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Various DNA damage checkpoint control mechanisms in eukaryotic cells help maintain genomic integrity. Among these, NBS1, a key component of the MRE11-RAD50-NBS1 (MRN) complex, is an essential protein involved in the DNA damage response (DDR). In this study, we discovered that DNA damage-binding protein 1 (DDB1) interacts with NBS1. DDB1 is a DDR sensor protein found in UV-induced DNA replication blocks. Through pull-down and immunoprecipitation assays conducted in Xenopus egg extracts and human cell lines, we demonstrated a specific interaction between NBS1 and DDB1. DDB1 was also found to associate with several proteins that interact with NBS1, including DNA topoisomerase 2-binding protein 1 (TopBP1) and Mediator of DNA damage checkpoint protein 1 (MDC1). Notably, the interaction between DDB1 and NBS1 is disrupted in MDC1-depleted egg extracts, indicating that MDC1 is necessary for this interaction. Furthermore, the depletion of DDB1 leads to increased Chk1 activation upon DNA damage. These novel findings regarding the interaction between NBS1 and DDB1 provide new insights into how DDB1 regulates DNA damage pathways. [ABSTRACT FROM AUTHOR]

Cell division cycle 6 (CDC6) is essential for the initiation of DNA replication in eukaryotic cells and contributes to the development of various human tumors. Polycystic ovarian syndrome (PCOS) is a reproductive endocrine disease in women of childbearing age, with a significant risk of endometrial cancer (EC). However, the role of CDC6 in the progression of PCOS to EC is unclear. Therefore, we examined CDC6 expression in patients with PCOS and EC. We evaluated the relationship between CDC6 expression and its prognostic value, potential biological functions, and immune infiltrates in patients with EC. In vitro analyses were performed to investigate the effects of CDC6 knockdown on EC proliferation, migration, invasion, and apoptosis. CDC6 expression was significantly upregulated in patients with PCOS and EC. Moreover, this protein caused EC by promoting the aberrant infiltration of macrophages into the immune microenvironment in patients with PCOS. A functional enrichment analysis revealed that CDC6 exerted its pro-cancer and pro-immune cell infiltration functions via the PI3K-AKT pathway. Moreover, it promoted EC proliferation, migration, and invasion but inhibited apoptosis. This protein significantly reduced EC survival when mutated. These findings demonstrate that CDC6 regulates the progression of PCOS to EC and promotes immune infiltration. [ABSTRACT FROM AUTHOR]

We have previously shown that 2-thiouridine (S2U), either as a single nucleoside or as an element of RNA chain, is effectively desulfurized under applied in vitro oxidative conditions. The chemically induced desulfuration of S2U resulted in two products: 4-pyrimidinone nucleoside (H2U) and uridine (U). Recently, we investigated whether the desulfuration of S2U is a natural process that also occurs in the cells exposed to oxidative stress or whether it only occurs in the test tube during chemical reactions with oxidants at high concentrations. Using different types of eukaryotic cells, such as baker's yeast, human cancer cells, or modified HEK293 cells with an impaired antioxidant system, we confirmed that 5-substituted 2-thiouridines are oxidatively desulfurized in the wobble position of the anticodon of some tRNAs. The quantitative LC-MS/MS-MRMhr analysis of the nucleoside mixtures obtained from the hydrolyzed tRNA revealed the presence of the desulfuration products of mcm5S2U: mcm5H2U and mcm5U modifications. We also observed some amounts of immature cm5S2U, cm5H2U and cm5U products, which may have indicated a disruption of the enzymatic modification pathway at the C5 position of 2-thiouridine. The observed process, which was triggered by oxidative stress in the living cells, could impair the function of 2-thiouridine-containing tRNAs and alter the translation of genetic information. [ABSTRACT FROM AUTHOR]

Autophagy maintains the stability of eukaryotic cells by degrading unwanted components and recycling nutrients and plays a pivotal role in muscle regeneration by regulating the quiescence, activation, and differentiation of satellite cells. Effective muscle regeneration is vital for maintaining muscle health and homeostasis. However, under certain disease conditions, such as aging, muscle regeneration can fail due to dysfunctional satellite cells. Dysregulated autophagy may limit satellite cell self-renewal, hinder differentiation, and increase susceptibility to apoptosis, thereby impeding muscle regeneration. This review explores the critical role of autophagy in muscle regeneration, emphasizing its interplay with apoptosis and recent advances in autophagy research related to diseases characterized by impaired muscle regeneration. Additionally, we discuss new approaches involving autophagy regulation to promote macrophage polarization, enhancing muscle regeneration. We suggest that utilizing cell therapy and biomaterials to modulate autophagy could be a promising strategy for supporting muscle regeneration. We hope that this review will provide new insights into the treatment of muscle diseases and promote muscle regeneration. [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]

Actin filaments, as key components of the cytoskeleton, have aroused great interest due to their numerous functional roles in eukaryotic cells, including intracellular electrical signaling. The aim of this research is to characterize the alternating current (AC) conduction characteristics of both globular and polymerized actin and quantitatively compare their values to those theoretically predicted earlier. Actin filaments have been demonstrated to act as conducting bionanowires, forming a signaling network capable of transmitting ionic waves in cells. We performed conductivity measurements for different concentrations of actin, considering both unpolymerized and polymerized actin to identify potential differences in their electrical properties. These measurements revealed two relevant characteristics: first, the polymerized actin, arranged in filaments, has a lower impedance than its globular counterpart; second, an increase in the actin concentration leads to higher conductivities. Furthermore, from the data collected, we developed a quantitative model to represent the electrical properties of actin in a buffer solution. We hypothesize that actin filaments can be modeled as electrical resistor–inductor–capacitor (RLC) circuits, where the resistive contribution is due to the viscous ion flows along the filaments; the inductive contribution is due to the solenoidal flows along and around the helix-shaped filament and the capacitive contribution is due to the counterion layer formed around each negatively charged filament. [ABSTRACT FROM AUTHOR]

