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ABSTRACT Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome, also named the vacuole in plants. However, the molecular mechanisms underlying the fusion of autophagosomes with the vacuole remain unclear. Here, we report the functional characterization of a rice (Oryza sativa) mutant with defects in storage protein transport in endosperm cells and accumulation of numerous autophagosomes in root cells. Cytological and immunocytochemical experiments showed that this mutant exhibits a defect in the fusion between autophagosomes and vacuoles. The mutant harbors a loss‐of‐function mutation in the rice homolog of Arabidopsis thaliana MONENSIN SENSITIVITY1 (MON1). Biochemical and genetic evidence revealed a synergistic interaction between rice MON1 and AUTOPHAGY‐RELATED 8a in maintaining normal growth and development. In addition, the rice mon1 mutant disrupted storage protein sorting to protein storage vacuoles. Furthermore, quantitative proteomics verified that the loss of MON1 function influenced diverse biological pathways including autophagy and vacuolar transport, thus decreasing the transport of autophagic and vacuolar cargoes to vacuoles. Together, our findings establish a molecular link between autophagy and vacuolar protein transport, and offer insights into the dual functions of the MON1–CCZ1 (CAFFEINE ZINC SENSITIVITY1) complex in plants. [ABSTRACT FROM AUTHOR]

As an emerging class of RNA molecules, circular RNAs play pivotal roles in various biological processes, thereby determining their three-dimensional (3D) structure is crucial for a deep understanding of their biological significances. Similar to linear RNAs, the development of computational methods for circular RNA 3D structure prediction is challenging, especially considering the inherent flexibility and potentially long length of circular RNAs. Here, we introduce an extension of our previous IsRNA2 model, named IsRNAcirc, to enable circular RNA 3D structure predictions through coarse-grained molecular dynamics simulations. The workflow of IsRNAcirc consists of four main steps, including input preparation, end closure, structure prediction, and model refinement. Our results demonstrate that IsRNAcirc can provide reasonable 3D structure predictions for circular RNAs, which significantly reduce the locally irrational elements contained in the initial input. Moreover, for a validation test set comprising 34 circular RNAs, our IsRNAcirc can generate 3D models with better scores than the template-based 3dRNA method. These findings demonstrate that our IsRNAcirc method is a promising tool to explore the structural details along with intricate interactions of circular RNAs. Author summary: Knowledge of the 3D structure of circular RNA molecules can help us better understand their roles in eukaryotic cells. However, experimental determination of circular RNA 3D structure remains challenging, with currently only one complete circular RNA 3D structure available in the Protein Data Bank. Thus, extending from their linear counterparts, computational methods that can predict circular RNA 3D structure are urgently needed. In particular, physics-based de novo 3D structure prediction methods are promising due to their powerful sampling capability in the conformational space, which avoid the problem of limited available templates in template-based approaches. We have developed IsRNAcirc, a new physics-based method that employs an accurate coarse-grained force field and replica-exchange molecular dynamics simulations to predict the 3D structure of circular RNAs. IsRNAcirc is shown to significantly reduce the number of locally irrational structures in the input trials, which are caused by the lack of suitable templates and the improper assembly of fragments. Furthermore, in the absence of experimental reference structures, the predicted 3D models were validated using the popular scoring functions, such as DFIRE-RNA and rsRNASP, demonstrating that IsRNAcirc outperforms the template-based 3dRNA method in generating low-energy conformations. [ABSTRACT FROM AUTHOR]

Nuclear speckles are nuclear membraneless organelles in higher eukaryotic cells playing a vital role in gene expression. Using an in situ reverse transcription-based sequencing method, we study nuclear speckle-associated human transcripts. Our data indicate the existence of three gene groups whose transcripts demonstrate different speckle localization properties: stably enriched in nuclear speckles, transiently enriched in speckles at the pre-messenger RNA stage, and not enriched. We find that stably enriched transcripts contain inefficiently excised introns and that disruption of nuclear speckles specifically affects splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, indicating a tight interplay between transcript speckle enrichment, genome organization, and splicing efficiency. Collectively, our data highlight a role of nuclear speckles in both co-and posttranscriptional splicing regulation. Last, we show that genes with stably enriched transcripts are over-represented among genes with heat shock-up-regulated intron retention, hinting at a connection between speckle localization and cellular stress response. [ABSTRACT FROM AUTHOR]

Mitotic anaphase onset is a key cellular process tightly regulated by multiple kinases. The involvement of mitogen-activated protein kinases (MAPKs) in this process has been established in Xenopus egg extracts. However, the detailed regulatory cascade remains elusive, and it is also unknown whether the MAPK-dependent mitotic regulation is evolutionarily conserved in the single-cell eukaryotic organisms such as fission yeast (Schizosaccharomyces pombe). Here, we show that two MAPKs in S. pombe indeed act in concert to restrain anaphase-promoting complex/cyclosome (APC/C) activity upon activation of the spindle assembly checkpoint (SAC). One MAPK, Pmk1, binds to and phosphorylates Slp1Cdc20, the co-activator of APC/C. Phosphorylation of Slp1Cdc20 by Pmk1, but not by Cdk1, promotes its subsequent ubiquitylation and degradation. Intriguingly, Pmk1-mediated phosphorylation event is also required to sustain SAC under environmental stress. Thus, our study establishes a new underlying molecular mechanism of negative regulation of APC/C by MAPK upon stress stimuli, and provides a previously unappreciated framework for regulation of anaphase entry in eukaryotic cells. [ABSTRACT FROM AUTHOR]

Disulfidptosis is a novel discovered form of programmed cell death (PCD) that diverges from apoptosis, necroptosis, ferroptosis, and cuproptosis, stemming from disulfide stress-induced cytoskeletal collapse. In cancer cells exhibiting heightened expression of the solute carrier family 7 member 11 (SLC7A11), excessive cystine importation and reduction will deplete nicotinamide adenine dinucleotide phosphate (NADPH) under glucose deprivation, followed by an increase in intracellular disulfide stress and aberrant disulfide bond formation within actin networks, ultimately culminating in cytoskeletal collapse and disulfidptosis. Disulfidptosis involves crucial physiological processes in eukaryotic cells, such as cystine and glucose uptake, NADPH metabolism, and actin dynamics. The Rac1-WRC pathway-mediated actin polymerization is also implicated in this cell death due to its contribution to disulfide bond formation. However, the precise mechanisms underlying disulfidptosis and its role in tumors are not well understood. This is probably due to the multifaceted functionalities of SLC7A11 within cells and the complexities of the downstream pathways driving disulfidptosis. This review describes the critical roles of SLC7A11 in cells and summarizes recent research advancements in the potential pathways of disulfidptosis. Moreover, the less-studied aspects of this newly discovered cell death process are highlighted to stimulate further investigations in this field. [ABSTRACT FROM AUTHOR]

