Higher Transcript Levels Research Articles

Characterization and expression profiling of fadd gene in response to exogenous Aeromonas hydrophila or Edwardsiella tarda challenge in the hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂)

In mammals, fas-associated protein with death domain (FADD) is involved in the process of cell apoptosis and plays a key role in innate immune signaling. Nevertheless, its detailed molecular mechanisms underlying apoptosis and immune responses to exogenous bacterial infections in teleosts remain largely unknown. In this study, a group of 60 hybrid yellow catfish (with the body weight of 25 ± 0.5 g) were used in subsequent experiments, we examined the expression profiling of fadd gene through comparative genomics and comparative immunological methods. Our results showed that fadd in the hybrid yellow catfish (hycfadd) exhibited similar gene and spatial structures to those in other vertebrates, and formed an independent clade in phylogeny. An expression pattern analysis revealed that hycfadd widely transcribed in various tissues, with the highest transcription level in the liver. Furthermore, expression profiling of hycfadd when intraperitoneally infected with 50 μL of exogenous Aeromonas hydrophila (2.0 × 107 CFU/mL) or Edwardsiella tarda (2.0 × 107 CFU/mL) within 48 h were significantly up-regulated in the kidney, spleen, liver and intestine. Important genes in the toll like receptor (tlr) 1-tlr2- myeloid differentiation primary response 88 (MyD88)-fadd-caspase (casp) 8 cascades of TLR signaling pathway in liver were significantly up-regulated after the A. hydrophila stimulation, suggesting that apoptosis through the TLR signaling pathway may have been triggered and activated, which were further verified in the liver, kidney, spleen, intestine and gill by a TUNEL assay. Overall, this study provides solid evidence for the bacterial induction of fadd-related apoptosis in teleosts.

A seamless connection from the burst sequence to the start codon is essential for polyhedrin hyperexpression in alphabaculoviruses

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of polyhedrin (POLH), and high-level transcription of the polh gene has been exploited to express foreign proteins in insect cells. While making Bombyx mori nucleopolyhedrovirus (BmNPV) polh mutants using a conventional transfer vector-based method, we noticed that a virus with a short sequence insertion just before the polh start codon produces fewer very small OBs. Detailed analysis of several BmNPV mutants revealed that insertions between the burst sequence and start codon markedly decrease POLH accumulation and polh transcription. We further confirmed this decrease using recombinant viruses expressing a reporter gene driven by the polh promoter. These findings underscore the critical importance of a seamless connection from the burst sequence to the start codon for baculovirus polh hyperexpression.

A member of the CAP protein superfamily, Hc-CAP-15, is important for the parasitic-stage development of Haemonchus contortus

BackgroundThe CAP superfamily proteins are distributed widely in eukaryotes and play crucial roles in various biological processes. However, very little is known about their functions in parasitic nematodes, including Haemonchus contortus, a socioeconomically important parasitic nematode. We have therefore studied a member of the CAP protein family of H. contortus, named Hc-CAP-15, with the aim to explore its roles in regulating the parasitic developmental process.MethodsThe conservation and phylogenetic relationships, spatial expression and temporal transcription profiles of Hc-CAP/cap-15, as well its biological function during parasite development were investigated using bioinformatics, immunofluorescence, real-time PCR and RNA interference (RNAi).ResultsHc-CAP-15 was found to be a single-domain CAP protein consisting of four conserved motifs that is localized in the cuticle, intestine and oocyte of adult worms. Hc-cap-15 was transcribed at all developmental stages of H. contortus, with the highest transcription level in parasitic fourth-stage larvae (L4s). Silencing of Hc-cap-15 resulted in a significant increase in the body length of L4s.ConclusionsThe results suggested that Hc-CAP-15 is important for the development of H. contortus. Our findings provide a basis for further study of the functions of the CAP family proteins in H. contortus and related parasitic nematodes.Graphical

A novel humanized tri-receptor transgenic mouse model of HAdV infection and pathogenesis.