The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a "homologous peptide". Three β-barrel PFTs were used for a detailed comparative analysis. Two of them—HlyII and cytotoxin K2 (CytK2)—are synthesized in Bacillus cereus sensu lato; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these β-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs. [ABSTRACT FROM AUTHOR]

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2′-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity. [ABSTRACT FROM AUTHOR]

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein–protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania. [ABSTRACT FROM AUTHOR]

14-3-3 proteins are widely distributed in eukaryotic cells and play an important role in plant growth, development, and stress tolerance. This study revealed nine 14-3-3 genes from the genome of Nitraria sibirica Pall., a halophyte with strong salt tolerance. The physicochemical properties, multiple sequence alignment, gene structure and motif analysis, and chromosomal distributions were analyzed, and phylogenetic analysis, cis-regulatory elements analysis, and gene transcription and expression analysis of Ns14-3-3s were conducted. The results revealed that the Ns14-3-3 gene family consists of nine members, which are divided into two groups: ε (four members) and non-ε (five members). These members are acidic hydrophilic proteins. The genes are distributed randomly on chromosomes, and the number of introns varies widely among the two groups. However, all genes have similar conserved domains and three-dimensional protein structures. The main differences are found at the N-terminus and C-terminus. The promoter region of Ns14-3-3s contains multiple cis-acting elements related to light, plant hormones, and abiotic stress responses. Transcriptional profiling and gene expression pattern analysis revealed that Ns14-3-3s were expressed in all tissues, although with varying patterns. Under salt stress conditions, Ns14-3-3 1a, Ns14-3-3 1b, Ns14-3-3 5a, and Ns14-3-3 7a showed significant changes in gene expression. Ns14-3-3 1a expression decreased in all tissues, Ns14-3-3 7a expression decreased by 60% to 71% in roots, and Ns14-3-3 1b expression increased by 209% to 251% in stems. The most significant change was observed in Ns14-3-3 5a, with its expression in stems increasing by 213% to 681%. The yeast two-hybrid experiments demonstrated that Ns14-3-3 5a interacts with NsVP1 (vacuolar H+-pyrophosphatase). This result indicates that Ns14-3-3 5a may respond to salt stress by promoting ionic vacuole compartmentalization in stems and leaves through interactions with NsVP1. In addition, N. sibirica has a high number of stems, allowing it to compartmentalize more ions through its stem and leaf. This may be a contributing factor to its superior salt tolerance compared to other plants. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are lipid bilayers derived from cell membranes, released by both eukaryotic cells and bacteria into the extracellular environment. During production, EVs carry proteins, nucleic acids, and various compounds, which are then released. While Gram-positive bacteria were traditionally thought incapable of producing EVs due to their thick peptidoglycan cell walls, recent studies on membrane vesicles (MVs) in Gram-positive bacteria have revealed their significant role in bacterial physiology and disease progression. This review explores the current understanding of MVs in Gram-positive bacteria, including the characterization of their content and functions, as well as their interactions with host and bacterial cells. It offers a fresh perspective to enhance our comprehension of Gram-positive bacterial EVs. [ABSTRACT FROM AUTHOR]

Transglutaminase type 2 (TG2) is the most ubiquitously expressed and well characterized member of the transglutaminase family. It is a ubiquitous multifunctional enzyme implicated in the regulation of several cellular pathways that support the survival, death, and general homeostasis of eukaryotic cells. Due to its multiple localizations both inside and outside the cell, TG2 participates in the regulation of many crucial intracellular signaling cascades in a tissue- and cell-specific manner, making this enzyme an important player in disease development and progression. Moreover, TG2 is capable of modulating the tumor microenvironment, a process of dynamic tissue remodeling and biomechanical events, resulting in changes which influence tumor initiation, growth, and metastasis. Even if generally related to the Ca2+-dependent post-translational modification of proteins, a number of different biological functions have been ascribed to TG2, like those of a peptide isomerase, protein kinase, guanine nucleotide binder, and cytosolic–nuclear translocator. With respect to cancer, TG2′s role is controversial and highly debated; it has been described both as an anti- and pro-apoptotic factor and is linked to all the processes of tumorigenesis. However, numerous pieces of evidence support a tissue-specific role of TG2 so that it can assume both oncogenic and tumor-suppressive roles. [ABSTRACT FROM AUTHOR]

The steadily increasing number of drug-resistant bacterial species has prompted the search for alternative treatments, resulting in a growing interest in bacteriophages. Although they are viruses infecting bacterial cells, bacteriophages are an extremely important part of the human microbiota. By interacting with eukaryotic cells, they are able to modulate the functioning of many systems, including the immune and nervous systems, affecting not only the homeostasis of the organism, but potentially also the regulation of pathological processes. Therefore, the aim of this review is to answer the questions of (i) how animal/human immune systems respond to bacteriophages under physiological conditions and under conditions of reduced immunity, especially during bacterial infection; (ii) whether bacteriophages can induce negative changes in brain functioning after crossing the blood–brain barrier, which could result in various disorders or in an increase in the risk of neurodegenerative diseases; and (iii) how bacteriophages can modify gut microbiota. The crucial dilemma is whether administration of bacteriophages is always beneficial or rather if it may involve any risks. [ABSTRACT FROM AUTHOR]

