gene to a length of 2.5 kbp also had no significant effect on the level of eGFP expression or long-term stability. The EBVTR fragment significantly increased expression stability for both the CMV and EEF1A1 promoter-based plasmids, and the expression level drop during the two-month culture was more significant for both CMV promoter-based plasmids. Target protein expression stability for the truncated plasmid, based on the EEF1A1 gene and EBVTR fragment, is sufficient for common biopharmaceutical applications, making these plasmid vectors a viable alternative to conventional CMV promoter-based vectors.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

Interaction of lncRNA Gm2044 and EEF2 promotes estradiol synthesis in ovarian follicular granulosa cells

The functions and molecular mechanisms of long noncoding RNA (lncRNA) in reproduction have been widely studied at present. However, lncRNA regulating hormone synthesis in ovarian follicular granulosa cells has not been sufficiently studied. Our previous research demonstrated that lncRNA Gm2044 could promote estradiol synthesis in follicular granulosa cells. In this study, we identified 21 binding proteins of lncRNA Gm2044 in ovarian follicles using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). RNA immunoprecipitation (RNA IP) and reverse transcription PCR (RT-PCR) confirmed that lncRNA Gm2044 can interact with eukaryotic translation elongation factor 2 (EEF2) protein. Furthermore, we constructed lncRNA Gm2044 knockout mice using the CRISPR/Cas9 method. Although the follicular development and fertility of female lncRNA Gm2044 knockout mice were not affected, the serum estradiol concentration in female lncRNA Gm2044 knockout mice significantly decreased. Western blotting and ELISA revealed that lncRNA Gm2044 may promote the binding of EEF2 to Nr5a1 mRNA and then enhance the Nr5a1 mRNA translation, and the upregulated NR5A1 protein can strengthen estradiol synthesis. To determine the potential signaling pathway of lncRNA Gm2044 regulating estradiol synthesis, transcriptome sequencing was performed for ovaries of adult lncRNA Gm2044 knockout mice, which identified 565 significant up-regulated genes and 303 significant down-regulated genes, which were then analyzed with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and validated by molecular experiments. Understanding how lncRNA Gm2044/EEF2 protein regulates estradiol synthesis will help treat estrogen-related reproductive diseases.