Human adenovirus (HAdV) is a highly virulent respiratory pathogen that poses clinical challenges in terms of diagnostics and treatment. Currently, no effective therapeutic drugs or prophylactic vaccines are available for HAdV infections. One factor contributing to this deficiency is that existing animal models, including wild-type and single-receptor transgenic mice, are unsuitable for HAdV proliferation and pathology testing. In this study, a tri-receptor transgenic mouse model expressing the three best-characterized human cellular receptors for HAdV (hCAR, hCD46, and hDSG2) was generated and validated via analysis of transgene insertion, receptor mRNA expression, and protein abundance distribution. Following HAdV-7 infection, the tri-receptor mice exhibited high transcription levels at the early and late stages of the HAdV gene, as well as viral protein expression. Furthermore, the tri-receptor mice infected with HAdV exhibited dysregulated cytokine responses and multiple tissue lesions. This transgenic mouse model represents human HAdV infection and pathogenesis with more accuracy than any other reported animal model. As such, this model facilitates the comprehensive investigation of HAdV pathogenesis as well as the evaluation of potential vaccines and therapeutic modalities for HAdV.

A novel antisense lncRNA, ARBAG harboring an RNA destabilizing GWAS variant for C-peptide dictates the transcript isoforms of GABRA6 in cerebellum.

Human disease-associated genetic variations often map to long non-coding RNA (lncRNA) genes; however, elucidation of their functional impact is challenging. We previously identified a new genetic variant rs4454083 (A/G) residing in exon of an uncharacterized lncRNA ARBAG that strongly associates with plasma levels of C-peptide, a hormone that regulates insulin bioavailability. On the opposite strand, rs4454083 also corresponds to an intron of a cerebellum-specific GABA receptor subunit gene GABRA6 that mediates strengthening of inhibitory synapses by insulin. Here, we show that alleles of rs4454083 modulate transcript levels of the antisense gene, ARBAG, which then controls the expression of the sense gene, GABRA6. Predisposing to low C-peptide, GG (a minor allele genotype across ethnicities) stabilizes ARBAG lncRNA causing higher transcript levels in cerebellum. ARBAG lncRNA abundance leads to cleavage of GABRA6 mRNA at the complementary region, resulting in a dysfunctional GABRA6 protein that would not be recruited for synapse strengthening. Together, our findings in human cerebellar cell-line and induced Pluripotent Stem Cells (iPSCs) demonstrate biological role of a novel lncRNA in determining the ratio of mRNA isoforms of a protein-coding gene and the ability of an embedded variant in modulating lncRNA stability leading to inter-individual differences in protein expression.

Transgenic Microalgae Expressing Double-Stranded RNA as Potential Feed Supplements for Controlling White Spot Syndrome in Shrimp Aquaculture.

Viral infection of farmed fish and shellfish represents a major issue within the aquaculture industry. One potential control strategy involves RNA interference of viral gene expression through the oral delivery of specific double-stranded RNA (dsRNA). In previous work, we have shown that recombinant dsRNA can be produced in the chloroplast of the edible microalga Chlamydomonas reinhardtii and used to control disease in shrimp. Here, we report a significant improvement in antiviral dsRNA production and its use to protect shrimp against white spot syndrome virus (WSSV). A new strategy for dsRNA synthesis was developed that uses two convergent copies of the endogenous rrnS promoter to drive high-level transcription of both strands of the WSSV gene element in the chloroplast. Quantitative RT-PCR indicated that ~119 ng dsRNA was produced per liter of culture of the transgenic microalga. This represents an ~10-fold increase in dsRNA relative to our previous report. The engineered alga was assessed for its ability to prevent WSSV infection when fed to shrimp larvae prior to a challenge with the virus. The survival of shrimp given feed supplemented with dried alga containing the dsRNA was significantly enhanced (~69% survival) relative to a negative control (<10% survival). The findings suggest that this new dsRNA production platform could be employed as a low-cost, low-tech control method for aquaculture.