Differently from higher eukaryotic cells, in the yeast Saccharomyces cerevisiae there are two mitochondrial carrier proteins involved in the transport of citrate: Ctp1 and Yhm2. Very little is known about the physiological role of these proteins. Wild-type and mutant yeast strains deleted in CTP1 and YHM2 were grown in media supplemented with a fermentable (glucose) or a nonfermentable (ethanol) carbon source. To assess changes in Ctp1 and Yhm2 mRNA expression levels, real-time PCR was performed after total RNA extraction. In the wild-type strain, the metabolic switch from the exponential to the stationary phase is associated with an increase in the expression level of the two citrate transporters. In addition, the results obtained in the mutant strains suggest that the presence of a single citrate transporter can partially compensate for the absence of the other. Ctp1 and Yhm2 differently contribute to fermentative and respiratory metabolism. Moreover, the two mitochondrial carriers represent a link between the Krebs cycle and the glyoxylate cycle, which play a key role in the metabolic adaptation strategies of S. cerevisiae. [ABSTRACT FROM AUTHOR]

Biofilm-related ocular infections can lead to vision loss and are difficult to treat with antibiotics due to challenges with application and increasing microbial resistance. In turn, the design and testing of new synthetic drugs is a time- and cost-consuming process. Therefore, in this work, for the first time, we assessed the in vitro efficacy of the plant-based abietic acid molecule, both alone and when introduced to a polymeric cellulose carrier, against biofilms formed by Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in standard laboratory settings as well as in a self-designed setting using the topologically challenging surface of the artificial eye. These analyses were performed using the standard microdilution method, the biofilm-oriented antiseptic test (BOAT), a modified disk-diffusion method, and eyeball models. Additionally, we assessed the cytotoxicity of abietic acid against eukaryotic cell lines and its anti-staphylococcal efficacy in an in vivo model using Galleria mellonella larvae. We found that abietic acid was more effective against Staphylococcus than Pseudomonas (from two to four times, depending on the test applied) and that it was generally more effective against the tested bacteria (up to four times) than against the fungus C. albicans at concentrations non-cytotoxic to the eukaryotic cell lines and to G. mellonella (256 and 512 µg/mL, respectively). In the in vivo infection model, abietic acid effectively prevented the spread of staphylococcus throughout the larvae organisms, decreasing their lethality by up to 50%. These initial results obtained indicate promising features of abietic acid, which may potentially be applied to treat ocular infections caused by pathogenic biofilms, with higher efficiency manifested against bacterial than fungal biofilms. [ABSTRACT FROM AUTHOR]

Numerous studies have shown that oxidative modifications of guanine (7,8-dihydro-8-oxoguanine, 8-oxoG) can affect cellular functions. 7,8-Dihydro-8-oxoadenine (8-oxoA) is another abundant paradigmatic ambiguous nucleobase but findings reported on the mutagenicity of 8-oxoA in bacterial and eukaryotic cells are incomplete and contradictory. Although several genotoxic studies have demonstrated the mutagenic potential of 8-oxoA in eukaryotic cells, very little biochemical and bioinformatics data about the mechanism of 8-oxoA-induced mutagenesis are available. In this review, we discuss dual coding properties of 8-oxoA, summarize historical and recent genotoxicity and biochemical studies, and address the main protective cellular mechanisms of response to 8-oxoA. We also discuss the available structural data for 8-oxoA bypass by different DNA polymerases as well as the mechanisms of 8-oxoA recognition by DNA repair enzymes. [ABSTRACT FROM AUTHOR]

Mitochondria are double-membrane organelles that are involved in energy production, apoptosis, and signaling in eukaryotic cells. Several studies conducted over the past decades have correlated mitochondrial dysfunction with various diseases, including cerebral ischemia, myocardial ischemia-reperfusion, and cancer. Mitochondrial transplantation entails importing intact mitochondria from healthy tissues into diseased tissues with damaged mitochondria to rescue the injured cells. In this review, the different mitochondrial transplantation techniques and their clinical applications have been discussed. In addition, the challenges and future directions pertaining to mitochondrial transplantation and its potential in the treatment of diseases with defective mitochondria have been summarized. [ABSTRACT FROM AUTHOR]

Mitochondria are double-membrane organelles within eukaryotic cells that act as cellular power houses owing to their ability to efficiently generate the ATP required to sustain normal cell function. Also, they represent a "hub" for the regulation of a plethora of processes, including cellular homeostasis, metabolism, the defense against oxidative stress, and cell death. Mitochondrial dysfunctions are associated with a wide range of human diseases with complex pathologies, including metabolic diseases, neurodegenerative disorders, and cancer. Therefore, regulating dysfunctional mitochondria represents a pivotal therapeutic opportunity in biomedicine. Marine ecosystems are biologically very diversified and harbor a broad range of organisms, providing both novel bioactive substances and molecules with meaningful biomedical and pharmacological applications. Recently, many mitochondria-targeting marine-derived molecules have been described to regulate mitochondrial biology, thus exerting therapeutic effects by inhibiting mitochondrial abnormalities, both in vitro and in vivo, through different mechanisms of action. Here, we review different strategies that are derived from marine organisms which modulate specific mitochondrial processes or mitochondrial molecular pathways and ultimately aim to find key molecules to treat a wide range of human diseases characterized by impaired mitochondrial function. [ABSTRACT FROM AUTHOR]

Mitochondria carry out various vital roles in eukaryotic cells, including ATP energy synthesis, the regulation of apoptosis, Fe-S cluster formation, and the metabolism of fatty acids, amino acids, and nucleotides. Throughout evolution, mitochondria lost most of their ancestor's genome but kept the replication, transcription, and translation machinery. Protein biosynthesis in mitochondria is specialized in the production of highly hydrophobic proteins encoded by mitochondria. These proteins are components of oxidative phosphorylation chain complexes. The coordination of protein synthesis must be precise to ensure the correct assembly of nuclear-encoded subunits for these complexes. However, the regulatory mechanisms of mitochondrial translation in human cells are not yet fully understood. In this study, we examined the contribution of the SLIRP protein in regulating protein biosynthesis in mitochondria. Using a click-chemistry approach, we discovered that deletion of the SLIRP gene disturbs mitochondrial translation, leading to the dysfunction of complexes I and IV, but it has no significant effect on complexes III and V. We have shown that this protein interacts only with the small subunit of the mitochondrial ribosome, which may indicate its involvement in the regulation of the mitochondrial translation initiation stage. [ABSTRACT FROM AUTHOR]

