Intrahost evolution leading to distinct lineages in the upper and lower respiratory tracts during SARS-CoV-2 prolonged infection.

Accumulating evidence points to persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in immunocompromised individuals as a source of novel lineages. While intrahost evolution of the virus in chronically infected patients has previously been reported, existing knowledge is primarily based on samples from the nasopharynx. In this study, we investigate the intrahost evolution and genetic diversity that accumulated during a prolonged SARS-CoV-2 infection with the Omicron BF.7 sublineage, which is estimated to have persisted for >1 year in an immunosuppressed patient. Based on the sequencing of eight samples collected at six time points, we identified 87 intrahost single-nucleotide variants, 2 indels, and a 362-bp deletion. Our analysis revealed distinct viral genotypes in the nasopharyngeal (NP), endotracheal aspirate, and bronchoalveolar lavage samples. This suggests that NP samples may not offer a comprehensive representation of the overall intrahost viral diversity. Our findings not only demonstrate that the Omicron BF.7 sublineage can further diverge from its already exceptionally mutated state but also highlight that patients chronically infected with SARS-CoV-2 can develop genetically specific viral populations across distinct anatomic compartments. This provides novel insights into the intricate nature of viral diversity and evolution dynamics in persistent infections.

Hybrid Protocells Based on Coacervate-Templated Fatty Acid Vesicles Combine Improved Membrane Stability with Functional Interior Protocytoplasm.

Prebiotically-plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self-assembly and coacervate droplets formed by liquid-liquid phase separation. Both types of structures form spontaneously and can be related to cellular compartmentalization motifs in today's living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation and membranes providing superior boundaries. Herein, protocell models constructed by spontaneous encapsulation of coacervate droplets by mixed fatty acid/phospholipid and by purely fatty acid membranes are described. Coacervate-supported membranes form over a range of coacervate and lipid compositions, with membrane properties impacted by charge-charge interactions between coacervates and membranes. Vesicles formed by coacervate-templated membrane assembly exhibit profoundly different permeability than traditional fatty acid or blended fatty acid/phospholipid membranes without a coacervate interior, particularly in the presence of magnesium ions (Mg2+). While fatty acid and blended membrane vesicles are disrupted by the addition of Mg2+, the corresponding coacervate-supported membranes remain intact and impermeable to externally-added solutes. With the more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, can be performed inside the protocell model due to the simple diffusion of FDA and then following with the coacervate-mediated abiotic hydrolysis to fluorescein.

How does NaCl enhance Cd tolerance in Inula crithmoides L? Insights into Cd uptake, compartmentalization, and chelation.

This study investigated the effect of NaCl on the uptake, translocation, compartmentalization, and chelation of cadmium (Cd) in the halophyte Inula crithmoides. Seedlings were subjected hydroponically for 21 days to 25 and 50 µM Cd applied alone or combined with 100 mM NaCl. Findings revealed that, Cd alone induced intense chlorosis and necrosis and altered plant development resulting in diminished biomass production. However, NaCl alleviated Cd-induced toxicity by increasing biomass accumulation, associated with restoration of photosynthesis activity. At the level of whole plant, NaCl reduced Cd concentration in different organs as well as its translocation toward the shoots. At the cellular level, Na Cl changed the cell-compartmentalization of Cd in the shoots and roots by inducing a preferential accumulation into the soluble fraction (vacuole). NaCl increased the chelation of Cd to chloride and nitrate. As compared to Cd alone, salt addition to Cd-treated plants enhanced significantly succinic acid concentration in the leaves suggesting a possible role of this acid in Cd-chelation. Globally, NaCl alleviated Cd-induced phytotoxicity in this halophyte by reducing Cd absorption, translocation and increased Cd fixation to organic acids as well as through the changes in Cd2+ cell compartmentalization. Obtained data suggested that this fast growing halophyte could be used to rehabilitate Cd polluted saline soils.

LINE1 and PRC2 control nucleolar organization and repression of the 8C state in human ESCs.