Epstein–Barr virus microRNA miR-BART2-5p accelerates nasopharyngeal carcinoma metastasis by suppressing RNase Ⅲ endonuclease DICER1

The development and progression of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. NPC is usually asymptomatic until it spreads to other sites, and more than 70% of cases are classified as locally advanced disease at diagnosis. EBV-positive nasopharyngeal cancer tissues express only limited viral latent proteins, but express high levels of the EBV-encoded BamHI-A rightward transcript (BART) miRNA molecules. Here, we report that EBV-miRNA-BART2-5p (BART2-5p) promotes NPC cell invasion and metastasis invivo and invitro but has no effect on NPC cell proliferation and apoptosis. In addition, BART2-5p altered the mRNA and miRNA expression profiles of NPC cells. The development of human tumors has been reported to be associated with altered miRNAs expression, and overall miRNAs expression is reduced in many types of tumors. We found that BART2-5p downregulated the expression of several miRNAs that could exert oncogenic functions. Mechanistically, BART2-5p directly targets the RNase III endonuclease DICER1, inhibiting its function of cleaving double-stranded stem-loop RNA into short double-stranded RNA, which in turn causes altered expression of a series of key epithelial-mesenchymal transition molecules, and reverting DICER1 expression can rescue this phenotype. Furthermore, analysis from clinical samples showed a negative correlation between BART2-5p and DICER1 expression. According to our study, high expression of BART2-5p in tissues and plasma of patients with NPC is associated with poor prognosis. Our results suggest that, BART2-5p can accelerate NPC metastasis through modulating miRNA profiles which are mediated by DICER1, implying a novel role of EBV miRNAs in the pathogenesis of NPC.

Fatty acid- and retinol-binding protein 6 does not control worm fatty acid content in Caenorhabditis elegans but might play a role in Haemonchus contortus parasitism

BackgroundNematodes have lost the ability to synthesise necessary lipids de novo and have complementally evolved the capacity to acquire fatty acids and their derivatives from a diet or host animal. Nematode-specific fatty acid- and retinol-binding protein (FAR) family is one approach that facilitates lipid acquisition, representing an Achilles heel and potential target against roundworms of socioeconomic significance. However, little is known about their detailed functional roles in either free-living or parasitic nematodes.MethodsA genome-wide identification and curation were performed to screen the FAR family members of Haemonchus contortus. Their transcription patterns in worms were also analysed to identify the targets. Ligand binding assay and molecular docking were conducted to verify the fatty acid binding activities of FAR proteins of interest. RNA interference (RNAi) and heterologous expression (rescuing) experiments were designed to explore the potential roles of the selected FAR protein in nematodes. Localisation of the protein was shown in sections of paraffin-embedded worms after an immunohistochemistry (IHC) assay.ResultsHere, an orthologue of far-6 in the model organism Caenorhabditis elegans (Ce-far-6) was functionally characterised in a parasitic nematode, H. contortus (Hc-far-6). It is demonstrated that knockdown of Ce-far-6 gene did not affect worm fat content, reproduction, or lifespan, but decreased worm body length at an early life stage of C. elegans. In particular, the Ce-far-6 mutant associated phenotype was completely rescued by Hc-far-6, suggesting a conserved functional role. Surprisingly, there were distinct tissue expression patterns of FAR-6 in the free-living C. elegans and parasitic H. contortus. High transcriptional level of Hc-far-6 and dominant expression of FAR-6 in the intestine of the parasitic stage of H. contortus link this gene/protein to nematode parasitism.ConclusionsThese findings substantially enhance our understanding of far genes and the associated lipid biology of this important parasitic nematode at a molecular level, and the approaches established are readily applicable to the studies of far genes in a broad range of parasites.Graphical

Effects of exogenous indole-3-acetic acid on the density of trichomes, expression of artemisinin biosynthetic genes, and artemisinin biosynthesis in Artemisia annua.

Artemisinin is the most practical medication for the treatment of malaria, but is only very minimally synthesized in Artemisia annua, significantly less than the market needs. In this study, indole-3-acetic acid (IAA) was used to investigate its effects on trichomes, artemisinin accumulation, and biosynthetic gene expression in A. anuua. The results showed that exogenous IAA could contribute to the growth and development of A. annua and increase the density of trichomes. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that artemisinin and dihydroartemisinic acid (DHAA) contents were increased by 1.9-fold (1.1mg/g) and 2.1-fold (0.51mg/g) after IAA treatment in comparison with control lines (CK), respectively. Furthermore, quantitative real-time PCR results showed that AaADS, AaCYP71AV1, AaALDH1, and AaDBR2, four critical enzyme genes for the biosynthesis of artemisinin, had relatively high transcription levels in leaves of A. annua treated with IAA. In summary, this study indicated that exogenous IAA treatment was a feasible strategy to enhance artemisinin production, which paves the way for further metabolic engineering of artemisinin biosynthesis.