In a eukaryotic cell, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is usually maintained within a specific range. This suggests the presence of a negative feedback loop mechanism preventing extensive mtDNA replication and depletion. However, the experimental data on this hypothetical mechanism are limited. In this study, we suggested that deletions in mtDNA, known to increase mtDNA abundance, can disrupt this mechanism, and thus, increase cell-to-cell variance in the mtDNA copy numbers. To test this, we generated Saccharomyces cerevisiae rho− strains with large deletions in the mtDNA and rho0 strains depleted of mtDNA. Given that mtDNA contributes to the total DNA content of exponentially growing yeast cells, we showed that it can be quantified in individual cells by flow cytometry using the DNA-intercalating fluorescent dye SYTOX green. We found that the rho− mutations increased both the levels and cell-to-cell heterogeneity in the total DNA content of G1 and G2/M yeast cells, with no association with the cell size. Furthermore, the depletion of mtDNA in both the rho+ and rho− strains significantly decreased the SYTOX green signal variance. The high cell-to-cell heterogeneity of the mtDNA amount in the rho− strains suggests that mtDNA copy number regulation relies on full-length mtDNA, whereas the rho− mtDNAs partially escape this regulation. [ABSTRACT FROM AUTHOR]

Bacteria use cell surface proteins to mediate host–pathogen interactions. Proteins responsible for cell adhesion, including E-cadherin, serve as receptors for entry into the host cell. We have previously shown that an increase in eukaryotic cell sensitivity to Serratia grimesii correlates with an increase in E-cadherin expression. On the other hand, Serratia proteamaculans invasion involves the EGFR, which can interact with E-cadherin on the surface of host cells. Therefore, we investigated the role of E-cadherin in Serratia invasion into M-HeLa and Caco-2 cells. Bacterial infection increased E-cadherin expression in both cell lines. Moreover, E-cadherin was detected in the Caco-2 cells in a full-length form and in the M-HeLa cells in only a truncated form in response to incubation with bacteria. Transfection with siRNA targeting E-cadherin inhibited S. proteamaculans invasion only into the Caco-2 cells. Thus, only full-length E-cadherin is involved in S. proteamaculans invasion. On the other hand, transfection with siRNA targeting E-cadherin inhibited S. grimesii invasion into both cell lines. Thus, not only may full-length E-cadherin but also truncated E-cadherin be involved in S. grimesii invasion. Truncated E-cadherin can be formed as a result of cleavage by bacterial proteases or the Ca2+-activated cellular protease ADAM10. The rate of Ca2+ accumulation in the host cells depends on the number of bacteria per cell upon infection. During incubation, Ca2+ accumulates only when more than 500 S. grimesii bacteria are infected per eukaryotic cell, and only under these conditions does the ADAM10 inhibitor reduce the sensitivity of the cells to bacteria. An EGFR inhibitor has the same quantitative effect on S. grimesii invasion. Apparently, as a result of infection with S. grimesii, Ca2+ accumulates in the host cells and may activate the ADAM10 sheddase, which can promote invasion by cleaving E-cadherin and, as a result, triggering EGFR signaling. Thus, the invasion of S. proteamaculans can only be promoted by full-length E-cadherin, and S. grimesii invasion can be promoted by both full-length and truncated E-cadherin. [ABSTRACT FROM AUTHOR]

There is a great interest in describing new molecules to be used as therapeutic targets in various diseases, particularly those that play a role in inflammatory responses and infection control. Mygalin is a synthetic analogue of spermidine, and previous studies have demonstrated its bactericidal effect against Escherichia coli, as well as its ability to modulate the inflammatory response of macrophages against lipopolysaccharide (LPS). However, the mechanisms through which mygalin regulates this inflammatory response remain poorly characterized. A set of platforms using molecular docking analysis was employed to analyze various properties of mygalin, including toxicity, biodistribution, absorption, and the prediction of its anti-inflammatory properties. In in vitro assays, we evaluated the potential of mygalin to interact with products of the inflammatory response, such as reactive oxygen species (ROS) and antioxidant activity, using the BMDM cell. The in silico analyses indicated that mygalin is not toxic, and can interact with proteins from the kinase group, and enzymes and receptors in eukaryotic cells. Molecular docking analysis showed interactions with key amino acid residues of COX-2, iNOS and 5-LOX enzymes. In vitro, assays demonstrated a significant reduction in the expression of iNOS and COX-2 induced by LPS, along with a decrease in the oxidative stress caused by the treatment with PMA, all without altering cell viability. Mygalin exhibited robust antioxidant activity in DPPH assays, regardless of the dose used, and inhibited heat-induced hemolysis. These studies suggest that mygalin holds promise for further investigation as a new molecule with anti-inflammatory and antioxidant properties. [ABSTRACT FROM AUTHOR]

Na+/H+ exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility. [ABSTRACT FROM AUTHOR]

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection. [ABSTRACT FROM AUTHOR]