The mechanisms that ensure developmental progression in the early human embryo remain largely unknown. Here, we show that the family of long interspersed nuclear element 1 (LINE1) transposons prevents the reversion of naive human embryonic stem cells (hESCs) to 8-cell-like cells (8CLCs). LINE1 RNA contributes to maintenance of H3K27me3 levels, particularly at chromosome 19 (Chr19). Chr19 is enriched for key 8C regulators, H3K27me3, and genes derepressed upon LINE1 knockdown or PRC2 inhibition. Moreover, Chr19 is strongly associated with the nucleolus in hESCs but less in 8CLCs. Direct inhibition of PRC2 activity induces the 8C program and leads to a relocalization of Chr19 away from the nucleolus. LINE1 KD or PRC2 inhibition induces nucleolar stress, and disruption of nucleolar architecture is sufficient to de-repress the 8C program. These results indicate that LINE1 RNA and PRC2 maintain H3K27me3-mediated gene repression and 3D nuclear organization to prevent developmental reversion of hESCs.

Transcription factor AtNAC002 positively regulates Cu toxicity tolerance in Arabidopsis thaliana.

Copper (Cu) is an essential micronutrient for plant growth and development, but environmental Cu pollution has become increasingly severe, adversely affecting both ecosystems and crop productivity. In this study, we identified the AtNAC002 gene as a positive regulator of Cu toxicity in Arabidopsis thaliana. We found that AtNAC002 expression was induced by Cu excess, and the atnac002 mutant was Cu-sensitive, accumulating more Cu than the wild-type. Additionally, atnac002 mutants exhibit reduced activities of antioxidant enzymes (SOD, POD, and CAT), leading to increased levels of reactive oxygen species and malondialdehyde, which decrease Cu resistance. AtNAC002 might play a role in vacuolar and mitochondrial Cu compartmentalization by regulating genes involved in Cu detoxification, specifically COX11 and HCC1. Furthermore, AtNAC002 was implicated in flavone and flavanol biosynthesis, with the atnac002 mutant showing reduced flavonoid content. Our findings suggest that AtNAC002 is integral to the regulation of Cu toxicity tolerance in A. thaliana. This knowledge is critical for advancing our understanding and offers potential molecular breeding targets to enhance plant performance under Cu excess, which is significant for improving global food security and forest restoration.

Tissue-Specific Regulation of Vesicular Trafficking Mediated by Rab-GEF Complex MON1/CCZ1 From Solanum chilense Increases Salt Stress Tolerance in Arabidopsis thaliana.

Salt stress constrains the development and growth of plants. To tolerate it, mechanisms of endocytosis and vacuolar compartmentalization of Na+ are induced. In this work, the genes that encode a putative activator of vesicular trafficking called MON1/CCZ1 from Solanum chilense, SchMON1 and SchCCZ1, were co-expressed in roots of Arabidopsis thaliana to determine whether the increase in prevacuolar vesicular trafficking also increases the Na+ compartmentalization capacity and tolerance. Initially, we demonstrated that both SchMON1 and SchCCZ1 genes rescued the dwarf phenotype of both A. thaliana mon1-1 and ccz1a/b mutants associated with the loss of function, and both proteins colocalized with their functional targets, RabF and RabG, in endosomes. Transgenic A. thaliana plants co-expressing these genes improved salt stress tolerance compared to wild type plants, with SchMON1 contributing the most. At the sub-cellular level, co-expression of SchMON1/SchCCZ1 reduced ROS levels and increased endocytic activity, and number of acidic structures associated with autophagosomes. Notably, greater Na+ accumulation in vacuoles of cortex and endodermis was evidenced in the SchMON1 genotype. Molecular analysis of gene expression in each genotype supported these results. Altogether, our analysis shows that root activation of prevacuolar vesicular trafficking mediated by MON1/CCZ1 emerges as a promising physiological molecular mechanism to increase tolerance to salt stress in crops of economic interest.

Phase Separation Mediated Sub-Nuclear Compartmentalization of Androgen Receptors.