ENTPD1 (CD39) and NT5E (CD73) expression in human glioblastoma: an in silico analysis.

Glioblastoma (GB) is the most common primary brain tumor in adults and carries a dismal prognosis, despite the best available treatment. The 2021 WHO Classification of CNS tumors incorporated molecular profiling to better define the characteristics and prognosis of tumor types and subtypes. These recent advances in diagnosis have not yet resulted in breakthrough therapies capable of shifting the treatment paradigm. NT5E/CD73 is a cell surface enzyme that participates in a complex purinergic pathway in synergy with ENTPD1/CD39 producing extracellular adenosine (ADO) from ATP. ADO promotes tumor progression by inducing immunosuppression, stimulating adhesion, invasion, and angiogenesis. In this study, we performed an in silico analysis of 156 human glioblastoma samples in an unexplored public database to investigate the transcriptional levels of NT5E and ENTPD1. The analysis revealed a significant increase in transcription levels of the genes under study in GB samples versus non-tumor brain tissue samples, in concordance with previous studies. High transcriptional levels of NT5E or ENTPD1 were independently related to a decrease in overall survival (p = 5.4e-04; 1.1e-05), irrespective of the IDH mutation status. NT5E transcriptional levels were significantly higher in GB IDH wild-type patients compared to GB IDH-mutant; however, ENTPD1 levels showed no significant difference, p ≤ 0.001. This in silico study indicates the need for a deeper understanding of the purinergic pathway relation to GB development, also inspiring future population studies that could explore ENTPD1 and NT5E not only as prognostic markers but also as potential therapeutic targets.

Analysis on driving factors of microbial community succession in Jiuyao of Shaoxing Huangjiu (Chinese yellow rice wine)

The microbial ecosystem of fermented food is greatly disturbed by human activities.Jiuyao is important saccharification starter for brewing huangjiu. The interaction between environmental factors and microorganisms significantly affected the microbial community structure at different stages of Jiuyao manufacturing. This study combined environmental factor analysis and high-throughput sequencing technology to comprehensively analyze the specific changes of microbial community and environmental factors in each fermentation stage of Jiuyao production and their correlation. The results showed that the activities of liquefaction enzyme, glycosylation enzyme and acid protease reached the highest value on the 8 th day (192 h) after the beginning of fermentation, and the cellulase activity reached the highest value at the end of fermentation. Pediococcus（37.5 %-58.2 %）, Weissella（9.2 %-27.0 %） and Pelomonas（0.1 %-12.1 %） were the main microbial genera in the genus bacteria, and Saccharomycopsis（37.1 %-52.0 %）, Rhizopus（12.5 %-31.0 %） and Saccharomyces（4.0 %-20.5 %） were the main microbial genera in the genus fungi. The results of correlation analysis showed that the microbial communities in Jiuyao were closely related to environmental factors. Most microbial communities were positively correlated with temperature, but negatively correlated with ambient humidity, CO2 concentration, acidity and water content of Jiuyao. In addition, the transcription levels of enzymes related to microbial glucose metabolism in Jiuyao were higher in the late stage of Jiuyao fermentation. Interestingly, these enzymes had high transcription levels in fungi such as Saccharomycopsis, Rhizopus and Saccharomyces, as well as in bacteria such as Pediococcus and Lactobacillus. This study provides a reference for revealing the succession rule of microbial community structure caused by environmental factors during the preparation of Jiuyao in Shaoxing Huangjiu.

Lactate oxidation is linked to energy conservation and to oxygen detoxification via a putative terminal cytochrome oxidase in Methanosarcina acetivorans

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35–40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.

Investigation of Compatibility between DNA Replication, Transcription, and Translation for in Vitro Central Dogma.

