Eukaryotic Research Articles

Structure and distribution of sensor histidine kinases in the fungal kingdom.

Two-component systems (TCSs) are diverse cell signaling pathways that play a significant role in coping with a wide range of environmental cues in both prokaryotic and eukaryotic organisms. These transduction circuitries are primarily governed by histidine kinases (HKs), which act as sensing proteins of a broad variety of stressors. To date, nineteen HK groups have been previously described in the fungal kingdom. However, the structure and distribution of these prominent sensing proteins were hitherto investigated in a limited number of fungal species. In this study, we took advantage of recent genomic resources in fungi to refine the fungal HK classification by deciphering the structural diversity and phylogenetic distribution of HKs across a large number of fungal clades. To this end, we browsed the genome of 91 species representative of different fungal clades, which yielded 726 predicted HK sequences. A domain organization analysis, coupled with a robust phylogenomic approach, led to an improved categorization of fungal HKs. While most of the compiled sequences were categorized into previously described fungal HK groups, some new groups were also defined. Overall, this study provides an improved overview of the structure, distribution, and evolution of HKs in the fungal kingdom.

Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of β-barrel pore-forming toxins (β-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane β-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif (CRM) in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

Effect of nystatin on invasion of <i>Serratia grimesii</i> and <i>Serratia proteamaculans</i> bacteria into epithelial cells

BACKGROUND: Bacteria use various endocytic pathways during entering non-phagocytic cells. The involvement of caveolae/lipid rafts in bacterial invasion has been demonstrated for many bacterial pathogens. However, for bacteria of the genus Serratia, the involvement of membrane microdomains in the process of bacterial internalization has been poorly studied. AIM: To evaluate the involvement of caveolae/lipid rafts in the invasion of S. grimesii and S. proteamaculans bacteria into non-phagocytic epithelial Caco-2 and M-HeLa cells using nystatin. MATERIALS AND METHODS: M-HeLa and Caco-2 epithelial cells were incubated with 50 μM nystatin for 1 hour at 37 ºC, after which they were infected with the bacteria S. grimesii strain 30063 and S. proteamaculans strain 94, the multiplicity of infection was 100 bacteria per cell. The number of intracellular bacteria was assessed using gentamicin protection assay. The level of caveolin-1 in cells was visualized using confocal microscopy and Western blotting. The expression of Toll-like receptors genes were measured by real-time RT-PCR. RESULTS: Treatment of epithelial cells with nystatin reduces the internalization of S. grimesii and S. proteamaculans into M-HeLa cells by 30% and does not affect penetration into Caco-2 cells. At the same time, nystatin does not affect the redistribution / the integrity impairment of lipid rafts and does not lead to the cytoskeleton reorganization of eukaryotic cells. The addition of nystatin increases the level of caveolin-1 in M-HeLa cells (caveolin-1 is not expressed in Caco-2), which leads to a change plasma membrane fluidity. Nystatin promotes the secretion of proinflammatory cytokines interleukin-6 and interleukin-8 in both cell lines. Infection of M-HeLa cells pretreated with nystatin with the studied bacteria leads to an increase in the expression of tlr2 and tlr4 genes, but does not exceed the level of their expression in control samples. Therefore, it is impossible to speak unambiguously about the participation of Toll-like receptors in the invasion of Serratia bacteria. CONCLUSIONS: The results obtained suggest that the interaction of bacteria with eukaryotic cells induces the expression of caveolin-1, which leads to a change plasma membrane components mobility. This may be due to the fact that β1-integrin is involved in the invasion of the studied bacteria, which should be stabilized at the plasma membrane upon binding of the ligand due to the formation of a cholesterol- and sphingolipid-rich membrane microenvironment.

Knockdown of PAIP1 Inhibits Breast Cancer Cell Proliferation by Regulating Cyclin E2 mRNA Stability.