Small nucleolar RNAs (snoRNAs) constitute a prevalent class of noncoding RNAs localized within the nucleoli of eukaryotic cells. Their involvement in diverse diseases underscores the significance of forecasting associations between snoRNAs and diseases. However, conventional experimental techniques for such predictions suffer limitations in scalability, protracted timelines, and suboptimal success rates. Consequently, efficient computational methodologies are imperative to realize the accurate predictions of snoRNA–disease associations. Herein, we introduce GCLSDA—graph Convolutional Network and contrastive learning predict snoRNA disease associations. GCLSDA is an innovative framework that combines graph convolution networks and self-supervised learning for snoRNA–disease association prediction. Leveraging the repository of MNDR v4.0 and ncRPheno databases, we construct a robust snoRNA–disease association dataset, which serves as the foundation to create bipartite graphs. The computational prowess of the light graph convolutional network (LightGCN) is harnessed to acquire nuanced embedded representations of both snoRNAs and diseases. With careful consideration, GCLSDA intelligently incorporates contrast learning to address the challenging issues of sparsity and over-smoothing inside correlation matrices. This combination not only ensures the precision of predictions but also amplifies the model's robustness. Moreover, we introduce the augmentation technique of random noise to refine the embedded snoRNA representations, consequently enhancing the precision of predictions. Within the domain of contrast learning, we unite the tasks of contrast and recommendation. This harmonization streamlines the cross-layer contrast process, simplifying the information propagation and concurrently curtailing computational complexity. In the area of snoRNA–disease associations, GCLSDA constantly shows its promising capacity for prediction through extensive research. This success not only contributes valuable insights into the functional roles of snoRNAs in disease etiology, but also plays an instrumental role in identifying potential drug targets and catalyzing innovative treatment modalities. [ABSTRACT FROM AUTHOR]

All eukaryotic cells, including oocytes, utilize an engine called cyclin-dependent kinase (Cdk) to drive the cell cycle. Cdks are activated by a co-factor called cyclin, which regulates their activity. The key Cdk–cyclin complex that regulates the oocyte cell cycle is known as Cdk1–cyclin B1. Recent studies have elucidated the roles of other cyclins, such as B2, B3, A2, and O, in oocyte cell cycle regulation. This review aims to discuss the recently discovered roles of various cyclins in mouse oocyte cell cycle regulation in accordance with the sequential progression of the cell cycle. In addition, this review addresses the translation and degradation of cyclins to modulate the activity of Cdks. Overall, the literature indicates that each cyclin performs unique and redundant functions at various stages of the cell cycle, while their expression and degradation are tightly regulated. Taken together, this review provides new insights into the regulatory role and function of cyclins in oocyte cell cycle progression. [ABSTRACT FROM AUTHOR]

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

Surfaces in highly anthropized environments are frequently contaminated by both harmless and pathogenic bacteria. Accidental contact between these contaminated surfaces and people could contribute to uncontrolled or even dangerous microbial diffusion. Among all possible solutions useful to achieve effective disinfection, ultraviolet irradiations (UV) emerge as one of the most "Green" technologies since they can inactivate microorganisms via the formation of DNA/RNA dimers, avoiding the environmental pollution associated with the use of chemical sanitizers. To date, mainly UV-C irradiation has been used for decontamination purposes, but in this study, we investigated the cytotoxic potential on contaminated surfaces of combined UV radiations spanning the UV-A, UV-B, and UV-C spectrums, obtained with an innovative UV lamp never conceived so far by analyzing its effect on a large panel of collection and environmental strains, further examining any possible adverse effects on eukaryotic cells. We found that this novel device shows a significant efficacy on different planktonic and sessile bacteria, and, in addition, it is compatible with eukaryotic skin cells for short exposure times. The collected data strongly suggest this new lamp as a useful device for fast and routine decontamination of different environments to ensure appropriate sterilization procedures. [ABSTRACT FROM AUTHOR]

CD8+ T cells and Natural Killer (NK) cells are cytotoxic lymphocytes important in the response to intracellular pathogens and cancer. Their activity depends on the integration of a large set of intracellular and environmental cues, including antigenic signals, cytokine stimulation and nutrient availability. This integration is achieved by signaling hubs, such as the mechanistic target of rapamycin (mTOR). mTOR is a conserved protein kinase that controls cellular growth and metabolism in eukaryotic cells and, therefore, is essential for lymphocyte development and maturation. However, our current understanding of mTOR signaling comes mostly from studies performed in transformed cell lines, which constitute a poor model for comprehending metabolic pathway regulation. Therefore, it is only quite recently that the regulation of mTOR in primary cells has been assessed. Here, we review the signaling pathways leading to mTOR activation in CD8+ T and NK cells, focusing on activation by cytokines. We also discuss how this knowledge can contribute to immunotherapy development, particularly for cancer treatment. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS. [ABSTRACT FROM AUTHOR]

The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea. This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa. The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein–protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1PAS∆). Here, we demonstrated that BcWCL1PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1PAS∆, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics. [ABSTRACT FROM AUTHOR]

The recognized antimicrobial activity of silver nanoparticles is a well-studied property, especially when designing and developing biomaterials with medical applications. As biological activity is closely related to the physicochemical characteristics of a material, aspects such as particle morphology and dimension should be considered. Microfluidic systems in continuous flow represent a promising method to control the size, shape, and size distribution of synthesized nanoparticles. Moreover, using microfluidics widens the synthesis options by creating and controlling parameters that are otherwise difficult to maintain in conventional batch procedures. This study used a microfluidic platform with a cross-shape design as an innovative method for synthesizing silver nanoparticles and varied the precursor concentration and the purging speed as experimental parameters. The compositional and microstructural characterization of the obtained samples was carried out by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Four formulations of alginate-based hydrogels with the addition of hyaluronic acid and silver nanoparticles were obtained to highlight the antimicrobial activity of silver nanoparticles and the efficiency of such a composite in wound treatment. The porous structure, swelling capacity, and biological properties were evaluated through physicochemical analysis (FT-IR and SEM) and through contact with prokaryotic and eukaryotic cells. The results of the physicochemical and biological investigations revealed desirable characteristics for performant wound dressings (i.e., biocompatibility, appropriate porous structure, swelling rate, and degradation rate, ability to inhibit biofilm formation, and cell growth stimulation capacity), and the obtained materials are thus recommended for treating chronic and infected wounds. [ABSTRACT FROM AUTHOR]