The androgen receptor (AR), a member of the nuclear steroid hormone receptor family of transcription factors, plays a crucial role not only in the development of the male phenotype but also in the development and growth of prostate cancer. While AR structure and AR interactions with coregulators and chromatin have been studied in detail, improving our understanding of AR function in gene transcription regulation, the spatio-temporal organization and the role of microscopically discernible AR foci in the nucleus are still underexplored. This review delves into the molecular mechanisms underlying AR foci formation, focusing on liquid-liquid phase separation and its role in spatially organizing ARs and their binding partners within the nucleus at transcription sites, as well as the influence of 3D-genome organization on AR-mediated gene transcription.

Subcellular compartmentalized localization of transmembrane proteins essential for production of fungal cyclic peptide cyclochlorotine.

Fungal biosynthetic gene clusters often include genes encoding transmembrane proteins, which have been mostly thought to be transporters exporting the products. However, there is little knowledge about subcellular compartmentalization of transmembrane proteins essential for biosynthesis. Fungal mycotoxin cyclochlorotine is synthesized by non-ribosomal peptide synthetase, which is followed by modifications with three transmembrane UstYa-family proteins. Heterologous expression in Aspergillus oryzae revealed that total biosynthesis of cyclochlorotine requires additional two transporter proteins. Here, we investigated subcellular localizations of the five transmembrane proteins under heterologous expression in A. oryzae. Enhanced green fluorescent protein (EGFP) fusions to the transmembrane proteins, which were confirmed to normally function in cyclochlorotine production, were expressed together with organellar markers. All the transmembrane proteins exhibited localizations commonly in line of the trans-Golgi, endosomes, and vacuoles. This study suggests that subcellular compartmentalization of UstYa family proteins and transporters allows corporative functions of delivering intermediates and subsequent modifications, completing cyclochlorotine biosynthesis.

Compartmentalized signaling in the soma: Coordination of electrical and protein kinase A signaling at neuronal ER-plasma membrane junctions.

Neuronal information processing depends on converting membrane depolarizations into compartmentalized biochemical signals that can modify neuronal activity and structure. However, our understanding of how neurons translate electrical signals into specific biochemical responses remains limited, especially in the soma where gene expression and ion channel function are crucial for neuronal activity. Here, I emphasize the importance of physically compartmentalizing action potential-triggered biochemical reactions within the soma. Emerging evidence suggests that somatic endoplasmic reticulum-plasma membrane (ER-PM) junctions are specialized organelles that coordinate electrical and biochemical signaling. The juxtaposition of ion channels and signaling proteins at a prominent subset of these sites enables compartmentalized calcium and cAMP-dependent protein kinase (PKA) signaling. I explore the hypothesis that these PKA-containing ER-PM junctions serve as critical sites for translating membrane depolarizations into PKA signals and identify key gaps in knowledge of the assembly, regulation, and neurobiological functions of this somatic signaling system.

Intracellular Construction of Organelle-like Compartments Facilitates Metabolic Flux in Escherichia coli.

The formation of well-designed synthetic compartments or membraneless organelles for applications in synthetic biology and cellular engineering has aroused enormous interest. However, establishing stable and robust intracellular compartments in bacteria remains a challenge. Here, we use the structured DIX domains derived from Wnt signaling pathway components, more specifically, Dvl2 and Axin1, as building blocks to generate intracellular synthetic compartments in Escherichia coli. Moreover, the aggregation behaviors and physical properties of the DIX-based compartments can be tailored by genetically embedding a specific dimeric domain into the DIX domains. Then, a pair of interacting motifs, consisting of the aforementioned dimeric domain and its corresponding binding ligand, was incorporated to modify the client recruitment pattern of the synthetic compartments. As a proof of concept, the human milk oligosaccharide lacto-N-tetraose (LNT) biosynthesis pathway was selected as a model metabolic pathway. The fermentation results demonstrated that the co-compartmentalization of sequential pathway enzymes into intracellular compartments created by DIX domain, or by the DIX domain in conjunction with interacting motifs, prominently enhanced the metabolic flux and increased LNT production. These synthetic protein compartments may provide a feasible and effective tool to develop versatile organelle-like compartments in bacteria for applications in cellular engineering and synthetic biology.

