Eukaryotic Research Articles

Peroxisome inter-organelle cooperation in Drosophila

Within many cellular organelles biochemical functions are compartmentalized, which facilitates optimized enzymatic environments. However, processing and or storage of metabolites in the same pathway can occur in multiple organelles. Thus, spatially separated organelles would need to cooperate functionally. Coordination would also be needed between organelles in different specialized cells, with shared metabolites passed via circulation. Peroxisomes are membrane-bounded organelles responsible for cellular redox and lipid metabolism in eukaryotic cells. Studies using single cells suggest peroxisomes coordinate with other organelles including mitochondria, ER (endoplasmic reticulum), lysosomes, and lipid droplets. Some of these coordinated functions require, or are at least enhanced by, direct contact between peroxisomes and other organelles. Peroxisome dysfunction in humans leads to multiorgan effects including neurological, metabolic, developmental, and age-related diseases. Thus, increased understanding of peroxisome coordination with other organelles, especially those specialized cells in various organs is essential. Drosophila melanogaster (fruit fly) has emerged recently as an effective animal model for understanding peroxisomes. Here we review current knowledge of genetic pathways regulating coordination between peroxisomes with other organelles in flies, speculating about analogous roles for conserved Drosophila genes encoding proteins with known organelle coordinating roles in other species.

Is stochastic thermodynamics the key to understanding the energy costs of computation?

The relationship between the thermodynamic and computational properties of physical systems has been a major theoretical interest since at least the 19th century. It has also become of increasing practical importance over the last half-century as the energetic cost of digital devices has exploded. Importantly, real-world computers obey multiple physical constraints on how they work, which affects their thermodynamic properties. Moreover, many of these constraints apply to both naturally occurring computers, like brains or Eukaryotic cells, and digital systems. Most obviously, all such systems must finish their computation quickly, using as few degrees of freedom as possible. This means that they operate far from thermal equilibrium. Furthermore, many computers, both digital and biological, are modular, hierarchical systems with strong constraints on the connectivity among their subsystems. Yet another example is that to simplify their design, digital computers are required to be periodic processes governed by a global clock. None of these constraints were considered in 20th-century analyses of the thermodynamics of computation. The new field of stochastic thermodynamics provides formal tools for analyzing systems subject to all of these constraints. We argue here that these tools may help us understand at a far deeper level just how the fundamental thermodynamic properties of physical systems are related to the computation they perform.

Phytochemical profiling and biological evaluation of the residues from industrial hemp (Cannabis sativa L.) inflorescences trimming: Focus on water extract

The study focused on investigating the phytochemical composition and bio-pharmacological properties of a water extract obtained from industrial hemp by-products, after trimming female inflorescences of Cannabis sativa L., cultivar Kompolti. The extract was found to be rich in phenolic compounds, particularly gallic acid, catechin, coumaric acid, ferulic acid, and benzoic acid. It also contained significant amounts of cannabidiolic acid and cannabigerolic acid as the main terpenophenols. To assess the biological potential of the extract, various ecotoxicological assays were conducted. The extract did not show significant phytotoxic effects on seedling germination in the allelopathic assay. In the brine shrimp (Artemia salina) lethality test, the LC50 value was 1.726 mg/mL, and in the Daphnia magna test, the extract showed no cardiotoxicity effects at the same concentration; thus, confirming its biocompatibility with these eukaryotic organisms. Additionally, the extract did not induce cytotoxicity in the murine C2C12 cell line till to the concentration of 1000 μg/mL. In isolated mouse prostate specimens, the extract (10–1000 μg/mL) also prevented LPS-induced gene expression of pro-inflammatory cytokines, namely tumor necrosis factor (TNF)α, interleukin (IL)-6, and IL-1β. Furthermore, the extract exhibited inhibitory effects on the growth of pathogenic bacterial, fungal, and dermatophyte species commonly associated with prostatitis. These results suggest that the residual powder from female inflorescence trimming can be considered an innovative plant material from the industrial hemp supply chain that deserves to be valorised in terms of recycling and upcycling; thus, reducing the environmental impact of the by-products and also opening the way for innovative health-promoting agents.

