Eukaryotic Systems Research Articles

Reversible crowdedness of pH-responsive and host–guest active polymersomes: Mimicking µm-sized cell structures

The structure–function characteristics of isolated artificial organelles (AOs) in protocells are mainly known, but there are few reports on clustered or aggregated AOs. To imitate µm-sized complex and heterogeneous cell structures, approaches are needed that enable reversible changes in the aggregation state of colloidal structures in response to chemical, biological, and external stimuli. To construct adaptive organelle-like or cell-like reorganization characteristics, we present an advanced crosslinking strategy to fabricate clustered polymersomes as a platform based on host–guest interactions between azobenzene-containing polymersomes (Azo-Psomes) and a β-cyclodextrin-modified polymer (β-CD polymer) as a crosslinker. First, the reversible (dis)assembly of clustered Azo-Psomes is carried out by the alternating input of crosslinker and adamantane-PEG3000 as a decrosslinker. Moreover, cluster size dependence is demonstrated by environmental pH. These offer the controlled fabrication of various homogeneous and heterogeneous Azo-Psomes structures, including the size regulation and visualization of clustered AOs through a fluorescent enzymatic cascade reaction. Finally, a temperature-sensitive crosslinking agent with β-CD units can promote the coaggregation of Azo-Psomes mediated by temperature changes. Overall, these (co–)clustered Azo-Psomes and their successful transformation in AOs may provide new features for modelling biological systems for eukaryotic cells and systems biology.

Structural view on autophagosome formation.

Autophagy is a conserved intracellular degradation system in eukaryotes, involving the sequestration of degradation targets into autophagosomes, which are subsequently delivered to lysosomes (or vacuoles in yeasts and plants) for degradation. In budding yeast, starvation-induced autophagosome formation relies on approximately 20 core Atg proteins, grouped into six functional categories: the Atg1/ULK complex, the phosphatidylinositol-3 kinase complex, the Atg9 transmembrane protein, the Atg2-Atg18/WIPI complex, the Atg8 lipidation system, and the Atg12-Atg5 conjugation system. Additionally, selective autophagy requires cargo receptors and other factors, including a fission factor, for specific sequestration. This review covers the 30-year history of structural studies on core Atg proteins and factors involved in selective autophagy, examining X-ray crystallography, NMR, and cryo-EM techniques. The molecular mechanisms of autophagy are explored based on protein structures, and future directions in the structural biology of autophagy are discussed, considering the advancements in the era of AlphaFold.

DNA vaccine incorporated poly (lactic-co-glycolic) acid (PLGA) microspheres offer enhanced protection against Aeromonas hydrophila infection

Encapsulation of DNA vaccines onto carriers enhances the immunogenicity of an antigen. Specifically, biodegradable polymers offer sustained release of vaccines which is crucial for any targeted delivery approach. Poly (lactic-co-glycolic) acid (PLGA) microspheres were used to load a DNA vaccine having a targeted gene of outer membrane protein (OMP) of Aeromonas hydrophila to clone and construct a DNA vaccine using a eukaryotic expression vector system (pVAX1-OMP DNA) and delivery in Carassius auratus against A. hydrophila infection. PLGA microspheres were prepared by emulsion technique oil-in-water and characterized by a High-Resolution Scanning Electron Microscope (HR-SEM). The results of PLGA-pVAX1-OMP DNA microspheres shows that average of 100–150 μm particle size and a loading efficiency (LE) of 68.8 %. Results indicate that C. auratus fed with PLGA-pVAX1-OMP DNA microspheres revealed a significant improvement in innate immune response, which includes, myeloperoxidase activity, respiratory burst and total immunoglobulin level compared with control group fish. The immune-related gene, IL1β, IL10, TGF, c-type, and g-type lysozyme also showed significantly higher expression after immunization. Furthermore, dietary supplementation of the PLGA-pVAX1-OMP DNA (G III) group exhibited a significantly higher survival rate (78 %) than the control group of fish. These results help us to understand the of mechanism of DNA vaccine administrated feed through PLGA nanoparticles resistance to infection by regulating systemic and innate immunity in Carassius auratus.

Dracorhodin targeting CMPK2 attenuates inflammation: A novel approach to sepsis therapy.