The landscape of lncRNAs in cell granules: Insights into their significance in cancer.

Cellular compartmentalization, achieved through membrane-based compartments, is a fundamental aspect of cell biology that contributes to the evolutionary success of cells. While organelles have traditionally been the focus of research, membrane-less organelles (MLOs) are emerging as critical players, exhibiting distinct morphological features and unique molecular compositions. Recent research highlights the pivotal role of long noncoding RNAs (lncRNAs) in MLOs and their involvement in various cellular processes across different organisms. In the context of cancer, dysregulation of MLO formation, influenced by altered lncRNA expression, impacts chromatin organization, oncogenic transcription, signaling pathways, and telomere lengthening. This review synthesizes the current understanding of lncRNA composition within MLOs, delineating their functions and exploring how their dysregulation contributes to human cancers. Environmental challenges in tumorigenesis, such as nutrient deprivation and hypoxia, induce stress granules, promoting cancer cell survival and progression. Advancements in biochemical techniques, particularly single RNA imaging methods, offer valuable tools for studying RNA functions within live cells. However, detecting low-abundance lncRNAs remains challenging due to their limited expression levels. The correlation between lncRNA expression and pathological conditions, particularly cancer, should be explored, emphasizing the importance of single-cell studies for precise biomarker identification and the development of personalized therapeutic strategies. This article is categorized under: RNA Export and Localization > RNA Localization RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Lysosomes accumulate at the perinuclear region of muscle cells during chick myogenesis.

Lysosomes are involved in a myriad of cellular functions, such as degradation of macromolecules, endocytosis and exocytosis, modulation of several signaling pathways, and regulation of cell metabolism. To fulfill these diverse functions, lysosomes can undergo several dynamic changes in their content, size, pH, and location within cells. Here, we studied some of these parameters during embryonic chick skeletal muscle cells. We used an anti-lysosome-associated membrane protein 2 (LAMP2) antibody to specifically determine the intracellular localization of lysosomes in these cells. Our data shows that lysosomes are highly enriched in the perinuclear region of chick embryonic muscle cells. We also showed that the wingless signaling pathway (Wnt)/ß-catenin signaling pathway can modulate the location of LAMP2 in chick myogenic cells. Our results highlight the role of lysosomes during muscle differentiation and particularly the presence of a subcellular population of lysosomes that are concentrated in the perinuclear region of muscle cells.

Human Tendon-on-Chip: Unveiling the Effect of Core Compartment-T Cell Spatiotemporal Crosstalk at the Onset of Tendon Inflammation.

The lack of representative in vitro models recapitulating human tendon (patho)physiology is among the major factors hindering consistent progress in the knowledge-based development of adequate therapies for tendinopathy.Here, an organotypic 3D tendon-on-chip model is designed that allows studying the spatiotemporal dynamics of its cellular and molecular mechanisms.Combining the synergistic effects of a bioactive hydrogel matrix with the biophysical cues of magnetic microfibers directly aligned on the microfluidic chip, it is possible to recreate the anisotropic architecture, cell patterns, and phenotype of tendon intrinsic (core) compartment. When incorporated with vascular-like vessels emulating the interface between its intrinsic-extrinsic compartments, crosstalk with endothelial cells are found to drive stromal tenocytes toward a reparative profile. This platform is further used to study adaptive immune cell responses at the onset of tissue inflammation, focusing on interactions between tendon compartment tenocytes and circulating T cells.The proinflammatory signature resulting from this intra/inter-cellular communication induces the recruitment of T cells into the inflamed core compartment and confirms the involvement of this cellular crosstalk in positive feedback loops leading to the amplification of tendon inflammation.Overall, the developed 3D tendon-on-chip provides a powerful new tool enabling mechanistic studies on the pathogenesis of tendinopathy as well as for assessing new therapies.

Peptide-Induced Division of Polymersomes for Biomimetic Compartmentalization.