Polyadenylate-binding protein-interacting protein 1 (PAIP1) is a protein that modulates translation initiation in eukaryotic cells. Studies have shown that PAIP1 was overexpressed in various type of cancers, and drives cancer progression by promoting cancer cell proliferation, invasion, and migration. In our previous study, we identified that PAIP1 was overexpressed in breast cancer, and the expression was correlated with poor prognosis. However, the biological function of PAIP1 in breast cancer has not been clearly understood. In this study, we constructed PAIP1 specifically silenced breast cancer cells. Then, cell proliferation, cell cycle distribution, and apoptosis were detected in PAIP1 knockdown cells. RNA-seq analysis was performed to predict the downstream target of PAIP1, and the molecular mechanism was explored. As a results, we found that knockdown of PAIP1 repressed cell proliferation, induced cell cycle arrest, and triggers apoptosis. Xenograft mouse model showed that knockdown of PAIP1 inhibits cell growth in vivo. RNA-seq predicted that CCNE2 mRNA was one of the downstream targets of PAIP1. In addition, we identified that knockdown of PAIP1-inhibited cell proliferation through modulating cyclin E2 expression. Mechanically, knockdown of PAIP1 reduces the expression of cyclin E2 by regulating the mRNA stability of cyclin E2. Moreover, in breast cancer tissues, we found that the expression of PAIP1 was positively correlated with cyclin E2. Taken together, our findings establish the role and mechanism of PAIP1 in breast cancer progression, indicating that PAIP1 would be a new therapeutic target for breast cancer treatment.

How coat proteins shape autophagy in plant cells.

Autophagy is a membrane trafficking pathway through which eukaryotic cells target their own cytoplasmic constituents for degradation in the lytic compartment. Proper biogenesis of autophagic organelles requires a conserved set of autophagy-related (ATG) proteins and their interacting factors, such as signalling phospholipid phosphatidylinositol 3-phosphate (PI3P) and coat complex II (COPII). The COPII machinery, which was originally identified as a membrane coat involved in the formation of vesicles budding from the endoplasmic reticulum, contributes to the initiation of autophagic membrane formation in yeast, metazoan, and plant cells; however, the exact mechanisms remain elusive. Recent studies using the plant model species Arabidopsis thaliana have revealed that plant-specific PI3P effectors are involved in autophagy. The PI3P effector FYVE2 interacts with the conserved PI3P effector ATG18 and with COPII components, indicating an additional role for the COPII machinery in the later stages of autophagosome biogenesis. In this Update, we examined recent research on plant autophagosome biogenesis and proposed working models on the functions of the COPII machinery in autophagy, including its potential roles in stabilizing membrane curvature and sealing the phagophore.

The prolyl isomerase FKBP11 is a secretory translocon accessory factor.

Eukaryotic cells encode thousands of secretory and membrane proteins, many of which are cotranslationally translocated into or across the endoplasmic reticulum (ER). Nascent polypeptides entering the ER encounter a network of molecular chaperones and enzymes that facilitate their folding. A rate-limiting step for some proteins is the trans-to-cis isomerization of the peptide bond between proline and the residue preceding it. The human ER contains six prolyl isomerases, but the function, organization and substrate range of these proteins is not clear. Here we show that the metazoan-specific, prolyl isomerase FKBP11 binds to ribosome-translocon complexes (RTCs) in the ER membrane, dependent on its single transmembrane domain (TMD) and a conserved, positively charged region at its cytosolic C-terminus. High throughput mRNA sequencing shows selective engagement with ribosomes synthesizing secretory and membrane proteins with long translocated segments, and functional analysis shows reduced stability of two such proteins, EpCAM and PTTG1IP, in cells depleted of FKBP11. We propose that FKBP11 is a translocon accessory factor that acts on a broad range of soluble secretory and transmembrane proteins during their synthesis at the ER.

Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in various water and land environments and organisms, including insects and mammals. Some P. alcalifaciens strains encode gene homologs of virulence factors found in pathogenic Enterobacterales members, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are pathogenic determinants in P. alcalifaciens is not known. In this study, we investigated P. alcalifaciens-host interactions at the cellular level, focusing on the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS1b is widespread in Providencia spp. and encoded on the chromosome. A large plasmid that is present in a subset of P. alcalifaciens strains, primarily isolated from diarrheal patients, encodes for T3SS1a. We show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, lyses its internalization vacuole, and proliferates in the cytosol. This triggers caspase-4-dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS1a in entry, vacuole lysis, and cytosolic proliferation is host cell type-specific, playing a more prominent role in intestinal epithelial cells than in macrophages or insect cells. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa and induces mild epithelial damage with negligible fluid accumulation in a T3SS1a- and T3SS1b-independent manner. However, T3SS1b was required for the rapid killing of Drosophila melanogaster. We propose that the acquisition of two T3SS has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

PP1 phosphatase controls both daughter cell formation and amylopectin levels in Toxoplasma gondii.

Virulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell cycle is therefore key to their pathogenesis. Phosphorylation is a crucial posttranslational modification that regulates many aspects of the eukaryotic cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasite Toxoplasma gondii. Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the proper assembly of the daughter cells' inner membrane complex (IMC), the segregation of organelles, and nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is usually found in the latent bradyzoite form of the parasite. Using transcriptomics and phospho-proteomics, we show that TgPP1 mainly acts through posttranslational mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylates a protein bearing a starch-binding domain. Mutagenesis analysis reveals that the targeted phospho-sites are linked to the ability of the parasite to regulate amylopectin steady-state levels. Therefore, we show that TgPP1 has pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation.

Antagonistic insulin-like signaling influencing glucose regulation in C. elegans

The insulin/ IGF-signaling (IIS) is an evolutionary conserved signaling pathway within eukaryotic organisms that regulates glucose metabolism and cell division. The nematode, C. elegans is an excellent model for exploring IIS as they have 40 insulin-like peptides that act on a single receptor, DAF-2. We aim to explore the relationships between IIS and glucose metabolism, focusing on the antagonistic insulin-like peptide, INS-17 and its influence over FDGT-1 (facilitated glucose transporter 1). FDGT-1 is expressed throughout the nematode, including the basolateral membrane of intestinal cells and where it is theorized to export glucose to the surrounding somatic cells rather than absorb it from the intestinal tract. We hypothesize that IIS controls transcription of FDGT-1 and antagonistic peptides should lead to a decrease in FDGT-1 transcription, resulting in the increased lifespan and increased fat accumulation observed in daf-2(lf) loss of function and ins-17(oe) over expressing strains. In this study, survival assays are used to assess the impact which high-glucose content has on lifespan, along with Oil Red O (ORO) lipid staining to visualize lipid accumulation amongst conditions and protein quantification of FDGT-1:GFP to evaluate FDGT-1 protein levels. Our findings are consistent with the idea of low insulin signaling leading to extended lifespan as daf-2(lf) lived significantly longer than WT in both 0% and 2% glucose conditions and ins-17(oe) living significantly longer than wildtype in 2% glucose conditions. The ORO lipid staining revealed that high glucose conditions and low insulin signaling result in lipid accumulation within the nematode. Using the fdgt-1:gfp strain, we were able to determine an increase in the FDGT-1 in the presence of glucose, suggesting FDGT-1 expression is influenced by glucose. These findings aim to further our understanding of IIS regulating glucose uptake and metabolism, along with the significance of the FDGT-1 glucose transporter.

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

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

Stem cell-derived extracellular vesicles: a potential intervention for Bronchopulmonary Dysplasia.

Despite advances in neonatal care, the incidence of Bronchopulmonary Dysplasia (BPD) remains high among extreme preterm infants. The pathogenesis of BPD is multifactorial, with inflammation playing a central role. There is strong evidence that stem cell therapy reduces inflammatory changes and restores normal lung morphology in animal models of hyperoxia-induced lung injury. These therapeutic effects occur without significant engraftment of the stem cells in the host lung, suggesting more of a paracrine mechanism mediated by their secretome. In addition, there are multiple concerns with stem cell therapy which may be alleviated by administering only the effective vesicles instead of the cells themselves. Extracellular vesicles (EVs) are cell-derived components secreted by most eukaryotic cells. They can deliver their bioactive cargo (mRNAs, microRNAs, proteins, growth factors) to recipient cells, which makes them a potential therapeutic vehicle in many diseases, including BPD. The following review will highlight recent studies that investigate the effectiveness of EVs derived from stem cells in preventing or repairing injury in the preterm lung, and the potential mechanisms of action that have been proposed. Current limitations will also be discussed as well as suggestions for advancing the field and easing the transition towards clinical translation in evolving or established BPD. IMPACT: Extracellular vesicles (EVs) derived from stem cells are a potential intervention for neonatal lung diseases. Their use might alleviate the safety concerns associated with stem cell therapy. This review highlights recent studies that investigate the effectiveness of stem cell-derived EVs in preclinical models of bronchopulmonary dysplasia. It adds to the existing literature by elaborating on the challenges associated with EV research. It also provides suggestions to advance the field and ease the transition towards clinical applications. Optimizing EV research could ultimately improve the quality of life of extreme preterm infants born at vulnerable stages of lung development.

