Cytochrome c regulates hyphal morphogenesis by interfering with cAMP-PKA signaling in Candida albicans.

In the human fungal pathogen Candida albicans, invasive hyphal growth is a well-recognized virulence trait. We employed transposon-mediated genome-wide mutagenesis, revealing that inactivating CTM1 blocks hyphal growth. CTM1 encodes a lysine (K) methyltransferase, which trimethylates cytochrome c (Cyc1) at K79. Mutants lacking CTM1 or expressing cyc1K79A grow as yeast under hyphae-inducing conditions, indicating that unmethylated Cyc1 suppresses hyphal growth. Transcriptomic analyses detected increased levels of the hyphal repressor NRG1 and decreased levels of hyphae-specific genes in ctm1Δ/Δ and cyc1K79A mutants, suggesting cyclic AMP (cAMP)-protein kinase A (PKA) signaling suppression. Co-immunoprecipitation and in vitro kinase assays demonstrated that unmethylated Cyc1 inhibits PKA kinase activity. Surprisingly, hyphae-defective ctm1Δ/Δ and cyc1K79A mutants remain virulent in mice due to accelerated proliferation. Our results unveil a critical role for cytochrome c in maintaining the virulence of C. albicans by orchestrating proliferation, growth mode, and metabolism. Importantly, this study identifies a biological function for lysine methylation on cytochrome c.

Exopolysaccharide is the potential effector of Lactobacillus fermentum PS150, a hypnotic psychobiotic strain.

Psychobiotics are a class of probiotics that confer beneficial effects on the mental health of the host. We have previously reported hypnotic effects of a psychobiotic strain, Lactobacillus fermentum PS150 (PS150), which significantly shortens sleep latency in experimental mice, and effectively ameliorate sleep disturbances caused by either caffeine consumption or a novel environment. In the present study, we discovered a L. fermentum strain, GR1009, isolated from the same source of PS150, and found that GR1009 is phenotypically distinct but genetically similar to PS150. Compared with PS150, GR1009 have no significant hypnotic effects in the pentobarbital-induced sleep test in mice. In addition, we found that heat-killed PS150 exhibited hypnotic effects and altered the gut microbiota in a manner similar to live bacteria, suggesting that a heat-stable effector, such as exopolysaccharide (EPS), could be responsible for these effects. Our comparative genomics analysis also revealed distinct genetic characteristics in EPS biosynthesis between GR1009 and PS150. Furthermore, scanning electron microscopy imaging showed a sheet-like EPS structure in PS150, while GR1009 displayed no apparent EPS structure. Using the phenol-sulfate assay, we found that the sugar content value of the crude extract containing EPS (C-EPS) from PS150 was approximately five times higher than that of GR1009, indicating that GR1009 has a lower EPS production activity than PS150. Through the pentobarbital-induced sleep test, we confirmed the hypnotic effects of the C-EPS isolated from PS150, as evidenced by a significant reduction in sleep latency and recovery time following oral administration in mice. In summary, we utilized a comparative approach to delineate differences between PS150 and GR1009 and proposed that EPS may serve as a key factor that mediates the observed hypnotic effect.

A Review on Phytochemicals of the Genus Maytenus and Their Bioactive Studies.

The genus Maytenus is a member of the Celastraceae family, of which several species have long been used in traditional medicine. Between 1976 and 2021, nearly 270 new compounds have been isolated and elucidated from the genus Maytenus. Among these, maytansine and its homologues are extremely rare in nature. Owing to its unique skeleton and remarkable bioactivities, maytansine has attracted many synthetic endeavors in order to construct its core structure. In this paper, the current status of the past 45 years of research on Maytenus, with respect to its chemical and biological activities are discussed. The chemical research includes its structural classification into triterpenoids, sesquiterpenes and alkaloids, along with several chemical synthesis methods of maytansine or maytansine fragments. The biological activity research includes activities, such as anti-tumor, anti-bacterial and anti-inflammatory activities, as well as HIV inhibition, which can provide a theoretical basis for the better development and utilization of the Maytenus.