Despite all modern advances in medicine, an effective drug for treating sepsis has yet to be found. The discovery of CMPK2 spurred hopes for the treatment of sepsis. However, CMPK2-untapped target inhibitors are still an enormous obstacle that has hindered the CMPK2-centric treatment of sepsis. Here, we found that the CMPK2 gene is highly expressed in the whole blood of sepsis patients by RNA-Seq. First, recombinant CMPK2 was purified by a eukaryotic expression purification system, and the activity of recombinant CMPK2 was detected by the ADP-GLO assay. Second, we developed an affinity MS strategy combined with quantitative lysine reactivity profiling to discover CMPK2 ligands from the active ingredients of Chinese herbs. In addition, the dissociation constant Kd of the ligand and the target protein CMPK2 was further detected by microscale thermophoresis technology. Third, we used this strategy to identify a naturally sourced small molecule, dracorhodin (DP). Using mass spectrometry-based quantitative lysine reactivity profiling combined with a series of mutant tests, the results show that K265 acts as a bright hotspot of DP inhibition of CMPK2. Fourth, immune-histochemical staining, ELISAs, RT-qPCR, flow cytometry and immunoblotting were used to illustrate the potential function and related mechanism of DP in regulating sepsis injury. Our results suggest that DP exerts powerful anti-inflammatory effects by regulating the NLRP3 inflammasome via the lipopolysaccharide (LPS)-induced CMPK2 pathway. Strikingly, DP significantly attenuated LPS-induced sepsis in a mouse model, but its effect was weakened in mice with myeloid-specific Cmpk2 ablation. We provide a new framework that provides more valuable information for new therapeutic approaches to sepsis, including the establishment of screening strategies and the development of target drugs to provide a theoretical basis for ultimately improving clinical outcomes for sepsis patients. Collectively, these findings reveal that DP is a promising CMPK2 inhibitor for the treatment of sepsis.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Biochemical and Structural Characterization of a Uronic Acid Oxidase from Citrus sinensis

AbstractAldaric acids are attractive diacids that can be prepared by selective oxidation of carbohydrates. For this, effective biocatalysts are in demand. This work reports on the discovery, biochemical and structural characterization of a VAO‐type flavin‐containing carbohydrate oxidase from Citrus sinensis: URAOCs3. URAOCs3 could be overexpressed using prokaryotic and eukaryotic expression systems. Extensive biochemical characterization revealed that the enzyme displays a high thermostability and an exquisite selectivity for uronic acids, galacturonic acid and glucuronic acid. The enzyme was further investigated by determining the crystal structure. The selective oxidation of D‐galacturonic acid in a complex mixture was demonstrated, showing how URAOCs3 was found to be highly effective in selectively producing galactaric acid while leaving other carbohydrates untouched. In addition to the specific discovery of URAOCs3, these findings suggest that plant proteomes can be an interesting source for new biocatalysts.

RNA Polymerase II evolution and adaptations: Insights from Plasmodium and other parasitic protists

The C-terminal domain (CTD) of RNA polymerase II plays a crucial role in regulating transcription dynamics in eukaryotes. The phosphorylation of serine residues within the CTD controls transcription initiation, elongation, and termination. While the CTD is highly conserved across eukaryotes, lower eukaryotes like protists, including Plasmodium, exhibit some differences. In this study, we performed a comparative analysis of CTD in eukaryotic systems to understand why the parasites evolved in this particular manner. The Plasmodium falciparum RPB1 is exceptionally large and feature a gap between the first and second heptad repeats, resulting in fifteen canonical heptad repeats excluding the initial repeat. Analysis of this intervening sequence revealed sub motifs of heptads where two serine residues occupy the first and fourth positions (S1X2X3S4). These motifs lie in the intrinsically disordered region of RPB1, a characteristic feature of the CTD. Interestingly, the S1X2X3S4 sub-motif was also observed in early-divergingeukaryotes like Leishmania major, which lack canonical heptad repeats. Furthermore, eukaryotes across the phylogenetic tree revealed a sigmoid pattern of increasing serine frequency in the CTD, indicating that serine enrichment is a significant step in the evolution of heptad-rich RPB1. Based on these observations and analysis, we proposed an evolutionary model for RNA Polymerase II CTD, encompassing organisms previously deemed exceptions, notably Plasmodium species. Thus, our study provides novel insights into the evolution of the CTD and will prompt further investigations into the differences exhibited by Plasmodium RNA Pol II and determine if they confer a survival advantage to the parasite.