The DNA damage response is a highly conserved protective mechanism that enables cells to cope with various lesions in the genome. Extensive studies across different eukaryotic cells have identified the crucial roles played by components required for response to DNA damage. When compared to the essential signal transducers and repair factors in the DNA damage response circuitry, the negative regulators and underlying mechanisms of this circuitry have been relatively under-examined. In this study, we investigated Gst1, a putative glutathione transferase in the fungal pathogen Candida albicans. We found that under stress caused by the DNA damage agent MMS, GST1 expression was significantly upregulated, and this upregulation was further enhanced by the loss of the checkpoint kinases and DNA repair factors. Somewhat counterintuitively, deletion of GST1 conferred increased resistance to MMS, potentially via enhancing the phosphorylation of Rad53. Furthermore, overexpression of RAD53 or deletion of GST1 resulted in upregulated transcription of DNA damage repair genes, including CAS1, RAD7, and RAD30, while repression of RAD7 transcription in the GST1 deletion reversed the strain's heightened resistance to MMS. Finally, Gst1 physically interacted with Rad53, and their interaction weakened in response to MMS-induced stress. Overall, our findings suggest a negative regulatory role for GST1 in DNA damage response in C. albicans, and position Gst1 within the Rad53-mediated signaling pathway. These findings hold significant implications for understanding the mechanisms underlying the DNA damage response in this fungal pathogen and supply new potential targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

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Mitochondria are crucial organelles that play a central role in cellular metabolism and programmed cell death in eukaryotic cells. Mitochondrial autophagy (mitophagy) is a selective process where damaged mitochondria are encapsulated and degraded through autophagic mechanisms, ensuring the maintenance of both mitochondrial and cellular homeostasis. Excessive programmed cell death in neurons can result in functional impairments following cerebral ischemia and trauma, as well as in chronic neurodegenerative diseases, leading to irreversible declines in motor and cognitive functions. Neuroinflammation, an inflammatory response of the central nervous system to factors disrupting homeostasis, is a common feature across various neurological events, including ischemic, infectious, traumatic, and neurodegenerative conditions. Emerging research suggests that regulating autophagy may offer a promising therapeutic avenue for treating certain neurological diseases. Furthermore, existing literature indicates that various small molecule autophagy regulators have been tested in animal models and are linked to neurological disease outcomes. This review explores the role of mitophagy in programmed neuronal death and its connection to neuroinflammation. [ABSTRACT FROM AUTHOR]

Advanced glycation end-products (AGE) are a pervasive form of protein damage implicated in the pathogenesis of neurodegenerative disease, atherosclerosis and diabetes mellitus. Glycation is typically mediated by reactive dicarbonyl compounds that accumulate in all cells as toxic byproducts of glucose metabolism. Here, we show that AGE crosslinking is harnessed to activate an antibacterial phospholipase effector protein deployed by the type VI secretion system of Enterobacter cloacae. Endogenous methylglyoxal reacts with a specific arginine-lysine pair to tether the N- and C-terminal α-helices of the phospholipase domain. Substitutions at these positions abrogate both crosslinking and toxic phospholipase activity, but in vitro enzyme function can be restored with an engineered disulfide that covalently links the N- and C-termini. Thus, AGE crosslinking serves as a bona fide post-translation modification to stabilize phospholipase structure. Given the ubiquity of methylglyoxal in prokaryotic and eukaryotic cells, these findings suggest that glycation may be exploited more generally to stabilize other proteins. This alternative strategy to fortify tertiary structure could be particularly advantageous in the cytoplasm, where redox potentials preclude disulfide bond formation. Protein glycation is a ubiquitous form of damage caused by reactive dicarbonyl byproducts of metabolism. Here, Jensen et al. show that dicarbonyl-mediated protein crosslinking is harnessed to activate a phospholipase toxin, demonstrating that glycation can also be beneficial for protein function. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), induced by Mycobacterium tuberculosis (Mtb) infection, remains a major public health issue worldwide. Mtb has developed complicated strategies to inhibit the immunological clearance of host cells, which significantly promote TB epidemic and weaken the anti-TB treatments. Host-directed therapy (HDT) is a novel approach in the field of anti-infection for overcoming antimicrobial resistance by enhancing the antimicrobial activities of phagocytes through phagosomal maturation, autophagy and antimicrobial peptides. Autophagy, a highly conserved cellular event within eukaryotic cells that is effective against a variety of bacterial infections, has been shown to play a protective role in host defense against Mtb. In recent decades, the introduction of nanomaterials into medical fields open up a new scene for novel therapeutics with enhanced efficiency and safety against different diseases. The active modification of nanomaterials not only allows their attractive targeting effects against the host cells, but also introduce the potential to regulate the host anti-TB immunological mechanisms, such as apoptosis, autophagy or macrophage polarization. In this review, we introduced the mechanisms of host cell autophagy for intracellular Mtb clearance, and how functional nanomaterials regulate autophagy for disease treatment. Moreover, we summarized the recent advances of nanomaterials for autophagy regulations as novel HDT strategies for anti-TB treatment, which may benefit the development of more effective anti-TB treatments. [ABSTRACT FROM AUTHOR]

Capsular polysaccharide is an important virulence factor of Glaesserella parasuis. An acapsular mutant displays multiple phenotype variations, while the underlying mechanism for these variations is unknown. In this study, we created an acapsular mutant by deleting the wza gene in the capsule locus. We then used transcriptome analysis to compare the gene expression profiles of the wza deletion mutant with those of the parental strain to understand the possible reasons for the phenotypic differences. The mutant Δwza, which has a deleted wza gene, secreted less polysaccharide and lost its capsule structure. The Δwza exhibited increased autoagglutination, biofilm formation and adherence to eukaryotic cells, while the complementary strain C-Δwza partially restored the phenotype. Transcriptome analysis revealed several differentially expressed genes (DEGs) in Δwza, including up-regulated outer membrane proteins and proteins involved in peptidoglycan biosynthesis, suggesting that wza deletion affects the cell wall homeostasis of G. parasuis. Transcriptome analysis revealed the contribution of non-coding RNAs in the regulation of DEGs. Moreover, a new virulence-associated trimeric autotransporter, VtaA31 is upregulated in Δwza. It is responsible for enhanced autoagglutination but not for enhanced biofilm formation and adherence to eukaryotic cells in Δwza. In conclusion, these data indicate that wza affects the expression of multiple genes, especially those related to cell wall synthesis. Furthermore, they provide evidence that vtaA31 is involved in the autoagglutination of G. parasuis. [ABSTRACT FROM AUTHOR]