The Mysterious World of Non-Canonical Caps - What We Know and Why We Need New Sequencing Techniques.

It was long believed that viral and eukaryotic mRNA molecules are capped at their 5' end solely by the N7-methylguanosine cap, which regulates various aspects of the RNA life cycle, from its biogenesis to its decay. However, the recent discovery of a variety of non-canonical RNA caps derived from metabolites and cofactors - such as NAD, FAD, CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleoside polyphosphates - has expanded the known repertoire of RNA modifications. These non-canonical caps are found across all domains of life and can impact multiple aspects of RNA metabolism, including stability, translation initiation, and cellular stress responses. The study of these modifications has been facilitated by sophisticated methodologies such as liquid chromatography-mass spectrometry, which have unveiled their presence in both prokaryotic and eukaryotic organisms. The identification of these novel RNA caps highlights the need for advanced sequencing techniques to characterize the specific RNA types bearing these modifications and understand their roles in cellular processes. Unravelling the biological role of non-canonical RNA caps will provide insights into their contributions to gene expression, cellular adaptation, and evolutionary diversity. This review emphasizes the importance of these technological advancements in uncovering the complete spectrum of RNA modifications and their implications for living systems.

Drosophila ring chromosomes interact with sisters and homologs to produce anaphase bridges in mitosis.

Ring chromosomes are known in many eukaryotic organisms, including humans. They are typically associated with a variety of maladies, including abnormal development and lethality. Underlying these phenotypes are anaphase chromatin bridges that can lead to chromosome loss, nondisjunction and breakage. By cytological examination of ring chromosomes in Drosophila melanogaster we identified five causes for anaphase bridges produced by ring chromosomes. Catenation of sister chromatids appears to be the most common cause and these bridges frequently resolve during anaphase, presumably by the action of topoisomerase II. Sister chromatid exchange and chromosome breakage followed by sister chromatid union also produce anaphase bridges. Mitotic recombination with the homolog was rare, but was another route to generation of anaphase bridges. Most surprising, was the discovery of homolog capture, where the ring chromosome was connected to its linear homolog in anaphase. We hypothesize that this is a remnant of mitotic pairing and that the linear chromosome is connected to the ring by multiple wraps produced through the action of topoisomerase II during establishment of homolog pairing. In support, we showed that in a ring/ring homozygote the two rings are frequently catenated in mitotic metaphase, a configuration that requires breaking and rejoining of at least one chromosome.

Autophagy and proteasomes in thymic epithelial cells: essential bulk protein degradation systems for immune homeostasis maintenance

The thymus is a central organ that controls T cell development. Thymic epithelial cells (TECs) create a unique microenvironment essential for the differentiation of major histocompatibility complex (MHC)-restricted and self-tolerant T cells. TECs present a complex of self-peptides and MHC molecules (self-pMHCs) to immature T cells and regulate their survival and differentiation based on their affinity for self-pMHCs. The processing of self-peptides in TECs depends on bulk protein degradation systems, specifically autophagy and proteasomes. Studies using autophagy- and proteasome-deficient mouse models have demonstrated that these degradation systems in TECs are indispensable for maintaining immune homeostasis. Although autophagy and proteasomes are ubiquitous in nearly all eukaryotic cells, TECs exhibit unique characteristics in their autophagy and proteasome functions. Autophagy in TECs is constitutively active and independent of stress responses, while TEC proteasomes contain specialized catalytic subunits. This review summarizes the distinctive characteristics of autophagy and proteasomes in TECs and their roles in immune system regulation.