A collagen-binding SIRPαFc fusion protein for targeted cancer immunotherapy

Collagen is abundant but exposed in tumor due to the abnormal tumor blood vessels, thus is considered as a tumor-specific target. The A3 domain of von Willebrand factor (vWF A3) is a kind of collagen-binding domain (CBD) which could bind collagen specifically. Previously we reported a chemosynthetic CBD-SIRPαFc conjugate, which could block CD47 and derived tumor-targeting ability by CBD. CBD-SIRPαFc conjugate represented improved anti-tumor efficacy with increased MHC II+ M1 macrophages, but the uncertain coupling ratio remained a problem. Herein, we produced a vWF A3-SIRPαFc fusion protein through eukaryotic expression system. It was examined at both molecular and cellular levels with its collagen affinity, uninfluenced original affinity to targets and phagocytosis-promoting function compared to unmodified SIRPαFc. Living imaging showed that vWF A3-SIRPαFc fusion protein derived the improved accumulation and retention in tumor than SIRPαFc. In the MC38 allograft model, vWF A3-SIRPαFc demonstrated a superior tumor-suppressing effect, characterized by increased MHC II+ M1 macrophages and T cells (particularly CD4+ T cells). These results revealed that vWF A3-SIRPαFc fusion protein derived tumor-targeting ability, leading to improved anti-tumor immunotherapeutic efficacy compared to SIRPαFc. Altogether, vWF A3 improved the anti-tumor efficacy and immune-activating function of SIRPαFc, supporting targeting tumor collagen as a possible targeted strategy.

Salmonella delivers H9N2 influenza virus antigens via a prokaryotic and eukaryotic dual-expression vector and elicits bivalent protection against avian influenza and fowl typhoid

The H9N2 avian influenza virus significantly affects the health of poultry and humans. We identified a prokaryotic and eukaryotic dual-expression vector system, pJHL270, that can provide simultaneous MHC class I and II stimulation of the host immune system, and we designed vaccine antigens by selecting the consensus HA1 sequence and M2e antigens from H9N2 virus circulating in South Korea from 2000 to 2021. The genes were cloned into the pJHL270 vector, and the cloned plasmid was delivered by a live-attenuated Salmonella Gallinarum (SG) strain. The immunity and protective efficacy of the SG-based H9N2 vaccine construct, JOL2922, against avian influenza and fowl typhoid (FT) were evaluated. The Ptrc and CMV promoters conferred antigen expression in prokaryotic and eukaryotic cells to induce balanced Th-1/Th-2 immunity. Chickens immunized with JOL2922 yielded high antigen-specific humoral and mucosal immune responses. qRT-PCR revealed that the strain generated polyfunctional IFN-γ and IL-4 secretion in immunized chickens. Furthermore, a FACS analysis showed increased CD3CD4+ and CD3CD8+ T-cell subpopulations following immunization. Peripheral Blood Mononuclear Cells (PBMCs) harvested from the immunized chickens significantly increased MHC class I and II expression, 3.5-fold and 2.5-fold increases, respectively. Serum collected from the immunized groups had an evident hemagglutinin inhibition titer of ≥6 log2. Immunization reduced the lung viral titer by 3.8-fold within 5 days post-infection. The strain also generated SG-specific humoral and cellular immune responses. The immunized birds all survived a virulent SG wild-type challenge. In addition, the bacterial burden was reduced by 2.7-fold and 2.1-fold in spleen and liver tissue, respectively, collected from immunized chickens. Our data indicate that an attenuated SG strain successfully delivered the dual-expression vector system and co-stimulated MHC class I and II antigen presentation pathways via exogenous and endogenous antigen presentation, thereby triggering a balanced Th-1/Th-2-based immune response and conferring effective protection against avian influenza and FT.

Rational design of eukaryotic riboswitches that up-regulate IRES-mediated translation initiation with high switching efficiency through a kinetic trapping mechanism in vitro.

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

Probing archaeal cell biology: exploring the use of dyes in the imaging of Sulfolobus cells.

Archaea are key players in many critical ecological processes. In comparison to eukaryotes and bacteria, however, our understanding of both the cell biology and diversity of archaea remains limited. While archaea inhabit a wide range of environmental conditions, many species are extremophiles, surviving in extreme temperature, salt or pH conditions, making their cell biology hard to study. Recently, our understanding of archaeal cell biology has been advanced significantly by the advent of live cell imaging in extremis as well as the development of genetic tools to exogenously express fluorescent proteins in some mesophilic archaeal model systems, e.g., Haloferax volcanii. However, for most archaeal species, especially thermophilic species or emerging model systems without well characterized genetic tools, live cell imaging remains dependent on fluorescent chemical probes to label and track the dynamics of living cells. While a wide range of fluorescent stains and markers that label different components of the cell are available commercially, their use has usually been optimized for use in a small number of eukaryotic cell systems. Here we report the successes and failures of the application of membrane, DNA, S-layer and cytoplasm markers in live cell imaging of archaea, as well as the optimization of fixation and immunolabelling approaches. We have applied these markers to the thermoacidophilic archaeon Sulfolobus acidocaldarius, but expect some to work in other archaeal species. Furthermore, those procedures that failed in S. acidocaldarius may still prove useful for imaging archaea that grow at a more neutral pH and/or at a less extreme temperature.