Polymersomes are synthetic vesicles that mimic the architecture of cellular compartments such as the cell membrane and organelles. These biomimetic compartments facilitate the creation of cell-like chemical systems, including microreactors and synthetic organelles. However, the construction of hierarchical multi-compartment systems remains challenging and typically requires the encapsulation of pre-formed vesicles within a host compartment. Here, we report the formation of multicompartment polymersomes with a vesicle-in-vesicle architecture achieved through self-division induced by short peptides incorporated into the vesicle membrane. A phenylalanine-phenylalanine-methionine (FFM) tripeptide was designed and encapsulated into the polymersome via microfluidics. We demonstrate that vesicle self-division occurs due to peptide incorporation into the membrane in response to pH changes. This self-division creates internal vesicles capable of colocalizing enzymes. The hybrid polymer-peptide system described here provides a straightforward method for developing subcompartmentalized systems, paving the way for engineering microreactors with life-like properties.

Pseudomonas aeruginosa ExoY infection of pulmonary microvascular endothelial cells releases cyclic nucleotides into the extracellular compartment.

Type three secretion system (TTSS) competent Pseudomonas aeruginosa expressing soluble promiscuous cyclase, ExoY, generates cyclic nucleotides in pulmonary microvascular endothelial cells (PMVECs). Within cells, cyclic nucleotide signals are highly compartmentalized, but these second messengers are also released into the extracellular space. While agonist-stimulation of endogenous adenylyl cyclase (AC) or the presence of ExoY increases cyclic nucleotides, the proportion of the signal that is in the intracellular versus extracellular compartments is unresolved. Further, it is unclear whether P. aeruginosa primary infection or treatment with sterile media supernatants derived from a primary infection, alters beta-adrenergic agonist-induced elevations in cAMP in PMVECs. Herein, we determine that PMVECs release cAMP into the extracellular space constitutively, following beta-adrenergic stimulation of endogenous AC, and following infection with P. aeruginosa expressing ExoY. Surprisingly, in PMVECs, only a small proportion of cGMP is detected within the cell at baseline or following P. aeruginosa ExoY-infection with a larger proportion of total cGMP being detected extracellularly. Thus, the ability of lung endothelium to generate cyclic nucleotides may be underestimated by examining intracellular cyclic nucleotides alone, since a large portion is delivered into the extracellular compartment. In addition, P. aeruginosa infection or treatment with sterile media supernatants from a primary infection suppress the beta-adrenergic cAMP response, which is further attenuated by the expression of functional ExoY. These findings reveal an overabundance of extracellular cyclic nucleotides following infection with ExoY expressing TTSS competent P. aeruginosa.

Advances in multi-enzyme co-localization strategies for the construction of microbial cell factory.

Biomanufacturing, driven by technologies such as synthetic biology, offers significant potential to advance the bioeconomy and promote sustainable development. It is anticipated to transform traditional manufacturing and become a key industry in future strategies. Cell factories are the core of biomanufacturing. The advancement of synthetic biology and growing market demand have led to the production of a greater variety of natural products and increasingly complex metabolic pathways. However, this progress also presents challenges, notably the conflict between natural product production and chassis cell growth. This conflict results in low productivity and yield, adverse side effects, metabolic imbalances, and growth retardation. Enzyme co-localization strategies have emerged as a promising solution. This article reviews recent progress and applications of these strategies in constructing cell factories for efficient natural product production. It comprehensively describes the applications of enzyme-based compartmentalization, metabolic pathway-based compartmentalization, and synthetic organelle-based compartmentalization in improving product titers. The article also explores future research directions and the prospects of combining multiple strategies with advanced technologies.

De novo biosynthesis of betulinic acid in engineered Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupinane-type pentacyclic triterpenoid natural product derived from lupeol that has favorable anti-inflammatory and anti-tumor activities. Currently, BA is mainly produced via botanical extraction, which significantly limits its widespread use. In this study, we investigated the de novo synthesis of BA in Saccharomyces cerevisiae, and to facilitate the synthesis and storage of hydrophobic BA, we adopted a dual-engineering strategy involving peroxisomes and lipid droplets to construct the BA biosynthetic pathway. By expressing Betula platyphylla-derived lupeol C-28 oxidase (BPLO) and Arabidopsis-derived ATR1, we succeeded in developing a BA-producing strain and following multiple expression optimizations of the linker between BPLO and ATR1, the BA titer reached 77.53 mg/L in shake flasks and subsequently reached 205.74 mg/L via fed-batch fermentation in a 5-L bioreactor. In this study, we developed a feasible approach for the de novo synthesis of BA and its direct precursor lupeol in engineered S. cerevisiae.

