Systematic review on efficacy and safety of empirical versus pre-emptive antifungal therapy among children with febrile neutropenia reveals paucity of data.

BACKGROUND: Two approaches are used to manage invasive fungal disease (IFD) in febrile neutropenic patients viz. empirical therapy (without attempting to confirm the diagnosis), or pre-emptive therapy (after screening tests for IFD). OBJECTIVE: This systematic review was undertaken to compare these approaches in children. METHODS: We searched PubMed, EMBASE, Cochrane Library, Scopus, Web of Science, CINAHL, Clinical Trial Registries and grey literature, for randomized controlled trials (RCT) comparing empirical versus pre-emptive antifungal therapy in children with FN suspected to have IFD. We used the Cochrane Risk of bias 2 tool for quality assessment, and evaluated the certainty of evidence using the GRADE approach. RESULTS: We identified 7989 citations. Stepwise screening identified only one relevant RCT that administered empirical (n = 73) or pre-emptive (n = 76) antifungal therapy. There were no significant differences in all-cause mortality (RR 1.56, 95% CI: 0.46, 5.31), IFD mortality (RR 1.04, 95% CI:0.15, 7.20) and other clinically important outcomes such as duration of fever, duration of hospitalization and proportion requiring ICU admission. There were no safety data reported. The number of days of antifungal therapy was significantly lower in the pre-emptive therapy arm. The certainty of evidence for all outcomes was 'moderate'. CONCLUSIONS: This systematic review highlighted the paucity of data, comparing empirical versus pre-emptive antifungal therapy in children with febrile neutropenia having suspected invasive fungal disease. Data from a single included trial suggests that both approaches may be comparable in research settings. Robust trials are warranted to address the gap in existing knowledge about the optimal approach in clinical practice.

Antifungal efficacy of chitosan extracted from shrimp shell on strawberry (Fragaria × ananassa) postharvest spoilage fungi.

The strong demand for biological materials in the food industry places chitosan at the forefront of other biopolymers. The present study aims to evaluate the antifungal properties of chitosan extracted from shrimp shell waste (Parapenaeus longirostris) against post-harvest strawberry (Fragaria × ananassa) spoilage fungi. The physicochemical characteristics (DD, Mw, and solubility) of extracted chitosan were determined. In addition, functional characteristics were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The antifungal effect of chitosan on mycelial growth and spore germination of Aspergillus niger, Botrytis cinerea, Fusarium oxysporum, and Rhizopus stolonifer was evaluated. Yield, degree of deacetylation, molecular weight, and solubility were 21.86%, 83.50%, 180 kDa, and 80.10%, respectively. A degree of deacetylation of 81.27% was calculated from the FTIR spectrum and a crystallinity index of 79.83% was determined from the X-ray diffraction pattern. SEM images of extracted chitosan showed a combination of fibrous and porous structure. At 3% chitosan, mycelial growth inhibition rates of A. niger, B. cinerea, F. oxysporum, and R. stolonifer ranged from 81.37% to 92.70%. At the same chitosan concentration, the percentages of spore germination inhibition of the isolated fungi ranged from 65.47% to 71.48%. The antifungal activity was highly dose-dependent. As a natural polymer, chitosan offers a convincing alternative to synthetic antimicrobials for the post-harvest preservation of strawberries. Its potential lies in its ability to inhibit the growth of spoilage fungi.

Reducing Temperature of Fused Deposition Modelling 3D Printing for Linalool Fast Dissolving Oral Films by Increasing Printer Nozzle Diameter.

Oral thrush and throat infections can occur in a wide range of patients. Treatments are available; however, resistance to drugs is a major problem for treating oral and throat infections. Three-dimensional printing (3DP) of fast dissolving oral films (FDFs) of linalool oil may provide an alternative solution. Linalool oil FDFs were printed by fused deposition modelling across 1-18% w/w linalool content range with nozzle diameters of 0.4 or 1 mm at the temperature range of 150°C-185°C. The FDFs were evaluated for physicochemical and mechanical properties. Increasing the printer nozzle diameter to 1 mm allowed reducing the printing temperature from 185°C to 150°C; consequently, more linalool was quantified in the films with improved content uniformity. The higher linalool content in the films increased the film disintegration time and mechanical strength. FDFs containing 10% w/w linalool showed clear antifungal activity against Candida albicans. Raman spectroscopy suggested linalool separation from excipients at higher temperature printing. Viscoelastic measurements indicated that to achieve printing; the elastic modulus of molten filament needed to be higher than the loss modulus at low angular frequencies. In conclusion, increasing the printing nozzle diameter may avoid loss of the active ingredient by reducing the temperature of the 3DP process.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

A Burkholderia cenocepacia-like environmental isolate strongly inhibits the plant fungal pathogen Zymoseptoria tritici.