Hsp90 is a molecular chaperone of central importance for protein homeostasis in the cytosol of eukaryotic cells, with key functional and structural traits conserved from yeast to man. During evolution, Hsp90 has gained additional functional importance, leading to an increased number of interacting co-chaperones and client proteins. Here, we show that the overall conformational transitions coupled to the ATPase cycle of Hsp90 are conserved from yeast to humans, but cycle timing as well as the dynamics are significantly altered. In contrast to yeast Hsp90, the human Hsp90 is characterized by broad ensembles of conformational states, irrespective of the absence or presence of ATP. The differences in the ATPase rate and conformational transitions between yeast and human Hsp90 are based on two residues in otherwise conserved structural elements that are involved in triggering structural changes in response to ATP binding. The exchange of these two mutations allows swapping of the ATPase rate and of the conformational transitions between human and yeast Hsp90. Our combined results show that Hsp90 evolved to a protein with increased conformational dynamics that populates ensembles of different states with strong preferences for the N-terminally open, client-accepting states. Hsp90 is a molecular chaperone important for protein homeostasis. Here, the authors show that the conformational transitions during the Hsp90 ATPase cycle are conserved from yeast to humans, but a few mutations alter cycle timing and dynamics. [ABSTRACT FROM AUTHOR]

Over the past few years, numerous bacterial strains have become resistant to selected drugs from various therapeutic groups. A potential tool in the fight against these strains is antimicrobial photodynamic therapy (APDT). APDT acts in a non-specific manner by generating reactive oxygen species and radicals, thereby inducing multidimensional intracellular effects. Importantly, the chance that bacteria will develop defense mechanisms against APDT is considered to be low. In our research, we performed the synthesis and physicochemical characterization of curcumin derivatives enriched with morpholine motifs. The obtained compounds were assessed regarding photostability, singlet oxygen generation, aggregation, and acute toxicity toward prokaryotic Aliivibrio fischeri cells in the Microtox® test. The impact of the compounds on the survival of eukaryotic cells in the MTT assay was also tested (WM266-4, WM115—melanoma, MRC-5—lung fibroblasts, and PHDF—primary human dermal fibroblasts). Initial studies determining the photocytotoxicity, and thus the potential APDT usability, were conducted with the following microbial strains: Candida albicans, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa. It was noted that the exposure of bacteria to LED light at 470 nm (fluence: 30 J/cm2) in the presence of quaternized curcumin derivatives at the conc. of 10 µM led to a reduction in Staphylococcus aureus survival of over 5.4 log. [ABSTRACT FROM AUTHOR]

In the eukaryotic cells, the ubiquitin–proteasome system (UPS) plays a crucial role in the intracellular protein turnover. It is involved in several cellular functions such as the control of the regular cell cycle progression, the immune surveillance, and the homeostasis. Within the 20S proteasome barrel-like structure, the catalytic subunits, β1, β2 and β5, are responsible for different proteolytic activities: caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-L), respectively. The β5 subunit is particularly targeted for its role in antitumor activity: the synthesis of β5 subunit inhibitors could be a promising strategy for the treatment of solid and hematologic tumors. In the present work, we performed two combination studies of AM12, a recently developed synthetic proteasome inhibitor, with curcumin and quercetin, two nutraceuticals endowed of many pharmacological properties. We measured the combination index (CI), applying the Chou and Talalay method, comparing the two studies, from 50% to 90% of proteasome inhibition. In the case of the combination AM12 + curcumin, an increasing synergism was observed from 50% to 90% of proteasome inhibition, while in the case of the combination AM12 + quercetin an additive effect was observed only from 50% to 70% of β5 subunit inhibition. These results suggest that combining AM12 with curcumin is a more promising strategy than combining it with quercetin for potential therapeutic applications, especially in treating tumors. [ABSTRACT FROM AUTHOR]

There is a growing interest in the utilization of metal oxide nanoparticles as antimicrobial agents. This review will focus on titanium dioxide nanoparticles (TiO2 NPs), which have been demonstrated to exhibit high antimicrobial activity against bacteria and fungi, chemical stability, low toxicity to eukaryotic cells, and therefore high biocompatibility. Despite the extensive research conducted in this field, there is currently no consensus on how to enhance the antimicrobial efficacy of TiO2 NPs. The aim of this review is to evaluate the influence of various factors, including particle size, shape, composition, and synthesis parameters, as well as microbial type, on the antibacterial activity of TiO2 NPs against bacteria and fungi. Furthermore, the review offers a comprehensive overview of the methodologies employed in the synthesis and characterization of TiO2 NPs. The antimicrobial activity of TiO2 exhibits a weak dependence on the microorganism species. A tendency towards increased antibacterial activity is observed with decreasing TiO2 NP size. The dependence on the shape and composition is more pronounced. The most pronounced antimicrobial potential is exhibited by amorphous NPs and NPs doped with inorganic compounds. This review may be of interest to specialists in biology, medicine, chemistry, and other related fields. [ABSTRACT FROM AUTHOR]

Eukaryotic cells feature distinct membrane-enclosed organelles such as mitochondria and peroxisomes, each playing vital roles in cellular function and organization. These organelles are linked at membrane contact sites, facilitating interorganellar molecule and ion exchange. Most contact-forming proteins identified to date are membrane proteins or membrane-associated proteins, which can form very stable contacts. Recent findings suggest additional mechanistically distinct tethering events that arise from dual protein targeting. Proteins bearing targeting signals for multiple organelles, such as an N -terminal signal for mitochondria and a C -terminal signal for peroxisomes, function as tethers, fostering contacts by engaging targeting factors at both organelles. A number of dually targeted membrane proteins can contribute to contact site formation and transit from one organelle to the other as well. These interactions may enable the fine-tuning of organelle proximity, hence, adapting connections to meet varying physiological demands. [ABSTRACT FROM AUTHOR]

During eukaryotic cell division, a microtubule-based structure called the spindle exerts forces on chromosomes. The best-studied spindle forces, including those responsible for the separation of sister chromatids, are directed parallel to the spindle's long axis. By contrast, little is known about forces perpendicular to the spindle axis, which determine the metaphase plate configuration and thus the location of chromosomes in the subsequent nucleus. Using live-cell microscopy, we find that metaphase chromosomes are spatially anti-correlated in mouse oocyte spindles, evidence of previously unknown long-range forces acting perpendicular to the spindle axis. We explain this observation by showing that the spindle's microtubule network behaves as a nematic liquid crystal and that deformation of the nematic field around embedded chromosomes causes long-range repulsion between them. [ABSTRACT FROM AUTHOR]

The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here, we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1 and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes. The mechanism of nonsense-mediated mRNA decay (NMD) is thought to differ between yeast and metazoa, even though the Upf1 RNA helicase is universally conserved. This study uses the crystal structure of Upf1 in complex with Nmd4 to show that the metazoan SMG6 NMD factor binds to Upf1 using an evolutionarily conserved binding mode. [ABSTRACT FROM AUTHOR]