Recent advances in in vitro synthetic biology have made it possible to reconstitute various cellular functions in a test tube. However, the integration of these functions remains a major challenge. This study aimed to identify a suitable condition to achieve all three reactions that constitute the central dogma: transcription, translation, and DNA replication. Specifically, we investigated the effect of the concentrations of 11 nonprotein factors required for in vitro transcription, translation, and DNA replication on each of these reactions. Our results indicate that certain factors have opposing effects on the three reactions. For example, while dNTP is necessary for DNA replication, it inhibited translation, and both rNTP and tRNA, which are essential for transcription and translation, inhibited DNA replication with several DNA polymerases. We also found that these opposing effects were partially alleviated by optimizing the magnesium concentration. Using this knowledge, we successfully demonstrated transcription/translation-coupled DNA replication with higher levels of transcription and translation while maintaining a certain level of DNA replication. These findings not only provide useful insights for the development of a complex artificial system with the central dogma but also raise the question of how natural cells overcome the incompatibility between the three reactions.

Nomograms for predicting survival in patients with HER2 negative breast cancer.

e12528 Background: It is increasingly recognized that there is considerable heterogeneity in HER2-negative breast cancer. The clinicopathological characteristics differed between HER2-low and HER2-0 breast cancer evaluated by immunohistochemistry (IHC) or in situ hybridization (ISH). HER2-low breast cancer may benefit from novel antibody-drug conjugates. However, the immunohistochemical agreement of HER2-low is poor. We aimed to construct nomograms to predict the survival of HER2-negative breast cancer and to evaluate the effects of HER2 on survival at the RNA transcript level. Methods: A retrospective analysis was performed on breast cancer patients who were evaluated HER2-negative by IHC or ISH in the TCGA database. Univariate and multivariate cox regression analysis was used to evaluate whether the HER2 RNA transcript level and clinical parameters were independent predictors for overall survival (OS), progression-free survival (PFS), and Disease-Specific Survival (DSS), and nomograms were generated for the prediction of 1-, 2-, 3-, 5-year OS, PFS, and DSS based on significant parameters. Survival curves were drawn using the Kaplan-Meier methods to compare mortality outcomes according to HER2 RNA transcript level (75% vs. 25%). Results: A total of 769 HER2-negative patients were included in the analysis. The median age of patients was 56 years (range 26-30), and the majority were White (80.3%). Univariate and multivariate cox regression analysis showed that age, pN_stage, and pM_stage were independent predictors for OS, but HER2 was not. The C-index of a nomogram based on these three parameters was 0.723 (95%CI, 0.6555-1). HER2, pT_stage, pN_stage, and pM_stage were analyzed as independent predictors of PFS. HER2, pN_stage, and pM_stage were independent predictors of DSS. The C-indexes of established nomograms were 0.734 (95%CI, 0.672-1) and 0.775 (95%CI, 0.686-1) respectively. PFS and DSS were significantly better for patients with high HER2 RNA transcript levels (PFS, log-rank p = 0.00446, HR = 0.352; DSS, log-rank p = 0.0119, HR = 0.247). Conclusions: In the TCGA database, the HER2 RNA transcript level was an independent predictor for PFS and DSS in HER2-negative breast cancer. Stratification of patients at the RNA level can help clinicians optimize treatment for HER2-negative breast cancer.

Optimization of Constitutive Promoters Using a Promoter-Trapping Vector in Burkholderia pyrrocinia JK-SH007.

Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01-2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.

MacroH2A2 antagonizes epigenetic programs of stemness in glioblastoma

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

Transcription of HOX Genes Is Significantly Increased during Neuronal Differentiation of iPSCs Derived from Patients with Parkinson’s Disease

Parkinson’s disease (PD) is the most serious movement disorder, but the actual cause of this disease is still unknown. Induced pluripotent stem cell-derived neural cultures from PD patients carry the potential for experimental modeling of underlying molecular events. We analyzed the RNA-seq data of iPSC-derived neural precursor cells (NPCs) and terminally differentiated neurons (TDNs) from healthy donors (HD) and PD patients with mutations in PARK2 published previously. The high level of transcription of HOX family protein-coding genes and lncRNA transcribed from the HOX clusters was revealed in the neural cultures from PD patients, while in HD NPCs and TDNs, the majority of these genes were not expressed or slightly transcribed. The results of this analysis were generally confirmed by qPCR. The HOX paralogs in the 3′ clusters were activated more strongly than the genes of the 5′ cluster. The abnormal activation of the HOX gene program upon neuronal differentiation in the cells of PD patients raises the possibility that the abnormal expression of these key regulators of neuronal development impacts PD pathology. Further research is needed to investigate this hypothesis.