Antimicrobial Actions and Applications of Chitosan.

Chitosan is a naturally originating product that can be applied in many areas due to its biocompatibility, biodegradability, and nontoxic properties. The broad-spectrum antimicrobial activity of chitosan offers great commercial potential for this product. Nevertheless, the antimicrobial activity of chitosan varies, because this activity is associated with its physicochemical characteristics and depends on the type of microorganism. In this review article, the fundamental properties, modes of antimicrobial action, and antimicrobial effects-related factors of chitosan are discussed. We further summarize how microorganisms genetically respond to chitosan. Finally, applications of chitosan-based biomaterials, such as nanoparticles and films, in combination with current clinical antibiotics or antifungal drugs, are also addressed.

Synergistic Antifungal Activity of Chitosan with Fluconazole against Candida albicans, Candida tropicalis, and Fluconazole-Resistant Strains.

(1) Background: Few antifungal drugs are currently available, and drug-resistant strains have rapidly emerged. Thus, the aim of this study is to evaluate the effectiveness of the antifungal activity from a combinational treatment of chitosan with a clinical antifungal drug on Candida albicans and Candida tropicalis. (2) Methods: Minimum inhibitory concentration (MIC) tests, checkerboard assays, and disc assays were employed to determine the inhibitory effect of chitosan with or without other antifungal drugs on C. albicans and C. tropicalis. (3) Results: Treatment with chitosan in combination with fluconazole showed a great synergistic fungicidal effect against C. albicans and C. tropicalis, but an indifferent effect on antifungal activity when challenged with chitosan-amphotericin B or chitosan-caspofungin simultaneously. Furthermore, the combination of chitosan and fluconazole was effective against drug-resistant strains. (4) Conclusions: These findings provide strong evidence that chitosan in combination with fluconazole is a promising therapy against two Candida species and its drug-resistant strains.

Evaluation of Biofilm Formation in Candida tropicalis Using a Silicone-Based Platform with Synthetic Urine Medium.

Molecular mechanisms of biofilm formation in Candida tropicalis and current methods for biofilm analyses in this fungal pathogen are limited. (2) Methods: Biofilm biomass and crystal violet staining of the wild-type and each gene mutant strain of C. tropicalis were evaluated on silicone under synthetic urine culture conditions. (3) Results: Seven media were tested to compare the effects on biofilm growth with or without silicone. Results showed that biofilm cells of C. tropicalis were unable to form firm biofilms on the bottom of 12-well polystyrene plates. However, on a silicone-based platform, Roswell Park Memorial Institute 1640 (RPMI 1640), yeast nitrogen base (YNB) + 1% glucose, and synthetic urine media were able to induce strong biofilm growth. In particular, replacement of Spider medium with synthetic urine in the adherence step and the developmental stage is necessary to gain remarkably increased biofilms. Interestingly, unlike Candida albicans, the C. tropicalis ROB1 deletion strain but not the other five biofilm-associated mutants did not cause a significant reduction in biofilm formation, suggesting that the biofilm regulatory circuits of the two species are divergent. (4) Conclusions: This system for C. tropicalis biofilm analyses will become a useful tool to unveil the biofilm regulatory network in C. tropicalis.

Associations of the BRAF V600E Mutation and PAQR3 Protein Expression with Papillary Thyroid Carcinoma Clinicopathological Features.