Background: Kidney stone formation is a common disease that causes a significant threat to human health. The crystallization mechanism of calcium oxalate, the most common type of kidney stone, has been extensively researched, yet the damaging effects and mechanisms of calcium oxalate crystals on renal tubular epithelial cells remain incompletely elucidated. Regulated mitochondrial dynamics is essential for eukaryotic cells, but its role in the occurrence and progression of calcium oxalate (CaOx) nephrolithiasis is not yet understood. Methods: An animal model of calcium oxalate-related nephrolithiasis was established in adult male Sprague‒Dawley (SD) rats by continuously administering drinking water containing 1% ethylene glycol for 28 days. The impact of calcium oxalate crystals on mitochondrial dynamics and apoptosis in renal tubular epithelial cells was investigated using HK2 cells in vitro. Blood samples and bilateral kidney tissues were collected for histopathological evaluation and processed for tissue injury, inflammation, fibrosis, oxidative stress detection, and mitochondrial dynamics parameter analysis. Results: Calcium oxalate crystals caused higher levels of mitochondrial fission and apoptosis in renal tubular epithelial cells both in vivo and in vitro. Administration of a PPARγ agonist significantly alleviated mitochondrial fission and apoptosis in renal tubular epithelial cells, and improved renal function, accompanied by reduced levels of oxidative stress, increased antioxidant enzyme expression, alleviation of inflammation, and reduced fibrosis in vivo. Conclusion: Our results indicated that increased mitochondrial fission in renal tubular epithelial cells is a critical component of kidney injury caused by calcium oxalate stones, leading to the accumulation of reactive oxygen species within the tissue and the subsequent initiation of apoptosis. Regulating mitochondrial dynamics represents a promising approach for calcium oxalate nephrolithiasis. [ABSTRACT FROM AUTHOR]

Summary Arg‐any amino acid‐Leu‐Arg (RXLR) effectors are central oomycete virulence factors that suppress plant immunity. Relatively little is known about how they are processed post‐translationally before delivery into host cells. A range of molecular, cell and biochemical processes were used to investigate proteolytic processing of RXLR and Glu‐Glu‐Arg (EER) motifs in Phytophthora infestans effectors. Proteolytic cleavage at the RXLR motif occurred before secretion in all effectors tested, suggesting it is a general rule. Cleavage occurred between the leucine and the second arginine. There was no cleavage of a naturally occurring second RXLR motif in a structured region of Pi21388/AvrBlb1, or one introduced at a similar position in effector Pi04314, in keeping with the motif being positionally constrained, potentially to disordered regions closely following the signal peptide. Remarkably, independent proteolytic cleavage of the EER motif, often found immediately after the RXLR, was also observed, occurring immediately after the arginine. Full‐length effectors expressed in host plant Nicotiana benthamiana revealed that, although secreted, they were poorly processed, suggesting that RXLR and EER cleavage does not occur in all eukaryotic cells. We conclude that, whether possessing both RXLR and EER, or either motif alone, these effectors are likely generally proteolytically processed before secretion. [ABSTRACT FROM AUTHOR]

Despite widespread public interest in the health impact of exposure to microwave radiation, studies of the influence of microwave radiation on biological samples are often inconclusive or contradictory. Here we examine the influence of microwave radiation of frequencies 3.5 GHz, 20 GHz and 29 GHz on the growth of microtubules, which are biological nanotubes that perform diverse functions in eukaryotic cells. Since microtubules are highly polar and can extend several micrometres in length, they are predicted to be sensitive to non-ionizing radiation. Moreover, it has been speculated that tubulin dimers within microtubules might rapidly toggle between different conformations, potentially participating in computational or other cooperative processes. Our data show that exposure to microwave radiation yields a microtubule growth curve that is distorted relative to control studies utilizing a homogeneous temperature jump. However, this apparent effect of non-ionizing radiation is reproduced by control experiments using an infrared laser or hot air to heat the sample and thereby mimic the thermal history of samples exposed to microwaves. As such, no non-thermal effects of microwave radiation on microtubule growth can be assigned. Our results highlight the need for appropriate control experiments in biophysical studies that may impact on the sphere of public interest. [ABSTRACT FROM AUTHOR]

Polyamines, characterized by their polycationic nature, are ubiquitously present in all organisms and play numerous cellular functions. Among polyamines, spermidine stands out as the predominant type in both prokaryotic and eukaryotic cells. The PotD-PotABC protein complex in Escherichia coli, belonging to the adenosine triphosphate-binding cassette transporter family, is a spermidine-preferential uptake system. Here, we report structural details of the polyamine uptake system PotD-PotABC in various states. Our analyses reveal distinct "inward-facing" and "outward-facing" conformations of the PotD-PotABC transporter, as well as conformational changes in the "gating" residues (F222, Y223, D226, and K241 in PotB; Y219 and K223 in PotC) controlling spermidine uptake. Therefore, our structural analysis provides insights into how the PotD-PotABC importer recognizes the substrate-binding protein PotD and elucidates molecular insights into the spermidine uptake mechanism of bacteria. [ABSTRACT FROM AUTHOR]

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens that infect eukaryotic cells and reside within a host-derived vacuole known as the inclusion. To facilitate intracellular replication, these bacteria must engage in host-pathogen interactions to obtain nutrients and membranes required for the growth of the inclusion, thereby sustaining prolonged bacterial colonization. Autophagy is a highly conserved process that delivers cytoplasmic substrates to the lysosome for degradation. Pathogens have developed strategies to manipulate and/or exploit autophagy to promote their replication and persistence. This review delineates recent advances in elucidating the interplay between Chlamydia trachomatis infection and autophagy in recent years, emphasizing the intricate strategies employed by both the Chlamydia pathogens and host cells. Gaining a deeper understanding of these interactions could unveil novel strategies for the prevention and treatment of Chlamydia infection. [ABSTRACT FROM AUTHOR]

Eukaryotic cells encounter diverse threats jeopardizing their integrity, prompting the development of defense mechanisms against these stressors. Among these mechanisms, inflammasomes are well‐known for their roles in coordinating the inflammatory response against infections. Extensive research has unveiled their multifaceted involvement in cellular processes beyond inflammation. Recent studies emphasize the intricate relationship between the inflammasome and the DNA damage response (DDR). They highlight how the DDR participates in inflammasome activation and the reciprocal impact of inflammasome on DDR and genome integrity preservation. Moreover, novel functions of inflammasome sensors in DDR pathways have emerged, broadening our understanding of their roles. Finally, this review delves into identifying common signals that drive the activation of inflammasome sensors alongside activation cues for the DNA damage response, offering potential insights into shared regulatory pathways between these critical cellular processes. [ABSTRACT FROM AUTHOR]