NF-κB transcription factors regulate the expression of immunity-related genes in different cell lines of silkworm (Bombyx mori)

The Toll and immune deficiency (IMD) signaling pathways in insects are highly conserved in evolution and regulate the expression of antimicrobial peptides (AMPs) and other immune-related genes mainly through nuclear factor-kappa B (NF-κB) transcription factors. However, the differences of NF-κB transcription factors Rels and Relish in the expression regulation of AMPs and other immune-related genes in silkworm (Bombyx mori) have not been systematically reported. In this study, the BmRelA, BmRelB and BmRelish1 genes were cloned and their eukaryotic cell overexpression vectors were constructed. After the recombinant vectors were transfected into BmE and BmN cells, the expression of AMPs and immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that the expression of AMP genes Defensin2 and Gloverin2 was mainly regulated by Relish, the expression of Moricin was mainly regulated by RelA and RelB, and the expression of other AMP genes was jointly regulated by both. In addition, the expression levels of peptidoglycan recognition proteins (PGRPs), β-1, 3-glucan recognition proteins (βGRPs), lysozymes (Lys) and lysozyme-like proteins (LLPs), and nitric oxide synthase (NOS) were up-regulated to varying degrees in different cell lines in response to RelA, RelB and Relish1, suggesting that the expression of these immune-molecules was also regulated by Toll or IMD pathways in silkworm. Compared with the regulatory specificity of transcription factors in Drosophila Toll and IMD signaling pathways on the expression of AMPs, this study found that the regulatory patterns of Rels and Relish1 on the expression of AMPs in silkworm are more complex, which provides an experimental basis for further analysis of the effect mechanism and feedback mechanism of Toll and IMD pathways in insects.

LC3-associated phagocytosis and human diseases: Insights from mechanisms to therapeutic potential.

LC3-associated phagocytosis (LAP) is a distinct type of autophagy that involves the sequestration of extracellular material by phagocytes. Beyond the removal of dead cells and cellular debris from eukaryotic cells, LAP is also involved in the removal of a variety of pathogens, including bacteria, fungi, and viruses. These events are integral to multiple physiological and pathological processes, such as host defense, inflammation, and tissue homeostasis. Dysregulation of LAP has been associated with the pathogenesis of several human diseases, including infectious diseases, autoimmune diseases, and neurodegenerative diseases. Thus, understanding the molecular mechanisms underlying LAP and its involvement in human diseases may provide new insights into the development of novel therapeutic strategies for these conditions. In this review, we summarize and highlight the current consensus on the role of LAP and its biological functions in disease progression to propose new therapeutic strategies. Further studies are needed to illustrate the precise role of LAP in human disease and to determine new therapeutic targets for LAP-associated pathologies.

The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.

The 26S proteasome is the major compartmental protease in eukaryotic cells, responsible for the ATP-dependent turnover of obsolete, damaged, or misfolded proteins that are delivered for degradation through attached ubiquitin modifications. In addition to targeting substrates to the proteasome, ubiquitin was recently shown to promote degradation initiation by directly modulating the conformational switching of the proteasome, yet the underlying mechanisms are unknown. Here, we used biochemical, mutational, and single-molecule FRET-based approaches to show that the proteasomal deubiquitinase Rpn11 functions as an allosteric sensor and facilitates the early steps of degradation. After substrate recruitment to the proteasome, ubiquitin binding to Rpn11 interferes with conformation-specific interactions of the ubiquitin-receptor subunit Rpn10, thereby stabilizing the engagement-competent state of the proteasome and expediting substrate insertion into the ATPase motor for mechanical translocation, unfolding, and Rpn11-mediated deubiquitination. These findings explain how modifications with poly-ubiquitin chains or multiple mono-ubiquitins allosterically promote substrate degradation and allow up to four-fold faster turnover by the proteasome.