Olfactory receptor neurons are sensitive to stimulus onset asynchrony: implications for odor source discrimination.

In insects, olfactory receptor neurons (ORNs) are localized in sensilla. Within a sensillum, different ORN types are typically co-localized and exhibit nonsynaptic reciprocal inhibition through ephaptic coupling. This inhibition is hypothesized to aid odor source discrimination in environments where odor molecules (odorants) are dispersed by wind, resulting in turbulent plumes. Under these conditions, odorants from a single source arrive at the ORNs synchronously, while those from separate sources arrive asynchronously. Ephaptic inhibition is expected to be weaker for asynchronous arriving odorants from separate sources, thereby enhancing their discrimination. Previous studies have focused on ephaptic inhibition of sustained ORN responses to constant odor stimuli. This begs the question of whether ephaptic inhibition also affects transient ORN responses and if this inhibition is modulated by the temporal arrival patterns of different odorants. To address this, we recorded co-localized ORNs in the fruit fly Drosophila melanogaster and exposed them to dynamic odorant mixtures. We found reciprocal inhibition, strongly suggesting the presence of ephaptic coupling. This reciprocal inhibition does indeed modulate transient ORN responses and is sensitive to the relative timing of odor stimuli. Notably, the strength of inhibition decreases as the synchrony and correlation between arriving odorants decrease. These results support the hypothesis that ephaptic inhibition aids odor source discrimination.

Compartmentalization of pathway sequential enzymes into synthetic protein compartments for metabolic flux optimization in Escherichia coli.

Advancing the formation of artificial membraneless compartments with organizational complexity and diverse functionality remains a challenge. Typically, synthetic compartments or membraneless organelles are made up of intrinsically disordered proteins featuring low-complexity sequences or polypeptides with repeated distinctive short linear motifs. In order to expand the repertoire of tools available for the formation of synthetic membraneless compartments, here, a range of DIshevelled and aXin (DIX) or DIX-like domains undergoing head-to-tail polymerization were demonstrated to self-assemble into aggregates and generate synthetic compartments within E. coli cells. Then, synthetic complex compartments with diverse intracellular morphologies were generated by coexpressing different DIX domains. Further, we genetically incorporated a pair of interacting motifs, comprising a homo-dimeric domain and its anchoring peptide, into the DIX domain and cargo proteins, respectively, resulting in the alteration of both material properties and client recruitment of synthetic compartments. As a proof-of-concept, several human milk oligosaccharide biosynthesis pathways were chosen as model systems. The findings indicated that the recruitment of pathway sequential enzymes into synthetic compartments formed by DIX-DIX heterotypic interactions or by DIX domains embedded with specific interacting motifs efficiently boosted metabolic pathway flux and improved the production of desired chemicals. We propose that these synthetic compartment systems present a potent and adaptable toolkit for controlling metabolic flux and facilitating cellular engineering.

Making the Thermodynamic Cost of Active Inference Explicit.

When describing Active Inference Agents (AIAs), the term "energy" can have two distinct meanings. One is the energy that is utilized by the AIA (e.g., electrical energy or chemical energy). The second meaning is so-called Variational Free Energy (VFE), a statistical quantity which provides an upper bound on surprisal. In this paper, we develop an account of the former quantity-the Thermodynamic Free Energy (TFE)-and its relationship with the latter. We highlight the necessary tradeoffs between these two in a generic, quantum information-theoretic formulation, and the macroscopic consequences of those tradeoffs for the ways that organisms approach their environments. By making this tradeoff explicit, we provide a theoretical basis for the different metabolic strategies that organisms from plants to predators use to survive.