The integrated stress response (ISR) is a conserved pathway in eukaryotic cells that is activated in response to multiple sources of cellular stress. Although acute activation of this pathway restores cellular homeostasis, intense or prolonged ISR activation perturbs cell function and may contribute to neurodegeneration. DNL343 is an investigational CNS-penetrant small-molecule ISR inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. DNL343 reduced CNS ISR activity and neurodegeneration in a dose-dependent manner in two established in vivo models - the optic nerve crush injury and an eIF2B loss of function (LOF) mutant - demonstrating neuroprotection in both and preventing motor dysfunction in the LOF mutant mouse. Treatment with DNL343 at a late stage of disease in the LOF model reversed elevation in plasma biomarkers of neuroinflammation and neurodegeneration and prevented premature mortality. Several proteins and metabolites that are dysregulated in the LOF mouse brains were normalized by DNL343 treatment, and this response is detectable in human biofluids. Several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD), a neurodegenerative disease that is driven by eIF2B LOF and chronic ISR activation, supporting their potential translational relevance. This study demonstrates that DNL343 is a brain-penetrant ISR inhibitor capable of attenuating neurodegeneration in mouse models and identifies several biomarker candidates that may be used to assess treatment responses in the clinic. [ABSTRACT FROM AUTHOR]

Background: Type VI secretion system (T6SS) is widely present in Gram-negative bacteria and directly mediates antagonistic prokaryote interactions. PAAR (proline-alanine-alanine-arginine repeats) proteins have been proven essential for T6SS-mediated secretion and target cell killing. Although PAAR proteins are commonly found in A. baumannii, their biological functions are not fully disclosed yet. In this study, we investigated the functions of a PAAR protein termed TagP (T6SS-associated-gene PAAR), encoded by the gene ACX60_RS09070 outside the core T6SS locus of A. baumannii strain ATCC 17978. Methods: In this study, tagP null and complement A. baumannii ATCC 17978 strains were constructed. The influence of TagP on T6SS function was investigated through Hcp detection and bacterial competition assay; the influence on environmental fitness was studied through in vitro growth, biofilm formation assay, surface motility assay, survivability in various simulated environmental conditions; the influence on pathogenicity was explored through cell adhesion and invasion assays, intramacrophage survival assay, serum survival assay, and G. melonella Killing assays. Quantitative transcriptomic and proteomic analyses were utilized to observe the global impact of TagP on bacterial status. Results: Compared with the wildtype strain, the tagP null mutant was impaired in several tested phenotypes such as surface motility, biofilm formation, tolerance to adverse environments, adherence to eukaryotic cells, endurance to serum complement killing, and virulence to Galleria melonella. Notably, although RNASeq and proteomics analysis revealed that many genes were significantly downregulated in the tagP null mutant compared to the wildtype strain, there is no significant difference in their antagonistic abilities. We also found that Histonelike nucleoid structuring protein (H-NS) was significantly upregulated in the tagP null mutant at both mRNA and protein levels. Conclusions: This study enriches our understanding of the biofunction of PAAR proteins in A. baumannii. The results indicates that TagP involved in a unique modulation of fitness and virulence control in A. baumannii, it is more than a classic PAAR protein involved in T6SS, while how TagP play roles in the fitness and virulence of A. baumannii needs further investigation to clarify. [ABSTRACT FROM AUTHOR]

The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family. [ABSTRACT FROM AUTHOR]

Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates. [ABSTRACT FROM AUTHOR]

Post-translational SUMOylation of nuclear and cytosolic proteins maintains homeostasis in eukaryotic cells and orchestrates programmed responses to changes in metabolic demand or extracellular stimuli. In excitable cells, SUMOylation tunes the biophysical properties and trafficking of ion channels. Ion channel SUMOylation status is determined by the opposing enzyme activities of SUMO ligases and deconjugases. Phosphorylation also plays a permissive role in SUMOylation. SUMO deconjugases have been identified for several ion channels, but their corresponding E3 ligases remain unknown. This study shows PIAS3, a.k.a. KChAP, is a bona fide SUMO E3 ligase for Kv4.2 and HCN2 channels in HEK cells, and endogenous Kv4.2 and Kv4.3 channels in cardiomyocytes. PIAS3-mediated SUMOylation at Kv4.2-K579 increases channel surface expression through a rab11a-dependent recycling mechanism. PKA phosphorylation at Kv4.2-S552 reduces the current mediated by Kv4 channels in HEK293 cells, cardiomyocytes, and neurons. This study shows PKA mediated phosphorylation blocks Kv4.2-K579 SUMOylation in HEK cells and cardiomyocytes. Together, these data identify PIAS3 as a key downstream mediator in signaling cascades that control ion channel surface expression. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) modification stands out among various RNA modifications as the predominant form within eukaryotic cells, influencing numerous cellular processes implicated in disease development. m6A modification has gained increasing attention in the development of atherosclerosis and has become a research hotspot in recent years. Programmed cell death (PCD), encompassing apoptosis, autophagy, pyroptosis, ferroptosis, and necroptosis, plays a pivotal role in atherosclerosis pathogenesis. In this review, we delve into the intricate interplay between m6A modification and diverse PCD pathways, shedding light on their complex association during the onset and progression of atherosclerosis. Clarifying the relationship between m6A and PCD in atherosclerosis is of great significance to provide novel strategies for cardiovascular disease treatment. [ABSTRACT FROM AUTHOR]

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella’s T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein invasion plasmid antigen D (IpaD). Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement. [ABSTRACT FROM AUTHOR]

Eukaryotic cells must accurately transfer their genetic material and cellular components to their daughter cells. Initially, cells duplicate their chromosomes and subsequently segregate them toward the poles. The actomyosin ring, a crucial molecular machinery normally located in the middle of the cells and underneath the plasma membrane, then physically divides the cytoplasm and all components into two daughter cells, each ready to start a new cell cycle. This process, known as cytokinesis, is conserved throughout evolution. Defects in cytokinesis can lead to the generation of genetically unstable tetraploid cells, potentially initiating uncontrolled proliferation and cancer. This review focuses on the molecular mechanisms by which budding yeast cells build the actomyosin ring and the preceding steps involved in forming a scaffolding structure that supports the challenging structural changes throughout cytokinesis. Additionally, we describe how cells coordinate actomyosin ring contraction, plasma membrane ingression, and extracellular matrix deposition to successfully complete cytokinesis. Furthermore, the review discusses the regulatory roles of Cyclin-Dependent Kinase (Cdk1) and the Mitotic Exit Network (MEN) in ensuring the precise timing and execution of cytokinesis. Understanding these processes in yeast provides insights into the fundamental aspects of cell division and its implications for human health. [ABSTRACT FROM AUTHOR]