The BRAFV600E mutation is the most prevalent genetic event in patients with papillary thyroid cancer (PTC). However, no study has investigated the expression of PAQR3 in papillary thyroid tissues in relation to the BRAFV600E mutation and the clinicopathological features of PTC patients. Furthermore, the potential associations of the BRAFV600E mutation, PAQR3 expression and clinicopathological parameters in the cancerous tissues of PTC patients have not been investigated. This study was conducted on 60 patients with PTC who were treated surgically at our institution from 2017 to 2018. PCR was used to amplify DNA by the amplification refractory mutation system (ARMS) method to detect BRAFV600E gene mutations. In addition, immunohistochemical techniques were utilized to assess PAQR3 expression in tumor tissue sections. The BRAFV600E mutation was associated with lymph node metastasis (LNM, p < 0.05) but not with other clinicopathological features. Low PAQR3 expression was associated with extrathyroidal extension and LNM (χ2 = 7.143, p = 0.009; χ2 = 6.459, p = 0.014, respectively). Furthermore, a statistically significant association was observed between chronic lymphocytic thyroiditis and LNM (χ2 = 5.275, p = 0.0250). A linear relationship between the BRAFV600E mutation and PAQR3 protein expression has not been identified. These factors may be independent risk factors of extrathyroidal extension and LNM in PTC and be used to indicate the invasiveness of PTC tumors. Higher quality, multivariate analyses based on larger samples from around the world are urgently needed to further validate and revise our findings in the future.

A Potential Antifungal Effect of Chitosan Against Candida albicans Is Mediated via the Inhibition of SAGA Complex Component Expression and the Subsequent Alteration of Cell Surface Integrity.

Due to the high incidence of nosocomial Candida albicans infection, the first-line drugs for C. albicans infection have been heavily used, and the emergence of drug-resistant strains has gradually increased. Thus, a new antifungal drug or therapeutic method is needed. Chitosan, a product of chitin deacetylation, is considered to be potentially therapeutic for fungal infections because of its excellent biocompatibility, biodegradability and low toxicity. The biocidal action of chitosan against C. albicans shows great commercial potential, but the exact mechanisms underlying its antimicrobial activity are unclear. To reveal these mechanisms, mutant library screening was performed. ADA2 gene, which encodes a histone acetylation coactivator in the SAGA complex, was identified. Transmission electronic microscopy images showed that the surface of chitosan-treated ada2Δ cells was substantially disrupted and displayed an irregular morphology. Interestingly, the cell wall of ada2Δ cells was significantly thinner than that of wild-type cells, with a thickness similar to that seen in the chitosan-treated wild-type strain. Although ADA2 is required for chitosan tolerance, expression of ADA2 and several Ada2-mediated cell wall-related genes (ALS2, PGA45, and ACE2) and efflux transporter genes (MDR1 and CDR1) were significantly inhibited by chitosan. Furthermore, GCN5 encoding a SAGA complex catalytic subunit was inhibited by chitosan, and gcn5Δ cells exhibited phenotypes comparable to those of ada2Δ cells in response to chitosan and other cell surface-disrupting agents. This study demonstrated that a potential antifungal mechanism of chitosan against C. albicans operates by inhibiting SAGA complex gene expression, which decreases the protection of the cell surface against chitosan.

Identification and characterization of ORF19.1725, a novel gene contributing to the white cell pheromone response and virulence-associated functions in Candida albicans.

An epigenetic transition between white cells and opaque cells influences several properties of Candida albicans biology, including cellular morphology, biofilm formation, virulence, and sexual mating. In particular, these two cell types exhibit marked differences in their ability to undergo sex. A previous study identified the transcriptional regulator of pheromone response in both the white and opaque states as Cph1 because deletion of this gene abolished both pheromone-induced cell adhesion in white cells and sexual mating in opaque cells. To further explore how these cell types exhibit distinct biological outputs upon pheromone stimulation, we selected five Cph1-regulated genes with significant expression during the pheromone response in the white state but not the opaque state. These phase-specific pheromone-induced genes are ORF19.1539, ORF19.1725, ORF19.2430, ORF19.2691 and ORF19.5557. Deletion of each gene revealed that orf19.1539Δ, orf19.1725Δ, orf19.2430Δ and orf19.5557Δ showed significant decreases in pheromone-stimulated cell adhesion in the white state but retained normal mating competency in the opaque state, indicating that a particular role in white cell pheromone response is mediated by these four genes. Interestingly, the defects of orf19.1725Δ in pheromone-stimulated cell adhesion also abolished conventional biofilms and hyphal growth. Zebrafish egg infection assays further demonstrated that ORF19.1725 is involved in cell adhesion, penetration and virulence. Overall, four Cph1-regulated downstream targets were identified in the regulation of white cell pheromone response. We also clarified the roles of C. albicans ORF19.1725 in cell adhesion, hyphal growth, biofilm formation and virulence.