Dynamic cluster field modeling of collective chemotaxis

Collective migration of eukaryotic cells is often guided by chemotaxis, and is critical in several biological processes, such as cancer metastasis, wound healing, and embryogenesis. Understanding collective chemotaxis has challenged experimental, theoretical and computational scientists because cells can sense very small chemoattractant gradients that are tightly controlled by cell-cell interactions and the regulation of the chemoattractant distribution by the cells. Computational models of collective cell migration that offer a high-fidelity resolution of the cell motion and chemoattractant dynamics in the extracellular space have been limited to a small number of cells. Here, we present Dynamic cluster field modeling (DCF), a novel computational method that enables simulations of collective chemotaxis of cellular systems with O(1000)\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\mathcal {O}}(1000)$$\\end{document} cells and high-resolution transport dynamics of the chemoattractant in the time-evolving extracellular space. We illustrate the efficiency and predictive capabilities of our approach by comparing our numerical simulations with experiments in multiple scenarios that involve chemoattractant secretion and uptake by the migrating cells, cell migration in confined spaces, regulation of the attractant distribution by cell motion, and interactions of the chemoattractant with an enzyme. The proposed algorithm opens new opportunities to address outstanding problems that involve collective cell migration in the central nervous system, immune response and cancer metastasis.

PlasmidScope: a comprehensive plasmid database with rich annotations and online analytical tools.

Plasmids are extrachromosomal genetic molecules that replicate independent of chromosomes in bacteria, archaea, and eukaryotic organisms. They contain diverse functional elements and are capable of horizontal gene transfer among hosts. While existing plasmid databases have archived plasmid sequences isolated from individual microorganisms or natural environments, there is a need for a comprehensive, standardized, and annotated plasmid database to address the vast accumulation of plasmid sequences. Here, we propose PlasmidScope (https://plasmid.deepomics.org/), a plasmid database offering comprehensive annotations, automated online analysis, and interactive visualization. PlasmidScope harbors a substantial collection of 852 600 plasmids curated from 10 repositories. Along with consolidated background information, PlasmidScope utilizes 12 state-of-the-art tools and provides comprehensive annotations for the curated plasmids, covering genome completeness, topological structure, mobility, host source, tRNA, tmRNA, signal peptides, transmembrane proteins and CRISPR/Cas systems. PlasmidScope offers diverse functional annotations for its 25 231 059 predicted genes from 9 databases as well as corresponding protein structures predicted by ESMFold. In addition, PlasmidScope integrates online analytical modules and interactive visualization, empowering researchers to delve into the complexities of plasmids.

Current trends to design antimalarial drugs targeting N-myristoyltransferase.

Malaria is a disease caused by Plasmodium spp., of which Plasmodium falciparum and Plasmodium vivax are the most prevalent. Unfortunately, traditional and some current treatment regimens face growing protozoan resistance. Thus, searching for and exploring new drugs and targets is necessary. One of these is N-myristoyltransferase (NMT). This enzyme is responsible for the myristoylation of several protein substrates in eukaryotic cells, including Plasmodium spp., thus enabling the assembly of protein complexes and stabilization of protein-membrane interactions. Given the importance of this target in developing new antiparasitic drugs, this review aims to explore the recent advances in the design of antimalarial drugs to target Plasmodium NMT.

Stress granules and organelles: Coordinating cellular responses in health and disease.

Membrane-bound organelles and membraneless organelles (MLOs) coordinate various biological processes within eukaryotic cells. Among these, stress granules (SGs) are significant cytoplasmic MLOs that form in response to cellular stress, exhibiting liquid-like properties alongside stable substructures. SGs interact with diverse organelles, thereby influencing cellular pathways that are critical in both health and disease contexts. This review discusses the interplay between SGs and organelles and explores the methodologies employed to analyze interactions between SGs and other MLOs. Furthermore, it highlights the pivotal roles SGs play in regulating cellular responses and the pathogenesis of ALS. Gaining insights into these interactions is essential for deciphering the mechanisms underlying both physiological processes and pathological conditions.

Morphometric analysis of actin networks.