The exocyst complex, an evolutionarily conserved octameric protein assembly, plays a central role in the targeted binding and fusion of vesicles at the plasma membrane. In fungal cells, this transport system is essential for polarized growth, morphogenesis, cell wall maintenance and virulence. Recent advances have greatly improved our understanding of the role and regulation of the exocyst complex in fungi. This review synthesizes these developments and focuses on the intricate interplay between the exocyst complex, specific fungal cargos and regulatory proteins. Insights into thestructure of the exocyst and its functional dynamics have revealed new dimensions of its architecture and its interactions with the cellular environment. Furthermore, the regulation of exocyst activity involves complex signaling pathways and interactions with cytoskeletal elements that are crucial for its role in vesicle trafficking. By exploring these emerging themes, this review provides a comprehensive overview of the multifaceted functions of the exocyst complex in fungal biology. Understanding these mechanisms offers potential avenues for novel therapeutic strategies against fungal pathogens and insights into the general principles of vesicle trafficking in eukaryotic cells. The review therefore highlights the importance of the exocyst complex in maintaining cellular functions and its broader implications in fungal pathogenicity and cell biology. [ABSTRACT FROM AUTHOR]

Mitochondria play a crucial role in maintaining the energy status and redox homeostasis of eukaryotic cells. They are responsible for the metabolic efficiency of cells, providing both ATP and intermediate metabolic products. They also regulate cell survival and death under stress conditions by controlling the cell response or activating the apoptosis process. This functional diversity of mitochondria indicates their great importance for cellular metabolism. Hence, dysfunctions of these structures are increasingly recognized as an element of the etiology of many human diseases and, therefore, an extremely promising therapeutic target. Mitochondrial dysfunctions can be caused by mutations in both nuclear and mitochondrial DNA, as well as by stress factors or replication errors. Progress in knowledge about the biology of mitochondria, as well as the consequences for the efficiency of the entire organism resulting from the dysfunction of these structures, is achieved through the use of model organisms. They are an invaluable tool for analyzing complex cellular processes, leading to a better understanding of diseases caused by mitochondrial dysfunction. In this work, we review the most commonly used model organisms, discussing both their advantages and limitations in modeling fundamental mitochondrial processes or mitochondrial diseases. [ABSTRACT FROM AUTHOR]

Ubiquitination modifications permit the degradation of labelled target proteins with the assistance of proteasomes and lysosomes, which is the main protein degradation pathway in eukaryotic cells. Polyubiquitination modifications of proteins can also affect their functions. De-ubiquitinating enzymes reverse the process of ubiquitination via cleavage of the ubiquitin molecule, which is known as a de-ubiquitination. It was demonstrated that ubiquitination and de-ubiquitination play key regulatory roles in fatty acid transport, de novo synthesis, and desaturation in dairy mammary epithelial cells. In addition, natural plant extracts, such as stigmasterol, promote milk fat synthesis in epithelial cells via the ubiquitination pathway. This paper reviews the current research on ubiquitination and de-ubiquitination in dairy milk fat production, with a view to providing a reference for subsequent research on milk fat and exploring new directions for the improvement of milk quality. [ABSTRACT FROM AUTHOR]

Cold atmospheric plasma (CAP) devices generate reactive oxygen and nitrogen species, have antimicrobial and antiviral properties, but also affect the molecular and cellular mechanisms of eukaryotic cells. The aim of this study is to investigate CAP treatment in the upper respiratory tract (URT) to reduce the incidence of ventilator-associated bacterial pneumonia (especially superinfections with multi-resistant pathogens) or viral infections (e.g., COVID-19). For this purpose, the surface-microdischarge-based plasma intensive care (PIC) device was developed by terraplasma medical GmbH. This study analyzes the safety aspects using in vitro assays and molecular characterization of human oral keratinocytes (hOK), human bronchial–tracheal epithelial cells (hBTE), and human lung fibroblasts (hLF). A 5 min CAP treatment with the PIC device at the "throat" and "subglottis" positions in the URT model did not show any significant differences from the untreated control (ctrl.) and the corresponding pressurized air (PA) treatment in terms of cell morphology, viability, apoptosis, DNA damage, and migration. However, pro-inflammatory cytokines (MCP-1, IL-6, and TNFα) were induced in hBTE and hOK cells and profibrotic molecules (collagen-I, FKBP10, and αSMA) in hLF at the mRNA level. The use of CAP in the oropharynx may make an important contribution to the recovery of intensive care patients. The results indicate that a 5 min CAP treatment in the URT with the PIC device does not cause any cell damage. The extent to which immune cell activation is induced and whether it has long-term effects on the organism need to be carefully examined in follow-up studies in vivo. [ABSTRACT FROM AUTHOR]

N6‐methyladenosine (m6A) is the most abundant modification of RNA in eukaryotic cells. Previous studies have shown that m6A is pivotal in diverse diseases especially cancer. m6A corelates with the initiation, progression, resistance, invasion, and metastasis of cancer. However, despite these insights, a comprehensive understanding of its specific roles and mechanisms within the complex landscape of cancer is still elusive. This review begins by outlining the key regulatory proteins of m6A modification and their posttranslational modifications (PTMs), as well as the role in chromatin accessibility and transcriptional activity within cancer cells. Additionally, it highlights that m6A modifications impact cancer progression by modulating programmed cell death mechanisms and affecting the tumor microenvironment through various cancer‐associated immune cells. Furthermore, the review discusses how microorganisms can induce enduring epigenetic changes and oncogenic effect in microorganism‐associated cancers by altering m6A modifications. Last, it delves into the role of m6A modification in cancer immunotherapy, encompassing RNA therapy, immune checkpoint blockade, cytokine therapy, adoptive cell transfer therapy, and direct targeting of m6A regulators. Overall, this review clarifies the multifaceted role of m6A modification in cancer and explores targeted therapies aimed at manipulating m6A modification, aiming to advance cancer research and improve patient outcomes. [ABSTRACT FROM AUTHOR]

Simple Summary: This study focused on developing a monoclonal antibody against the F4ac subtype of the FaeG protein of enterotoxigenic Escherichia coli by screening an antibody library in immunized mice, providing a potential drug option for antibiotic replacement therapy in veterinary medicine. The researchers immunized mice with formaldehyde-inactivated ETEC and a soluble recombinant FaeG protein to generate an antibody library. Through fluorescence-activated cell sorting and a eukaryotic expression system, we screened and obtained anti-rFaeG IgG monoclonal antibodies that effectively inhibited ETEC's adhesion to IPEC-J2 cells. In vivo experiments confirmed the protective effect of these antibodies in mice challenged with ETEC, demonstrating their potential as a safe and effective therapeutic option. The misuse of antibiotics in veterinary medicine presents significant challenges, highlighting the need for alternative therapeutic approaches such as antibody drugs. Therefore, it is necessary to explore the application of antibody drugs in veterinary settings to reduce economic losses and health risks. This study focused on targeting the F4ac subtype of the FaeG protein, a key adhesion factor in enterotoxigenic Escherichia coli (ETEC) infections in piglets. By utilizing formaldehyde-inactivated ETEC and a soluble recombinant FaeG (rFaeG) protein, an antibody library against the FaeG protein was established. The integration of fluorescence-activated cell sorting (FACS) and a eukaryotic expression vector containing murine IgG Fc fragments facilitated the screening of anti-rFaeG IgG monoclonal antibodies (mAbs). The results demonstrate that the variable regions of the screened antibodies could inhibit K88-type ETEC adhesion to IPEC-J2 cells. Furthermore, in vivo neutralization assays in mice showed a significant increase in survival rates and a reduction in intestinal inflammation. This research underscores the potential of antibody-based interventions in veterinary medicine, emphasizing the importance of further exploration in this field to address antibiotic resistance and improve animal health outcomes. [ABSTRACT FROM AUTHOR]