Cpp1 phosphatase mediated signaling crosstalk between Hog1 and Cek1 mitogen-activated protein kinases is involved in the phenotypic transition in Candida albicans.

Cellular signaling pathways involved in cell growth and differentiation mediated by mitogen-activated protein kinase (MAPK) cascades have been well characterized in fungi. However, the mechanisms of signaling crosstalk between MAPKs to ensure signaling specificity are largely unknown. Previous work showed that activation of the Candida albicans Cek1 MAPK pathway resulted in opaque cell formation and filamentation, which mirrored the phenotypes to hog1Δ. Additionally, deleting the HOG1 gene stimulated Cek1p. Thus, we hypothesized that an unknown factor could act as a bridge between these two MAPKs. In Saccharomyces cerevisiae, the dual-specificity phosphatase (DSP) Msg5 specifically dephosphorylates Fus3p/Kss1p. C. albicans Cpp1, an ortholog of Msg5, has been shown to be important in regulating Cek1p. Compared with the wild-type strain, hog1Δ shows a ∼40% reduction in CPP1 expression. Consistent with previous reports, CPP1 deletion also resulted in Cek1 hyperphosphorylation, implicating Cpp1 as a regulator of the Hog1 and Cek1 cascades. Interestingly, both cpp1Δ and hog1Δ induced 100% opaque colony formation in MTL-homozygous strains grown on N-acetylglucosamine (NAG) plates, whereas the wild-type and complemented strains exhibited 80.9% and 77.1% white-to-opaque switching rates, respectively. CPP1 gene deletion also caused hyperfilamentous phenotypes in both white and opaque cells. These phenomena may be due to highly phosphorylated Cek1p, as deleting CEK1 in the cpp1Δ background generated nonfilamentous strains and reduced opaque colony formation. Taken together, we conclude that cpp1Δ and hog1Δ exhibited comparable phenotypes, and both are involved in regulating Cek1 phosphorylation, implicating Cpp1 phosphatase as a key intermediary between the Hog1 and Cek1 signal transduction pathways.

The conserved dual phosphorylation sites of the Candida albicans Hog1 protein are crucial for white-opaque switching, mating, and pheromone-stimulated cell adhesion.

Candida albicans is an opportunistic human pathogen capable of causing life-threatening infections in immunocompromised patients. C. albicans has a unique morphological transition between white and opaque phases. These two cells differ in virulence, mating capability, biofilm formation, and host-cell interaction. Previous studies revealed that deletion of the SSK2, PBS2, or HOG1 gene resulted in 100% opaque cell formation and suppressed the mating response. Thr-174 and Tyr-176 of the Hog1 protein are important phosphoacceptors and can be activated in response to stimuli. In this study, we first demonstrated the importance of two conserved phosphorylation sites in white-opaque switching, mating, and pheromone-stimulated cell adhesion. Six Hog1 point-mutated strains were generated, including nonphosphorylated strains (Hog1(T174A), Hog1(Y176F), and Hog1(T174A,Y176F)) and negatively charged phosphorylated strains (Hog1(T174D), Hog1(Y176D), and Hog1(T174D,Y176D)). Point mutation on Thr-174, Tyr-176 or in combination with the Hog1 protein in C. albicans MTL homozygous strains stimulated opaque cell formation at a frequency of 100%. Furthermore, mating projections of point-mutated strains were significantly shorter and their mating efficiencies and pheromone-stimulated cell adhesive numbers were lower than those of the wild-type. By investigating the effects of Hog1 phosphorylation in ssk1Δ and sln1Δ, we also demonstrate that the phosphorylation intensity of Hog1p is directly involved in the white-opaque switching. Taken together, the results of our study demonstrate that dual phosphorylation sites of C. albicans are crucial for white-opaque transition, sexual mating, and pheromone-induced cell adhesion.