This paper deals with the problems encountered in the study of eukaryotic cell membranes. A discussion on the structure and composition of membranes, lateral heterogeneity of membranes, lipid raft formation, and involvement of actin and cytoskeleton networks in the maintenance of membrane structure is included. Modern methods for the study of membranes and their constituent domains are discussed. Various simplified models of biomembranes and lipid rafts are presented. Computer modelling is considered as one of the most important methods. This is stated that from the study of the plasma membrane structure, it is desirable to proceed to the diverse membranes of all organelles of the cell. The qualitative composition and molar content of individual classes of polar lipids, free sterols and proteins in each of these membranes must be considered. A program to create an open access electronic database including results obtained from the membrane modelling of individual cell organelles and the key sites of the membranes, as well as models of individual molecules composing the membranes, has been proposed. [ABSTRACT FROM AUTHOR]

The existence of mitochondria in eukaryotic host cells as a remnant of former microbial organisms has been widely accepted, as has their fundamental role in several diseases and physiological aging. In recent years, it has become clear that the health, aging, and life span of multicellular hosts are also highly dependent on the still-residing microbiota, e.g., those within the intestinal system. Due to the common evolutionary origin of mitochondria and these microbial commensals, it is intriguing to investigate if there might be a crosstalk based on preserved common properties. In the light of rising knowledge on the gut–brain axis, such crosstalk might severely affect brain homeostasis in aging, as neuronal tissue has a high energy demand and low tolerance for according functional decline. In this review, we summarize what is known about the impact of both mitochondria and the microbiome on the host's aging process and what is known about the aging of both entities. For a long time, bacteria were assumed to be immortal; however, recent evidence indicates their aging and similar observations have been made for mitochondria. Finally, we present pathways by which mitochondria are affected by microbiota and give information about therapeutic anti-aging approaches that are based on current knowledge. [ABSTRACT FROM AUTHOR]

The morphology of the nucleus is roughly spherical in most eukaryotic cells. However, this organelle shape needs to change as the cell travels through narrow intercellular spaces during cell migration and during cell division in organisms that undergo closed mitosis, i.e., without dismantling the nuclear envelope, such as yeast. In addition, the nuclear morphology is often modified under stress and in pathological conditions, being a hallmark of cancer and senescent cells. Thus, understanding nuclear morphological dynamics is of uttermost importance, as pathways and proteins involved in nuclear shaping can be targeted in anticancer, antiaging, and antifungal therapies. Here, we review how and why the nuclear shape changes during mitotic blocks in yeast, introducing novel data that associate these changes with both the nucleolus and the vacuole. Altogether, these findings suggest a close relationship between the nucleolar domain of the nucleus and the autophagic organelle, which we also discuss here. Encouragingly, recent evidence in tumor cell lines has linked aberrant nuclear morphology to defects in lysosomal function. [ABSTRACT FROM AUTHOR]

Titanium implantation success may be compromised by Staphylococcus aureus surface colonization and posterior infection. To avoid this issue, different strategies have been investigated to promote an antibacterial character to titanium. In this work, two antibacterial agents (silver nanoparticles and a multifunctional antimicrobial peptide) were used to coat titanium surfaces. The modulation of the nanoparticle (≈32.1 ± 9.4 nm) density on titanium could be optimized, and a sequential functionalization with both agents was achieved through a two-step functionalization method by means of surface silanization. The antibacterial character of the coating agents was assessed individually as well as combined. The results have shown that a reduction in bacteria after 4 h of incubation can be achieved on all the coated surfaces. After 24 h of incubation, however, the individual antimicrobial peptide coating was more effective than the silver nanoparticles or their combination against Staphylococcus aureus. All tested coatings were non-cytotoxic for eukaryotic cells. [ABSTRACT FROM AUTHOR]

Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein–protein and lipid–protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces Serratia invasion. [ABSTRACT FROM AUTHOR]

The accumulation of protein aggregates is the hallmark of many neurodegenerative diseases. The dysregulation of protein homeostasis (or proteostasis) caused by acute proteotoxic stresses or chronic expression of mutant proteins can lead to protein aggregation. Protein aggregates can interfere with a variety of cellular biological processes and consume factors essential for maintaining proteostasis, leading to a further imbalance of proteostasis and further accumulation of protein aggregates, creating a vicious cycle that ultimately leads to aging and the progression of age-related neurodegenerative diseases. Over the long course of evolution, eukaryotic cells have evolved a variety of mechanisms to rescue or eliminate aggregated proteins. Here, we will briefly review the composition and causes of protein aggregation in mammalian cells, systematically summarize the role of protein aggregates in the organisms, and further highlight some of the clearance mechanisms of protein aggregates. Finally, we will discuss potential therapeutic strategies that target protein aggregates in the treatment of aging and age-related neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Mitochondria are organelles present in almost all eukaryotic cells, where they represent the main site of energy production. Mitochondria are involved in several important cell processes, such as calcium homeostasis, OXPHOS, autophagy, and apoptosis. Moreover, they play a pivotal role also in inflammation through the inter-organelle and inter-cellular communications, mediated by the release of mitochondrial damage-associated molecular patterns (mtDAMPs). It is currently well-documented that in addition to traditional endocrine and paracrine communication, the cells converse via extracellular vesicles (EVs). These small membrane-bound particles are released from cells in the extracellular milieu under physio-pathological conditions. Importantly, EVs have gained much attention for their crucial role in inter-cellular communication, translating inflammatory signals into recipient cells. EVs cargo includes plasma membrane and endosomal proteins, but EVs also contain material from other cellular compartments, including mitochondria. Studies have shown that EVs may transport mitochondrial portions, proteins, and/or mtDAMPs to modulate the metabolic and inflammatory responses of recipient cells. Overall, the relationship between EVs and mitochondria in inflammation is an active area of research, although further studies are needed to fully understand the mechanisms involved and how they may be targeted for therapeutic purposes. Here, we have reported and discussed the latest studies focused on this fascinating and recent area of research, discussing of tricky connection between mitochondria and EVs in inflammatory-related diseases. [ABSTRACT FROM AUTHOR]