The organization of cytoskeletal elements is pivotal for coordinating intracellular transport in eukaryotic cells. Several quantitative measures based on image analysis have been proposed to characterize morphometric features of fluorescently labeled actin networks. While helpful in detecting differences in actin organization between treatments or genotypes, the accuracy of these measures could not be rigorously assessed due to a lack of ground-truth data to which they could be compared. To overcome this limitation, we utilized coarse-grained computer simulations of actin filaments and cross-linkers to generate synthetic actin networks with varying levels of bundling. We converted the simulated networks into pseudofluorescence images similar to images obtained using confocal microscopy. Using both published and novel analysis procedures, we extracted a series of morphometric parameters and benchmarked them against analogous measures based on the ground-truth actin configurations. Our analysis revealed a set of parameters that reliably reports on actin network density, orientation, ordering, and bundling. Application of these morphometric parameters to root epidermal cells of Arabidopsis thaliana revealed subtle changes in network organization between wild-type and mutant cells. This work provides robust measures that can be used to quantify features of actin networks and characterize changes in actin organization for different experimental conditions.

RNA modification in normal hematopoiesis and hematologic malignancies.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic cells. Previous studies have shown that m6A plays a critical role under both normal physiological and pathological conditions. Hematopoiesis and differentiation are highly regulated processes, and recent studies on m6A mRNA methylation have revealed how this modification controls cell fate in both normal and malignant hematopoietic states. However, despite these insights, a comprehensive understanding of its complex roles between normal hematopoietic development and malignant hematopoietic diseases remains elusive. This review first provides an overview of the components and biological functions of m6A modification regulators. Additionally, it highlights the origin, differentiation process, biological characteristics, and regulatory mechanisms of hematopoietic stem cells, as well as the features, immune properties, and self-renewal pathways of leukemia stem cells. Last, the article systematically reviews the latest research advancements on the roles and mechanisms of m6A regulatory factors in normal hematopoiesis and related malignant diseases. More importantly, this review explores how targeting m6A regulators and various signaling pathways could effectively intervene in the development of leukemia, providing new insights and potential therapeutic targets. Targeting m6A modification may hold promise for achieving more precise and effective leukemia treatments.

CLE peptide signaling in plant-microbe interactions.

Cell-cell communication is essential for both unicellular and multicellular organisms. Secreted peptides that act as diffusive ligands are utilized by eukaryotic organisms to transduce information between cells to coordinate developmental and physiological processes. In plants, The CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes encode a family of secreted small peptides which play pivotal roles in stem cell homeostasis in various types of meristems. Accumulated evidence has revealed that CLE peptides mediate trans-kingdom interactions between plants and microbes, including pathogens and symbionts. This review highlights the emerging roles of CLE peptide signaling in plant-microbe interactions, focusing on their involvement in nodulation, immunity, and symbiosis with arbuscular mycorrhizal fungi. Understanding these interactions provides insights into the sophisticated regulatory networks to balance plant growth and defense, enhancing our knowledge of plant biology and potential agricultural applications.

Dominant and genome-wide formation of DNA:RNA hybrid G-quadruplexes in living yeast cells

Guanine-rich DNA forms G-quadruplexes (G4s) that play a critical role in essential cellular processes. Previous studies have mostly focused on intramolecular G4s composed of four consecutive guanine tracts (G-tracts) from a single strand. However, this structural form has not been strictly confirmed in the genome of living eukaryotic cells. Here, we report the formation of hybrid G4s (hG4s), consisting of G-tracts from both DNA and RNA, in the genome of living yeast cells. Analysis of Okazaki fragment syntheses and two other independent G4-specific detections reveal that hG4s can efficiently form with as few as a single DNA guanine-guanine (GG) tract due to the participation of G-tracts from RNA. This finding increases the number of potential G4-forming sites in the yeast genome from 38 to 587,694, a more than 15,000-fold increase. Interestingly, hG4s readily form and even dominate at G4 sites that are theoretically capable of forming the intramolecular DNA G4s (dG4s) by themselves. Compared to dG4s, hG4s exhibit broader kinetics, higher prevalence, and greater structural diversity and stability. Most importantly, hG4 formation is tightly coupled to transcription through the involvement of RNA, allowing it to function in a transcription-dependent manner. Overall, our study establishes hG4s as the overwhelmingly dominant G4 species in the yeast genome and emphasizes a renewal of the current perception of the structural form, formation mechanism, prevalence, and functional role of G4s in eukaryotic genomes. It also establishes a sensitive and currently the only method for detecting the structural form of G4s in living cells.