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

Eukaryotic cells show an astounding ability to remodel their shape and cytoskeleton and to migrate through pores and constrictions smaller than their nuclear diameter. However, the relation of nuclear deformation and migration dynamics in confinement remains unclear. Here, we study the mechanics and dynamics of mesenchymal cancer cell nuclei transitioning through three-dimensional compliant hydrogel channels. We find a biphasic dependence of migration speed and transition frequency on channel width, peaking at widths comparable to the nuclear diameter. Using confocal imaging and hydrogel bead displacement, we determine nuclear deformations and corresponding forces during confined migration. The nucleus deforms reversibly with a reduction in volume during confinement. With decreasing channel width, the nuclear shape during transmigration changes biphasically, concomitant with the transitioning dynamics. Our proposed physical model explains the observed nuclear shapes and transitioning dynamics in terms of the cytoskeletal force generation adapting from purely pullingbased to a combined pulling-and pushing-based mechanism with increasing nuclear confinement. [ABSTRACT FROM AUTHOR]

The current‐generation adenine base editor (ABE) ABE8e, which has evolved from the prokaryotic evolution system, exhibits high efficiency in mediating A‐to‐G conversion and is presumed to be promising for gene therapy. However, its much wider editing window and substantially higher off‐target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library‐assisted protein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off‐target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose four evolved variants that offered either similar or modestly higher efficiency within a narrower editing window of protospacer position ≈4–7 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off‐target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease‐suppressing mutations and disease‐correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant‐library‐assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing. [ABSTRACT FROM AUTHOR]

Mitochondria are crucial organelles responsible for energy generation in eukaryotic cells. Oxidative stress, calcium disorders, and mitochondrial DNA abnormalities can all cause mitochondrial dysfunction. It is now well documented that mitochondrial dysfunction significantly contributes to the pathogenesis of numerous illnesses. Hence, it is vital to investigate innovative treatment methods targeting mitochondrial dysfunction. Extracellular vesicles (EVs) are cell-derived nanovesicles that serve as intercellular messengers and are classified into small EVs (sEVs, < 200 nm) and large EVs (lEVs, > 200 nm) based on their sizes. It is worth noting that certain subtypes of EVs are rich in mitochondrial components (even structurally intact mitochondria) and possess the ability to transfer them or other contents including proteins and nucleic acids to recipient cells to modulate their mitochondrial function. Specifically, EVs can modulate target cell mitochondrial homeostasis as well as mitochondria-controlled apoptosis and ROS generation by delivering relevant substances. In addition, the artificial modification of EVs as delivery carriers for therapeutic goods targeting mitochondria is also a current research hotspot. In this article, we will focus on the ability of EVs to modulate the mitochondrial function of target cells, aiming to offer novel perspectives on therapeutic approaches for diverse conditions linked to mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Polyamines are ubiquitous biomolecules with a number of established functions in eukaryotic cells. In plant cells, polyamines have previously been linked to abiotic and biotic stress tolerance, as well as to the modulation of programmed cell death (PCD), with contrasting reports on their pro‐PCD and pro‐survival effects. Here, we used two well‐established platforms for the study of plant PCD, Arabidopsis thaliana suspension cultures cells and the root hair assay, to examine the roles of the polyamines spermine and spermidine in the regulation of PCD. Using these systems for precise quantification of cell death rates, we demonstrate that both polyamines can trigger PCD when applied exogenously at higher doses, whereas at lower concentrations they inhibit PCD induced by both biotic and abiotic stimuli. Furthermore, we show that concentrations of polyamines resulting in inhibition of PCD generated a transient ROS burst in our experimental system, and activated the expression of oxidative stress‐ and pathogen response‐associated genes. Finally, we examined PCD responses in existing Arabidopsis polyamine synthesis mutants, and identified a subtle PCD phenotype in Arabidopsis seedlings deficient in thermo‐spermine. The presented data show that polyamines can have a role in PCD regulation; however, that role is dose‐dependent and consequently they may act as either inhibitors, or inducers, of PCD in Arabidopsis. [ABSTRACT FROM AUTHOR]

The biological clock in eukaryotes controls daily rhythms in physiology and behavior. It displays a complex organization that involves the molecular transcriptional clock and the redox oscillator which may coordinately work to control cellular rhythms. The redox oscillator has emerged very early in evolution in adaptation to the environmental changes in O2 levels and has been shown to regulate daily rhythms in glycerolipid (GL) metabolism in different eukaryotic cells. GLs are key components of lipid droplets (LDs), intracellular storage organelles, present in all living organisms, and essential for energy and lipid homeostasis regulation and survival; however, the cell bioenergetics status is not constant across time and depends on energy demands. Thus, the formation and degradation of LDs may reflect a time-dependent process following energy requirements. This work investigated the presence of metabolic rhythms in LD content along evolution by studying prokaryotic and eukaryotic cells and organisms. We found sustained temporal oscillations in LD content in Pseudomonas aeruginosa bacteria and Caenorhabditis elegans synchronized by temperature cycles, in serum-shock synchronized human embryonic kidney cells (HEK 293 cells) and brain tumor cells (T98G and GL26) after a dexamethasone pulse. Moreover, in synchronized T98G cells, LD oscillations were altered by glycogen synthase kinase-3 (GSK-3) inhibition that affects the cytosolic activity of the metabolic oscillator or by knocking down LIPIN-1, a key GL synthesizing enzyme. Overall, our findings reveal the existence of metabolic oscillations in terms of LD content highly conserved across evolutionary scales notwithstanding variations in complexity, regulation, and cell organization. [ABSTRACT FROM AUTHOR]