A novel function for Hog1 stress-activated protein kinase in controlling white-opaque switching and mating in Candida albicans.

Candida albicans is a commensal in heathy people but has the potential to become an opportunistic pathogen and is responsible for half of all clinical infections in immunocompromised patients. Central to understanding C. albicans behavior is the white-opaque phenotypic switch, in which cells can undergo an epigenetic transition between the white state and the opaque state. The phenotypic switch regulates multiple properties, including biofilm formation, virulence, mating, and fungus-host interactions. Switching between the white and opaque states is associated with many external stimuli, such as oxidative stress, pH, and N-acetylglucosamine, and is directly regulated by the Wor1 transcriptional circuit. The Hog1 stress-activated protein kinase (SAPK) pathway is recognized as the main pathway for adapting to environmental stress in C. albicans. In this work, we first show that loss of the HOG1 gene in A: / A: and α/α cells, but not A: /α cells, results in 100% white-to-opaque switching when cells are grown on synthetic medium, indicating that switching is repressed by the A1: /α2 heterodimer that represses WOR1 gene expression. Indeed, switching in the hog1Δ strain was dependent on the presence of WOR1, as a hog1Δ wor1Δ strain did not show switching to the opaque state. Deletion of PBS2 and SSK2 also resulted in C. albicans cells switching from white to opaque with 100% efficiency, indicating that the entire Hog1 SAPK pathway is involved in regulating this unique phenotypic transition. Interestingly, all Hog1 pathway mutants also caused defects in shmoo formation and mating efficiencies. Overall, this work reveals a novel role for the Hog1 SAPK pathway in regulating white-opaque switching and sexual behavior in C. albicans.

The effect of chronic lymphocytic thyroiditis on patients with thyroid cancer.

BACKGROUND: The purpose of this study was to investigate the association between chronic lymphocytic thyroiditis (CLT) and malignant tumors of the thyroid. METHODS: A retrospective review of 647 patients who underwent thyroid surgery at the Department of Breast and Thyroid Surgery in Anhui Provincial Hospital, China in 2012 was performed. The clinicopathological characteristics of patients with thyroid malignancies and CLT were collected. CLT was diagnosed by histopathological method. RESULTS: Among 647 patients, 144 patients had thyroid malignancies and 108 patients had been diagnosed with CLT. Moreover, in total, 44 patients had thyroid malignancies coexistent with CLT: forty-one (93.2%) patients had been diagnosed with the papillary thyroid cancer (PTC); two (4.5%) patients suffered from medullary carcinoma; and one (2.3%) patient suffered from lymphoma. The morbidity of thyroid malignancies in patients with CLT was significantly higher than that in patients without CLT (40.7% versus 18.6%; P <0.001). A female preponderance was observed in the patients with CLT compared with those without CLT (P <0.001). There was no statistically significant difference in the tumor size (P = 0.073), multifocality (P = 0.0871), neck lymph node metastasis (P = 0.350), age (P = 0.316), microcarcinoma (P = 0.983) and tumor-node-metastasis (TNM) stage (P = 0.949) between the patients of thyroid malignancies with CLT and without CLT. CONCLUSIONS: Female predominance was observed in patients with CLT. CLT may have no effect on the progression of thyroid malignant tumor. Nevertheless, the influences of CLT on the prognosis of the thyroid carcinoma still need to be investigated with a larger sample size.