Aescin Inhibits Herpes simplex Virus Type 1 Induced Membrane Fusion.

Infections with Herpes simplex virus can cause severe ocular diseases and encephalitis. The present study aimed to investigate potential inhibitors of fusion between HSV-1 and the cellular membrane of the host cell. Fusion and entry of HSV-1 into the host cell is mimicked by a virus-free eukaryotic cell culture system by co-expression of the HSV-1 glycoproteins gD, gH, gL, and gB in presence of a gD receptor, resulting in excessive membrane fusion and polykaryocyte formation. A microscopic read-out was used for the screening of potential inhibitors, whereas luminometric quantification of cell-cell fusion was used in a reporter fusion assay. HSV-1 gB was tagged at its C-terminus with mCherry to express mCherry-gB in both assay systems for the visualization of the polykaryocyte formation. Reporter protein expression of SEAP was regulated by a Tet-On 3 G system. The saponin mixture aescin was identified as the specific inhibitor (IC50 7.4 µM, CC50 24.3 µM, SI 3.3) of membrane fusion. A plaque reduction assay on Vero cells reduced HSV-1 entry into cells and HSV-1 cell-to-cell spread significantly; 15 µM aescin decreased relative plaque counts to 41%, and 10 µM aescin resulted in a residual plaque size of 11% (HSV-1 17 syn+) and 2% (HSV-1 ANG path). Release of the HSV-1 progeny virus was reduced by one log step in the presence of 15 µM aescin. Virus particle integrity was mainly unaffected. Analytical investigation of aescin by UHPLC-MS revealed aescin IA and -IB and isoaescin IA and -IB as the main compounds with different functional activities. Aescin IA had the lowest IC50, the highest CC50, and an SI of > 4.6.

Eukaryotic viruses encode the ribosomal protein eL40

Viruses in the phylum Nucleocytoviricota are large, complex and have an exceptionally diverse metabolic repertoire. Some encode hundreds of products involved in the translation of mRNA into protein, but none was known to encode any of the proteins in ribosomes, the central engines of translation. With the discovery of the eL40 gene in FloV-SA2, we report the first example of a eukaryotic virus encoding a ribosomal protein and show that this gene is also present and expressed in other uncultivated marine giant viruses. FloV-SA2 also encodes a “group II” viral rhodopsin, a viral light-activated protein of unknown function previously only reported in metagenomes. FloV-SA2 is thus a valuable model system for investigating new mechanisms by which viruses manipulate eukaryotic cell metabolism.

Systematic discovery of antibacterial and antifungal bacterial toxins.

Microorganisms use toxins to kill competing microorganisms or eukaryotic cells. Polymorphic toxins are proteins that encode carboxy-terminal toxin domains. Here we developed a computational approach to identify previously undiscovered, conserved toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short toxins, showing that they cause cell death upon heterologous expression in either Escherichia coli or Saccharomyces cerevisiae. Five cognate immunity genes that neutralize the toxins were also discovered. The toxins are encoded by 2.2% of sequenced bacteria. A subset of the toxins exhibited potent antifungal activity against various pathogenic fungi but not against two invertebrate model organisms or macrophages. Experimental validation suggested that these toxins probably target the cell membrane or DNA or inhibit cell division. Further characterization and structural analysis of two toxin-immunity protein complexes confirmed DNase activity. These findings expand our knowledge of microbial toxins involved in inter-microbial competition that may have the potential for clinical and biotechnological applications.