This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at pH 4.0, 5.5, and 9.0, purified by 48-, 46-, and 51-fold and having a specific activity of 0.43, 0.55 and 0.36 E/mg protein, respectively. The kinetic parameters of preparations from cells cultured at extreme pH demonstrated: (1) an increase in the affinity for citrate and isocitrate; and (2) a shift in the pH optima to the acidic and alkaline side in accordance with the modulation of the medium pH. The regulatory properties of the enzyme from cells subjected to alkaline stress showed increased sensitivity to Fe2+ ions and high peroxide resistance. Reduced glutathione (GSH) stimulated AH, while oxidized glutathione (GSSG) inhibited AH. A more pronounced effect of both GSH and GSSG was noted for the enzyme obtained from cells grown at pH 5.5. The data obtained provide new approaches to the use of Y. lipolytica as a model of eukaryotic cells demonstrating the development of a stress-induced pathology and to conducting a detailed analysis of enzymatic activity for its correction. [ABSTRACT FROM AUTHOR]

A comet assay is a trusted and widely used method for assessing DNA damage in individual eukaryotic cells. However, it is time-consuming and requires extensive monitoring and sample manipulation by the user. This limits the throughput of the assay, increases the risk of errors, and contributes to intra- and inter-laboratory variability. Here, we describe the development of a device which automates high throughput sample processing for a comet assay. This device is based upon our patented, high throughput, vertical comet assay electrophoresis tank, and incorporates our novel, patented combination of assay fluidics, temperature control, and a sliding electrophoresis tank to facilitate sample loading and removal. Additionally, we demonstrated that the automated device performs at least as well as our "manual" high throughput system, but with all the advantages of a fully "walkaway" device, such as a decreased need for human involvement and a decreased assay run time. Our automated device represents a valuable, high throughput approach for reliably assessing DNA damage with the minimal operator involvement, particularly if combined with the automated analysis of comets. [ABSTRACT FROM AUTHOR]

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites. [ABSTRACT FROM AUTHOR]

In vertebrates, two homologous heterotetrameric AP1 complexes regulate the intracellular protein sorting via vesicles. AP-1 complexes are ubiquitously expressed and are composed of four different subunits: γ, β1, μ1 and σ1. Two different complexes are present in eukaryotic cells, AP1G1 (contains γ1 subunit) and AP1G2 (contains γ2 subunit); both are indispensable for development. One additional tissue-specific isoform exists for μ1A, the polarized epithelial cells specific to μ1B; two additional tissue-specific isoforms exist for σ1A: σ1B and σ1C. Both AP1 complexes fulfil specific functions at the trans-Golgi network and endosomes. The use of different animal models demonstrated their crucial role in the development of multicellular organisms and the specification of neuronal and epithelial cells. Ap1g1 (γ1) knockout mice cease development at the blastocyst stage, while Ap1m1 (μ1A) knockouts cease during mid-organogenesis. A growing number of human diseases have been associated with mutations in genes encoding for the subunits of adaptor protein complexes. Recently, a new class of neurocutaneous and neurometabolic disorders affecting intracellular vesicular traffic have been referred to as adaptinopathies. To better understand the functional role of AP1G1 in adaptinopathies, we generated a zebrafish ap1g1 knockout using CRISPR/Cas9 genome editing. Zebrafish ap1g1 knockout embryos cease their development at the blastula stage. Interestingly, heterozygous females and males have reduced fertility and showed morphological alterations in the brain, gonads and intestinal epithelium. An analysis of mRNA profiles of different marker proteins and altered tissue morphologies revealed dysregulated cadherin-mediated cell adhesion. These data demonstrate that the zebrafish model organism enables us to study the molecular details of adaptinopathies and thus also develop treatment strategies. [ABSTRACT FROM AUTHOR]

Among multiple cytoprotective mechanisms, eukaryotic cells exhibit a complex transcriptional program relying on the Nrf2 transcription factor, which is generally recruited upon biological stressors including oxidative-stress-based cellular insults. The relevance of this master regulator has remarkably emerged in recent years in several research fields such as cancer, inflammatory disorders and age-related neurological diseases. Here, we document the generation and characterization of a novel Nrf2/ARE pathway biosensor fish which exhibits a dynamic spatiotemporal expression profile during the early developmental stages. The transgenic line is responsive to known Nrf2 pathway modulators but also to Edaravone, which direct activity on the Nrf2 pathway has never been documented in a live transgenic fish model. We also show that the reporter is faithfully activated during fin regeneration, and its degree of expression is slightly affected in a glucocerebrosidase (Gba1) morphant zebrafish model. Therefore, this novel transgenic fish may represent a valuable tool to be exploited for the characterization of zebrafish models of human diseases, as well as for primary high-throughput drug screening. [ABSTRACT FROM AUTHOR]

Phage therapy has been successfully used as an experimental therapy in the treatment of multidrug-resistant strains of Staphylococcus aureus (MDRSA)-caused skin infections and is seen as the most promising alternative to antibiotics. However, in recent years a number of reports indicating that phages can interact with eukaryotic cells emerged. Therefore, there is a need to re-evaluate phage therapy in light of safety. It is important to analyze not only the cytotoxicity of phages alone but also the impact their lytic activity against bacteria may have on human cells. As progeny virions rupture the cell wall, lipoteichoic acids are released in high quantities. It has been shown that they act as inflammatory agents and their presence could lead to the worsening of the patient's condition and influence their recovery. In our work, we have tested if the treatment of normal human fibroblasts with staphylococcal phages will influence the metabolic state of the cell and the integrity of cell membranes. We have also analyzed the effectiveness of bacteriophages in reducing the number of MDRSA attached to human fibroblasts and the influence of the lytic activity of phages on cell viability. We observed that, out of three tested anti-Staphylococcal phages—vB_SauM-A, vB_SauM-C and vB_SauM-D—high concentrations (109 PFU/mL) of two, vB_SauM-A and vB_SauM-D, showed a negative impact on the viability of human fibroblasts. However, a dose of 107 PFU/mL had no effect on the metabolic activity or membrane integrity of the cells. We also observed that the addition of phages alleviated the negative effect of the MDRSA infection on fibroblasts' viability, as phages were able to effectively reduce the number of bacteria in the co-culture. We believe that these results will contribute to a better understanding of the influence of phage therapy on human cells and encourage even more studies on this topic. [ABSTRACT FROM AUTHOR]