Plasmid vector(s) in Bacillus thuringiensis harbor genes for insect pest control and for neglected infectious diseases in humans

Plasmids (circular DNA molecules) represent an ingenious strategy for horizontal gene transfer in prokaryotes and eukaryotic cells. Plasmids harbored in bacteria are responsible for the spread of traits such as antibiotic resistance, virulence factors, and the machinery for the horizontal gene transfer e.g., type IV secretion systems. Remarkably, Bacillus thuringiensis (Bt) cryptic plasmids encode and carry genes that, under the host environment, replicate and concomitate with sporulation, producing parasporal crystalline proteins of two major types, crystalline (Cry) and cytolytic (Cyt), the former toxic against different orders of insects such as Lepidopterans, Coleopterans, and Dipterans (Cry proteins, MW 50–130 KDa); Cyt proteins, produced by B. thuringiensis subspecies israelensis (Bti)(MW 27-kDa) are toxic against Dipterans, i.e., mosquitoes and black flies. The X-Ray tridimensional structure for both types of toxins, formed by three domains, mostly of beta sheets antiparallel (Domain II and Domain III) linked through loops of different lengths. Domain I is a bundle of alpha helices. This structure is characterized by five conserved blocks, implying a conservation in the mode of action. Cyt proteins possess two alpha helices and some beta sheets with a structure similar to the antimicrobial peptides. Indeed, the mode of action proposed is mediated by the toxin-lipid interaction that hypothetically could result in transmembrane ionic channel formation. Several pieces of evidence support the action of both toxins in insects and mammals. The question is to what extent these Bt/Bti plasmid-encoded Cry or Cyt genes can be applied as bioinsecticides individually or in combination with Lysinibacillus sphaericus. The feasibility of being considered a promising and safe biological strategy for crop pests and vector-borne neglected infectious diseases is an issue pinpointed in the present review.

Two Tetrahymena kinesin-9 family members exhibit slow plus-end-directed motility in vitro

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.

Application and research progress of MultiBac: A review.

Although the traditional Escherichia coli expression system has matured and is cost-effective, the posttranslation modifications of proteins expressed in eukaryotic cells differ significantly from those expressed in E coli. Insect cells have gradually entered the realm of researchers; however, the proteins synthesized by insect cells are somewhat different from those of mammals in terms of modification. Herein, we have introduced a relatively new method. MultiBac, We introduce the development process, characteristics, and applications of MultiBac technology. And provide new methods for basic researchers. MultiBac has evolved into an indispensable tool in the fields of biotechnology and pharmaceuticals, facilitating the efficient production of recombinant proteins and the study of complex protein complexes. Furthermore, its development has benefited from the integration of synthetic biology techniques, providing additional versatility. But it also has some disadvantages. MultiBac technology is poised to become a key tool in unlocking the mysteries of the protein world, propelling the life sciences ever forward. But researchers should consider its limitations when selecting the most appropriate expression system for their specific needs.

African Swine Fever Virus Modulates the Endoplasmic Reticulum Stress-ATF6-Calcium Axis to Facilitate Viral Replication.

ABSTRACTAfrican swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. The endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with the unfolded protein response (UPR) (downstream the ER stress) are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.

Quantitative mapping of proteasome interactomes and substrates using ProteasomeID.

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.