Extracellular arginine availability modulates eIF2α O-GlcNAcylation and heme oxygenase 1 translation for cellular homeostasis

BackgroundNutrient limitations often lead to metabolic stress during cancer initiation and progression. To combat this stress, the enzyme heme oxygenase 1 (HMOX1, commonly known as HO-1) is thought to play a key role as an antioxidant. However, there is a discrepancy between the level of HO-1 mRNA and its protein, particularly in cells under stress. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) is a recently discovered cellular signaling mechanism that rivals phosphorylation in many proteins, including eukaryote translation initiation factors (eIFs). The mechanism by which eIF2α O-GlcNAcylation regulates translation of HO-1 during extracellular arginine shortage (ArgS) remains unclear.MethodsWe used mass spectrometry to study the relationship between O-GlcNAcylation and Arg availability in breast cancer BT-549 cells. We validated eIF2α O-GlcNAcylation through site-specific mutagenesis and azido sugar N-azidoacetylglucosamine-tetraacylated labeling. We then evaluated the effect of eIF2α O-GlcNAcylation on cell recovery, migration, accumulation of reactive oxygen species (ROS), and metabolic labeling during protein synthesis under different Arg conditions.ResultsOur research identified eIF2α, eIF2β, and eIF2γ, as key O-GlcNAcylation targets in the absence of Arg. We found that O-GlcNAcylation of eIF2α plays a crucial role in regulating antioxidant defense by suppressing the translation of the enzyme HO-1 during Arg limitation. Our study showed that O-GlcNAcylation of eIF2α at specific sites suppresses HO-1 translation despite high levels of HMOX1 transcription. We also found that eliminating eIF2α O-GlcNAcylation through site-specific mutagenesis improves cell recovery, migration, and reduces ROS accumulation by restoring HO-1 translation. However, the level of the metabolic stress effector ATF4 is not affected by eIF2α O-GlcNAcylation under these conditions.ConclusionsOverall, this study provides new insights into how ArgS fine-tunes the control of translation initiation and antioxidant defense through eIF2α O-GlcNAcylation, which has potential biological and clinical implications.

COVID-19 and the Cardiovascular System: Requiem for a Medical Minotaur.

COVID-19 and the Cardiovascular System: Requiem for a Medical Minotaur.

The microRNA pathway core genes are indispensable for development and reproduction in the brown planthopper, Nilaparvata lugens.

MicroRNAs (miRNAs) are small single-stranded non-coding RNAs involved in a variety of cellular events by regulating gene expression at the post-transcriptional level. Several core genes in miRNA biogenesis have been reported to participate in a wide range of physiological events, in some insect species. However, the functional significance of miRNA pathway core genes in Nilaparvata lugens remains unknown. In the present study, we conducted a systematic characterisation of five core genes involved in miRNA biogenesis. We first performed spatiotemporal expression analysis and found that miRNA core genes exhibited similar expression patterns, with high expression levels in eggs and relatively high transcriptional levels in the ovaries and fat bodies of females. RNA interference experiments showed that injecting third-instar nymphs with dsRNAs targeting the miRNA core genes, NlAgo1, NlDicer1, and NlDrosha resulted in high mortality rates and various degrees of body melanism, moulting defects, and wing deformities. Further investigations revealed that the suppression of miRNA core genes severely impaired ovarian development and oocyte maturation, resulting in significantly reduced fecundity and disruption of intercellular spaces between follicle cells. Moreover, the expression profiles of miR-34-5p, miR-275-3p, miR-317-3p, miR-14, Let-7-1, and miR-2a-3p were significantly altered in response to the knockdown of miRNA core genes mixture, suggesting that they play essential roles in regulating miRNA-mediated gene expression. Therefore, our results provide a solid theoretical basis for the miRNA pathway in N. lugens and suggest that the NlAgo1, NlDicer1, and NlDrosha-dependent miRNA core genes are essential for the development and reproduction of this agricultural pest.