The fundamental question of how forces are generated in a motile cell, a lamellipodium, and a comet tail is the subject of this note. It is now well established that cellular motility results from the polymerization of actin, the most abundant protein in eukaryotic cells, into an interconnected set of filaments. We portray this process in a continuum mechanics framework, claiming that polymerization promotes a mechanical swelling in a narrow zone around the nucleation loci, which ultimately results in cellular or bacterial motility. To this aim, a new paradigm in continuum multi-physics has been designed, departing from the well-known theory of Larché–Cahn chemo-transport-mechanics. In this note, we set up the theory of network growth and compare the outcomes of numerical simulations with experimental evidence. [ABSTRACT FROM AUTHOR]

Poly (ADP-ribose) polymerase 1 (PARP1) is a multifunctional nuclear enzyme that catalyzes poly-ADP ribosylation in eukaryotic cells. In addition to maintaining genomic integrity, this nuclear enzyme is also involved in transcriptional regulation. PARP1 can trigger and maintain changes in the chromatin structure and directly recruit transcription factors. PARP1 also prevents DNA methylation. However, most previous reviews on PARP1 have focused on its involvement in maintaining genome integrity, with less focus on its transcriptional regulatory function. This article comprehensively reviews the transcriptional regulatory function of PARP1 and its application in disease treatment, providing new ideas for targeting PARP1 for the treatment of diseases other than cancer. [ABSTRACT FROM AUTHOR]

Metamonads are a diverse group of heterotrophic microbial eukaryotes adapted to living in hypoxic environments. All metamonads but one harbour metabolically altered 'mitochondrion-related organelles' (MROs) with reduced functions, however the degree of reduction varies. Here, we generate high-quality draft genomes, transcriptomes, and predicted proteomes for five recently discovered free-living metamonads. Phylogenomic analyses placed these organisms in a group we name the 'BaSk' (Barthelonids+Skoliomonads) clade, a deeply branching sister group to the Fornicata, a phylum that includes parasitic and free-living flagellates. Bioinformatic analyses of gene models shows that these organisms are predicted to have extremely reduced MRO proteomes in comparison to other free-living metamonads. Loss of the mitochondrial iron-sulfur cluster assembly system in some organisms in this group appears to be linked to the acquisition in their common ancestral lineage of a SUF-like minimal system Fe/S cluster pathway by lateral gene transfer. One of the isolates, Skoliomonas litria, appears to have lost all other known MRO pathways. No proteins were confidently assigned to the predicted MRO proteome of this organism suggesting that the organelle has been lost. The extreme mitochondrial reduction observed within this free-living anaerobic protistan clade demonstrates that mitochondrial functions may be completely lost even in free-living organisms. Mitochondria are essential cellular components that are found in animals, plants, fungi, and protists. This study reports what is believed to be the first example of complete mitochondrial loss in a free-living organism, providing insights into the evolutionary plasticity of eukaryotic cells. [ABSTRACT FROM AUTHOR]

Hierarchical compartmentalization responding to changes in intracellular and extracellular environments is ubiquitous in living eukaryotic cells but remains a formidable task in synthetic systems. Here we report a two-level compartmentalization approach based on a thermo-responsive aqueous two-phase system (TR-ATPS) comprising poly(N-isopropylacrylamide) (PNIPAM) and dextran (DEX). Liquid membraneless compartments enriched in PNIPAM are phase-separated from the continuous DEX solution via liquid-liquid phase separation at 25 °C and shrink dramatically with small second-level compartments generated at the interface, resembling the structure of colloidosome, by increasing the temperature to 35 °C. The TR-ATPS can store biomolecules, program the spatial distribution of enzymes, and accelerate the overall biochemical reaction efficiency by nearly 7-fold. The TR-ATPS inspires on-demand, stimulus-triggered spatiotemporal enrichment of biomolecules via two-level compartmentalization, creating opportunities in synthetic biology and biochemical engineering. Hierarchical compartmentalization responding to changes in intracellular and extracellular environments is ubiquitous in living eukaryotic cells but remains a formidable task in synthetic systems. Here the Authors report a two-level compartmentalization approach based on a thermo-responsive aqueous two-phase system comprising poly(N-isopropylacrylamide) and dextran. [ABSTRACT FROM AUTHOR]

The plasma membrane (PM) is constantly exposed to various stresses from the extracellular environment, such as heat and oxidative stress. These stresses often cause the denaturation of membrane proteins and destabilize PM integrity, which is essential for normal cell viability and function. For maintenance of PM integrity, most eukaryotic cells have the PM quality control (PMQC) system, which removes damaged membrane proteins by endocytosis. Removal of damaged proteins from the PM by ubiquitin-mediated endocytosis is a key mechanism for the maintenance of PM integrity, but the importance of the early endosome in the PMQC system is still not well understood. Here we show that key proteins in early/sorting endosome function, Vps21p (yeast Rab5), Vps15p (phosphatidylinositol-3 kinase subunit), and Vps3p/8p (CORVET complex subunits), are involved in maintaining PM integrity. We found that Vps21p-enriched endosomes change the localization in the vicinity of the PM in response to heat stress and then rapidly fuse and form the enlarged compartments to efficiently transport Can1p to the vacuole. Additionally, we show that the deubiquitinating enzyme Doa4p is also involved in the PM integrity and its deletion causes the mislocalization of Vps21p to the vacuolar lumen. Interestingly, in cells lacking Doa4p or Vps21p, the amounts of free ubiquitin are decreased, and overexpression of ubiquitin restored defective cargo internalization in vps9D cells, suggesting that defective PM integrity in vps9D cells is caused by lack of free ubiquitin. [ABSTRACT FROM AUTHOR]

Nonsense-mediated mRNA decay (NMD) is a highly conserved post-transcriptional gene expression regulatory mechanism in eukaryotic cells. NMD eliminates aberrant mRNAs with premature termination codons to surveil transcriptome integrity. Furthermore, NMD fine-tunes gene expression by destabilizing RNAs with specific NMD features. Thus, by controlling the quality and quantity of the transcriptome, NMD plays a vital role in mammalian development, stress response, and tumorigenesis. Deficiencies of NMD factors result in early embryonic lethality, while the underlying mechanisms are poorly understood. SMG5 is a key NMD factor. In this study, we generated an Smg5 conditional knockout mouse model and found that Smg5-null results in early embryonic lethality before E13.5. Furthermore, we produced multiple lines of Smg5 knockout mouse embryonic stem cells (mESCs) and found that the deletion of Smg5 in mESCs does not compromise cell viability. Smg5-null delays differentiation of mESCs. Mechanistically, our study reveals that the c-MYC protein, but not c-Myc mRNA, is upregulated in SMG5-deficient mESCs. The overproduction of c-MYC protein could be caused by enhanced protein synthesis upon SMG5 loss. Furthermore, SMG5-null results in dysregulation of alternative splicing on multiple stem cell differentiation regulators. Overall, our findings underscore the importance of SMG5-NMD in regulating mESC cell-state transition. [ABSTRACT FROM AUTHOR]