Protein SUMOylation and Its Functional Role in Nuclear Receptor Control

Post-translational protein modifications (PTMs) significantly enhance the functional diversity of proteins and are therefore important for the expansion and the dynamics of the cell’s proteome. In addition to structurally simpler PTMs, substrates also undergo modification through the reversible attachment of small proteins. The best understood PTM of this nature to date is the covalent conjugation of ubiquitin and ubiquitin-like proteins (UBLs) to their substrates. The protein family of small ubiquitin-like modifier (SUMO) is one of these UBLs that has received increasing scientific attention. The pathway of SUMOylation is highly conserved in all eukaryotic cells and is crucial for their survival. It plays an essential role in many biological processes, such as the maintenance of genomic integrity, transcriptional regulation, gene expression, and the regulation of intracellular signal transduction, and thereby influences DNA damage repair, immune responses, cell cycle progression, and apoptosis. Several studies have already shown that in this context protein SUMOylation is involved in the control mechanisms of various cellular receptors. This article unites data from different studies focusing on the investigation of the strictly conserved three-step enzyme cascade of protein SUMOylation and the functional analysis of the involved proteins E1, E2, and E3 and SUMOylation target proteins. Furthermore, this review highlights the role of nuclear receptor SUMOylation and its importance for the cellular functionality and disease development arising from defects in correct protein SUMOylation.

Microtubules and cardiovascular diseases: insights into pathology and therapeutic strategies

Microtubules, complex cytoskeletal structures composed of tubulin proteins in eukaryotic cells, have garnered recent attention in cardiovascular research. Investigations have focused on the post-translational modifications of tubulin, including acetylation and detyrosination. Perturbations in microtubule homeostasis have been implicated in various pathological processes associated with cardiovascular diseases such as heart failure, ischemic heart disease, and arrhythmias. Thus, elucidating the intricate interplay between microtubule dynamics and cardiovascular pathophysiology is imperative for advancing preventive and therapeutic strategies. Several natural compounds have been identified to potentially modulate microtubules, thereby exerting regulatory effects on cardiovascular diseases. This review synthesizes current literature to delineate the roles of microtubules in cardiovascular diseases and assesses the potential of natural compounds in microtubule-targeted therapies.

A user-friendly CRISPR/Cas9 system for mutagenesis of Neurosporacrassa

As a widely used eukaryotic model organism, Neurosporacrassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N.crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamineamidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N.crassa compared to traditional methods.

Navigating the fungal battlefield: cysteine-rich antifungal proteins and peptides from Eurotiales

Fungi are ubiquitous in the environment and play a key role in the decomposition and recycling of nutrients. On the one hand, their special properties are a great asset for the agricultural and industrial sector, as they are used as source of nutrients, producers of enzymes, pigments, flavorings, and biocontrol agents, and in food processing, bio-remediation and plant growth promotion. On the other hand, they pose a serious challenge to our lives and the environment, as they are responsible for fungal infections in plants, animals and humans. Although host immunity opposes invading pathogens, certain factors favor the manifestation of fungal diseases. The prevalence of fungal infections is on the rise, and there is an alarming increase in the resistance of fungal pathogens to approved drugs. The limited number of antimycotics, the obstacles encountered in the development of new drugs due to the poor tolerability of antifungal agents in patients, the limited number of unique antifungal targets, and the low species specificity contribute to the gradual depletion of the antifungal pipeline and newly discovered antifungal drugs are rare. Promising candidates as next-generation therapeutics are antimicrobial proteins and peptides (AMPs) produced by numerous prokaryotic and eukaryotic organisms belonging to all kingdom classes. Importantly, filamentous fungi from the order Eurotiales have been shown to be a rich source of AMPs with specific antifungal activity. A growing number of published studies reflects the efforts made in the search for new antifungal proteins and peptides (AFPs), their efficacy, species specificity and applicability. In this review, we discuss important aspects related to fungi, their impact on our life and issues involved in treating fungal infections in plants, animals and humans. We specifically highlight the potential of AFPs from Eurotiales as promising alternative antifungal therapeutics. This article provides insight into the structural features, mode of action, and progress made toward their potential application in a clinical and agricultural setting. It also identifies the challenges that must be overcome in order to develop AFPs into therapeutics.