Fungal phytopathogens cause significant reductions in agricultural yields annually, and overusing chemical fungicides for their control leads to environmental pollution and the emergence of resistant pathogens. Exploring natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We isolated and characterized a novel bacterial strain associated with the species Burkholderia cenocepacia, termed APO9, which strongly inhibits Zymoseptoria tritici, a commercially important pathogenic fungus causing Septoria tritici blotch in wheat. Additionally, this strain exhibits inhibitory activity against four other phytopathogens. We found that physical contact plays a crucial role for APO9's antagonistic capacity. Genome sequencing of APO9 and biosynthetic gene cluster (BGC) analysis identified nine classes of BGCs and three types of secretion systems (types II, III, and IV), which may be involved in the inhibition of Z. tritici and other pathogens. To identify genes driving APO9's inhibitory activity, we screened a library containing 1,602 transposon mutants and identified five genes whose inactivation reduced inhibition efficiency. One such gene encodes for a diaminopimelate decarboxylase located in a terpenoid biosynthesis gene cluster. Phylogenetic analysis revealed that while some of these genes are also found across the Burkholderia genus, as well as in other Betaproteobacteria, the combination of these genes is unique to the Burkholderia cepacia complex. These findings suggest that the inhibitory capacity of APO9 is complex and not limited to a single mechanism, and may play a role in the interaction between various Burkholderia species and various phytopathogens within diverse plant ecosystems. IMPORTANCE: The detrimental effects of fungal pathogens on crop yields are substantial. The overuse of chemical fungicides contributes not only to environmental pollution but also to the emergence of resistant pathogens. Investigating natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We discovered and examined a unique bacterial strain that demonstrates significant inhibitory activity against several phytopathogens. Our research demonstrates that this strain has a wide spectrum of inhibitory actions against plant pathogens, functioning through a complex mechanism. This plays a vital role in the interactions between plant microbiota and phytopathogens.

Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Candida parapsilosis has recently emerged as a major threat due to the worldwide emergence of fluconazole-resistant strains causing clonal outbreaks in hospitals and poses a therapeutic challenge due to the limited antifungal armamentarium. Here, we used precise genome editing using CRISPR-Cas9 to gain further insights into the contribution of mutations in ERG11, ERG3, MRR1, and TAC1 genes and the influence of allelic dosage to antifungal resistance in C. parapsilosis. Seven of the most common amino acid substitutions previously reported in fluconazole-resistant clinical isolates (including Y132F in ERG11) were engineered in two fluconazole-susceptible C. parapsilosis lineages (ATCC 22019 and STZ5). Each mutant was then challenged in vitro against a large array of antifungals, with a focus on azoles. Any possible change in virulence was also assessed in a Galleria mellonella model. We successfully generated a total of 19 different mutants, using CRISPR-Cas9. Except for R398I (ERG11), all remaining amino acid substitutions conferred reduced susceptibility to fluconazole. However, the impact on fluconazole in vitro susceptibility varied greatly according to the engineered mutation, the stronger impact being noted for G583R acting as a gain-of-function mutation in MRR1. Cross-resistance with newer azoles, non-medical azoles, but also non-azole antifungals such as flucytosine, was occasionally noted. Posaconazole and isavuconazole remained the most active in vitro. Except for G583R, no fitness cost was associated with the acquisition of fluconazole resistance. We highlight the distinct contributions of amino acid substitutions in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in C. parapsilosis.

Design, synthesis, antifungal evaluation and mechanism study of novel norbornene derivatives as potential laccase inhibitors.