The Case for the Target of Rapamycin Pathway as a Candidate Circadian Oscillator.

The molecular mechanisms that drive circadian (24 h) rhythmicity have been investigated for many decades, but we still do not have a complete picture of eukaryotic circadian systems. Although the transcription/translation feedback loop (TTFL) model has been the primary focus of research, there are many examples of circadian rhythms that persist when TTFLs are not functioning, and we lack any good candidates for the non-TTFL oscillators driving these rhythms. In this hypothesis-driven review, the author brings together several lines of evidence pointing towards the Target of Rapamycin (TOR) signalling pathway as a good candidate for a non-TTFL oscillator. TOR is a ubiquitous regulator of metabolism in eukaryotes and recent focus in circadian research on connections between metabolism and rhythms makes TOR an attractive candidate oscillator. In this paper, the evidence for a role for TOR in regulating rhythmicity is reviewed, and the advantages of TOR as a potential oscillator are discussed. Evidence for extensive feedback regulation of TOR provides potential mechanisms for a TOR-driven oscillator. Comparison with ultradian yeast metabolic cycles provides an example of a potential TOR-driven self-sustained oscillation. Unanswered questions and problems to be addressed by future research are discussed.

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway

Mechanosensitive mechanisms are often used to sense damage to tissue structure, stimulating matrix synthesis and repair. While this kind of mechanoregulatory process is well recognized in eukaryotic systems, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, direct compression and hydrostatic pressure. In all cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Reduction in cell envelope stiffness following removal of the endopeptidase ShyA led to large increases in cell envelope deformation and substantially increased VxrAB response, further supporting the responsiveness of VxrAB. Our findings demonstrate a mechanosensitive gene regulatory system in bacteria and suggest that mechanical signals may contribute to the regulation of cell wall homeostasis.

Reactive Oxygen Species: A Crosslink between Plant and Human Eukaryotic Cell Systems.

Reactive oxygen species (ROS) are important regulating factors that play a dual role in plant and human cells. As the first messenger response in organisms, ROS coordinate signals in growth, development, and metabolic activity pathways. They also can act as an alarm mechanism, triggering cellular responses to harmful stimuli. However, excess ROS cause oxidative stress-related damage and oxidize organic substances, leading to cellular malfunctions. This review summarizes the current research status and mechanisms of ROS in plant and human eukaryotic cells, highlighting the differences and similarities between the two and elucidating their interactions with other reactive substances and ROS. Based on the similar regulatory and metabolic ROS pathways in the two kingdoms, this review proposes future developments that can provide opportunities to develop novel strategies for treating human diseases or creating greater agricultural value.

Expression and purification of Drosophila OBP44a with the aids of LC-MS and NMR

The production of highly purified native soluble proteins in large quantities is crucial for studying protein structure and function. Odorant binding proteins (OBPs) are small, soluble, extracellular proteins with multiple disulfide bonds, whose functions include, but are not limited to, binding hydrophobic molecules and delivering them to their corresponding receptors expressed on insect olfactory receptor neurons. Expression of proteins with multiple disulfide bonds like OBPs usually results in insolubility and low yield, which has been a significant barrier to understanding their biological roles and physiological functions. In the E. coli system, expression of OBPs often results in insoluble inclusion bodies or a limited amount of periplasmic soluble proteins. Although expression of OBPs in eukaryotic systems such as Sf9 insect cells or yeast Pichia pastoris can increase the solubility of the protein, the process remains insufficient. Additionally, monitoring the purity and native apo state of the protein is critical for establishing the correct conformation of the protein. In this study, we employed an E. coli host with an altered intracellular environment to produce cytosolic soluble OBP44a protein, which yielded over 100 mg/L. We monitored the integrity of disulfide bonds throughout the purification process using LC-MS and used NMR to ensure the final product adopted a single conformation. Our study presents an efficient method for obtaining large quantities of soluble proteins in a single conformation, which enables extensive in vitro studies of secreted proteins like OBPs.

RNA processing and degradation mechanisms shaping the mitochondrial transcriptome of budding yeasts.