Widely used in biomedical and bioanalytical applications, the detonation nanodiamonds (NDs) are generally considered to be biocompatible and non-toxic to a wide range of eukaryotic cells. Due to their high susceptibility to chemical modifications, surface functionalisation is often used to tune the biocompatibility and antioxidant activity of the NDs. The response of photosynthetic microorganisms to redox-active NDs is still poorly understood and is the focus of the present study. The green microalga Chlamydomonas reinhardtii was used to assess the potential phytotoxicity and antioxidant activity of NDs hosting hydroxyl functional groups at concentrations of 5–80 μg NDs/mL. The photosynthetic capacity of microalgae was assessed by measuring the maximum quantum yield of PSII photochemistry and the light-saturated oxygen evolution rate, while oxidative stress was assessed by lipid peroxidation and ferric-reducing antioxidant capacity. We demonstrated that hydroxylated NDs might reduce cellular levels of oxidative stress, protect PSII photochemistry and facilitate the PSII repair under methyl viologen and high light associated stress conditions. Factors involved in this protection may include the low phytotoxicity of hydroxylated NDs in microalgae and their ability to accumulate in cells and scavenge reactive oxygen species. Our findings could pave the way for using hydroxylated NDs as antioxidants to improve cellular stability in algae-based biotechnological applications or semi-artificial photosynthetic systems. [ABSTRACT FROM AUTHOR]

Mitochondria play a key role in regulating host metabolism, immunity and cellular homeostasis. Remarkably, these organelles are proposed to have evolved from an endosymbiotic association between an alphaproteobacterium and a primitive eukaryotic host cell or an archaeon. This crucial event determined that human cell mitochondria share some features with bacteria, namely cardiolipin, N-formyl peptides, mtDNA and transcription factor A, that can act as mitochondrial-derived damage-associated molecular patterns (DAMPs). The impact of extracellular bacteria on the host act largely through the modulation of mitochondrial activities, and often mitochondria are themselves immunogenic organelles that can trigger protective mechanisms through DAMPs mobilization. In this work, we demonstrate that mesencephalic neurons exposed to an environmental alphaproteobacterium activate innate immunity through toll-like receptor 4 and Nod-like receptor 3. Moreover, we show that mesencephalic neurons increase the expression and aggregation of alpha-synuclein that interacts with mitochondria, leading to their dysfunction. Mitochondrial dynamic alterations also affect mitophagy which favors a positive feedback loop on innate immunity signaling. Our results help to elucidate how bacteria and neuronal mitochondria interact and trigger neuronal damage and neuroinflammation and allow us to discuss the role of bacterial-derived pathogen-associated molecular patterns (PAMPs) in Parkinson's disease etiology. [ABSTRACT FROM AUTHOR]

Mitochondrion is an important organelle of eukaryotic cells and a critical target of ionizing radiation (IR) outside the nucleus. The biological significance and mechanism of the non-target effect originating from mitochondria have received much attention in the field of radiation biology and protection. In this study, we investigated the effect, role, and radioprotective significance of cytosolic mitochondrial DNA (mtDNA) and its associated cGAS signaling on hematopoietic injury induced by IR in vitro culture cells and in vivo total body irradiated mice in this study. The results demonstrated that γ-ray exposure increases the release of mtDNA into the cytosol to activate cGAS signaling pathway, and the voltage-dependent anion channel (VDAC) may contribute to IR-induced mtDNA release. VDAC1 inhibitor DIDS and cGAS synthetase inhibitor can alleviate bone marrow injury and ameliorate hematopoietic suppression induced by IR via protecting hematopoietic stem cells and adjusting subtype distribution of bone marrow cells, such as attenuating the increase of the F4/80+ macrophage proportion in bone marrow cells. The present study provides a new mechanistic explanation for the radiation non-target effect and an alternative technical strategy for the prevention and treatment of hematopoietic acute radiation syndrome. [ABSTRACT FROM AUTHOR]

Biological rhythms are ubiquitous across organisms and coordinate key cellular processes. Oscillations of Mg2+ levels in cells are now well-established, and due to the critical roles of Mg2+ in cell metabolism, they are potentially fundamental for the circadian control of cellular activity. The identity of the transport proteins responsible for sustaining Mg2+ levels in eukaryotic cells remains hotly debated, and several are restricted to specific groups of higher eukaryotes. Here, using the eukaryotic minimal model cells of Ostreococcus tauri, we report two homologs of common descents of the Cyclin M (CNNM)/CorC protein family. Overexpression of these proteins leads to a reduction in the overall magnesium content of cells and a lengthening of the period of circadian gene expression rhythms. However, we observed a paradoxical increase in the magnesium content of the organelle fraction. The chemical inhibition of Mg2+ transport has a synergistic effect on circadian period lengthening upon the overexpression of one CNNM homolog, but not the other. Finally, both homologs rescue the deleterious effect of low extracellular magnesium on cell proliferation rates. Overall, we identified two CNNM proteins that directly affect Mg2+ homeostasis and cellular rhythms. [ABSTRACT FROM AUTHOR]