Yeast mitochondrial genes are expressed as polycistronic transcription units that contain RNAs from different classes and show great evolutionary variability. The promoters are simple, and transcriptional control is rudimentary. Posttranscriptional mechanisms involving RNA maturation, stability, and degradation are thus the main force shaping the transcriptome and determining the expression levels of individual genes. Primary transcripts are fragmented by tRNA excision by RNase P and tRNase Z, additional processing events occur at the dodecamer site at the 3' end of protein-coding sequences. groups I and II introns are excised in a self-splicing reaction that is supported by protein splicing factors encoded by the nuclear genes, or by the introns themselves. The 3'-to-5' exoribonucleolytic complex called mtEXO is the main RNA degradation activity involved in RNA turnover and processing, supported by an auxiliary 5'-to-3' exoribonuclease Pet127p. tRNAs and, to a lesser extent, rRNAs undergo several different base modifications. This complex gene expression system relies on the coordinated action of mitochondrial and nuclear genes and undergoes rapid evolution, contributing to speciation events. Moving beyond the classical model yeast Saccharomyces cerevisiae to other budding yeasts should provide important insights into the coevolution of both genomes that constitute the eukaryotic genetic system.

Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster).

Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. In this report, we show that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. Our research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.

Cysteamine-mediated blockade of the glycine cleavage system modulates epithelial cell inflammatory and innate immune responses to viral infection

Transient blockade of glycine decarboxylase (GLDC) can restrict de novo pyrimidine synthesis, which is a well-described strategy for enhancing the host interferon response to viral infection and a target pathway for some licenced anti-inflammatory therapies. The aminothiol, cysteamine, is produced endogenously during the metabolism of coenzyme A, and is currently being investigated in a clinical trial as an intervention in community acquired pneumonia resulting from viral (influenza and SARS-CoV-2) and bacterial respiratory infection. Cysteamine is known to inhibit both bacterial and the eukaryotic host glycine cleavage systems via competitive inhibition of GLDC at concentrations, lower than those required for direct antimicrobial or antiviral activity. Here, we demonstrate for the first time that therapeutically achievable concentrations of cysteamine can inhibit glycine utilisation by epithelial cells and improve cell-mediated responses to infection with respiratory viruses, including human coronavirus 229E and Influenza A. Cysteamine reduces interleukin-6 (IL-6) and increases the interferon-λ (IFN-λ) response to viral challenge and in response to liposomal polyinosinic:polycytidylic acid (poly I:C) simulant of RNA viral infection.

Salmonella-Delivered COBRA-HA1 Antigen Derived from H1N1 Hemagglutinin Sequences Elicits Broad-Spectrum Protection Against Influenza A Subtypes

A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines. In this study, a strategy called computationally optimized broadly reactive antigen (COBRA) was used to generate a consensus sequence of the hemagglutinin globular head portion (HA1) of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs. Constructs carrying different HA1 regions were delivered into eukaryotic cells by Salmonella-mediated bactofection using a Semliki Forest virus RNA-dependent RNA polymerase (RdRp)-based eukaryotic expression system, pJHL204. Recombinant protein expression was confirmed by Western blot and immunofluorescence assays. Mice immunized with the designed constructs produced a humoral response, with a significant increase in immunoglobulin G (IgG) levels, and a cell-mediated immune response, including a 1.5-fold increase in CD4+ and CD8+ T cells. Specifically, constructs #1 and #5 increased the production of interferon-γ (IFN-γ) producing CD4+ and CD8+ T cells, skewing the response toward the T helper type 1 cell (Th1) pathway. Additionally, interleukin-4 (IL-4)-producing T cells were upregulated 4-fold. Protective efficacy was demonstrated, with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer > 40 against the selected viral strains. The designed constructs conferred a broadly protective immune response, resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34, A/Brisbane/59/2007, A/California/07/2009, KBPV VR-92, and NCCP 43021 strains. This discovery revolutionizes influenza vaccine design and delivery; Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy. This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.

Protein arginine methylation in transcription and epigenetic regulation

Arginine methylation is a prevalent post-translational modification found in all eukaryotic systems. It involves the addition of a methyl group to the guanidino nitrogen atoms of arginine residues within proteins, and this process is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). In mammals, there exist nine PRMTs (PRMT1–9) that catalyze three distinct types of arginine methylation: monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine. These modifications play critical roles in numerous fundamental cellular processes, including transcription, RNA metabolism, genome maintenance, and signaling transduction. Aberrations in protein arginine methylation have been implicated in various human diseases, such as neurodevelopmental disorders and cancer. This review offers a general overview of arginine methylation, covering its deposition, its impact on protein function, and the diverse regulatory mechanisms involved. We specifically focus on an in-depth view of the role of arginine methylation in transcription and the epigenetic regulation of gene expression. Readers are directed towards additional reviews that encompass other aspects of arginine methylation biology.