BACKGROUND: To discover novel fungicide candidates, five series of novel norbornene hydrazide, bishydrazide, oxadiazole, carboxamide and acylthiourea derivatives (2a-2 t, 3a-3 f, 4a-4 f, 5a-5 f and 7a-7 f) were designed, synthesized and assayed for their antifungal activity toward seven representative plant fungal pathogens. RESULTS: In the in vitro antifungal assay, some title norbornene derivatives presented good antifungal activity against Botryosphaeria dothidea, Sclerotinia sclerotiorum and Fusarium graminearum. Especially, compound 2b exhibited the best inhibitory activity toward B. dothidea with the median effective concentration (EC50) of 0.17 mg/L, substantially stronger than those of the reference fungicides boscalid and carbendazim. The in vivo antifungal assay on apples revealed that 2b had significant curative and protective effects, both of which were superior to boscalid. In the preliminary antifungal mechanism study, 2b was able to injure the surface morphology of hyphae, destroy the cell membrane integrity and increase the intracellular reactive oxygen species (ROS) level of B. dothidea. In addition, 2b could considerably inhibit the laccase activity with the median inhibitory concentration (IC50) of 1.02 µM, much stronger than that of positive control cysteine (IC50 = 35.50 µM). The binding affinity and interaction mode of 2b with laccase were also confirmed by molecular docking. CONCLUSION: This study presented a promising lead compound for the study on novel laccase inhibitors as fungicidal agrochemicals, which demonstrate significant anti-B. dothidea activity and laccase inhibitory activity. This article is protected by copyright. All rights reserved.

The Emergence of Candida auris is Not Associated with Changes in Antifungal Prescription at Hospitals.

Purpose: This study describes the emergence of Candida auris in Hong Kong, focusing on the incidence and trends of different Candida species over time. Additionally, the study analyzes the relationship between C. auris and antifungal prescription, as well as the impact of outbreaks caused by C. auris. Patients and Methods: Data were collected from 43 public hospitals across seven healthcare networks (A to G) in Hong Kong, including Candida species culture and antifungal prescription information. Among 150,267 patients with 206,405 hospitalization episodes, 371,653 specimens tested positive for Candida species. Trends in Candida species and antifungal prescription were analyzed before (period 1: 2015 1Q to 2019 1Q) and after (period 2: 2019 2Q to 2023 2Q) the emergence of C. auris in Hong Kong. Results: Candida albicans was the most prevalent species, accounting for 57.1% (212,163/371,653) of isolations, followed by Candida glabrata (13.1%, 48,666), Candida tropicalis (9.2%, 34,261), and Candida parapsilosis (5.3%, 19,688). C. auris represented 2.0% of all Candida species isolations. Comparing period 2 to period 1, the trend of C. albicans remained stable, while C. glabrata, C. tropicalis, and C. parapsilosis demonstrated a slower increasing trend in period 2 than in period 1. Other species, including C. auris, exhibited a 1.1% faster increase in trend during period 2 compared to period 1. Network A, with the highest antifungal prescription, did not experience any outbreaks, while networks F and G had 40 hospital outbreaks due to C. auris in period 2. Throughout the study period, healthcare networks B to G had significantly lower antifungal prescription compared to network A, ranging from 54% to 78% less than that of network A. Conclusion: There is no evidence showing correlation between the emergence of C. auris and antifungal prescription in Hong Kong. Proactive infection control measures should be implemented to prevent nosocomial transmission and outbreak of C. auris.

Genotypic and phenotypic characteristics of Candida parapsilosis bloodstream isolates: Health Care Associated Infections in a teaching Hospital in Italy.

BACKGROUND: Candidemia is the most common healthcare associated invasive fungal infection. Over the last few decades, candidemia caused by Candida species other than Candida albicans, particularly the Candida parapsilosis complex, has emerged worldwide. The aims of this study were: to analyze the genotypic and phenotypic characteristics of C. parapsilosis strains isolated from blood cultures and the environment in a hospital in southern Italy, to study the possible source of infection and to correlate the isolated strains. METHODS: From April to October 2022, cases of candidemia due to C. parapsilosis in patients admitted to a hospital in the Apulia region were investigated. However, 119 environmental samples from the intensive care unit were collected for identification of the likely environmental reservoir of infection. Routine antifungal (amphotericin B, anidulafungin, fluconazole) susceptibility was performed on all isolates. Whole genome sequencing was performed to study the genotypic correlation of the isolates. Biofilm biomass and metabolic activity were also quantified for all isolates. RESULTS: A total of 43 C. parapsilosis isolates were cultured from the bloodstream of each patient in different departments, and seven surface samples were positive for C. parapsilosis. Most of the isolated yeasts (41/50; 85 %) were resistant to fluconazole and were genetically related to each other, suggesting an ongoing clonal outbreak of this pathogen. The fluconazole-susceptible isolates produced significantly more biofilm than did the resistant isolates. Metabolic activity was also higher for fluconazole-susceptible than resistant isolates. CONCLUSION: Cross-transmission of the microorganisms is suggested by the phenotypic similarity and genetic correlation between clinical and environmental strains observed in our study.

Preparing and evaluating the anti-microbial effect of Allium jesdianum mouthwash on some of the most common oral microorganisms.

Background: Due to the increasing resistance of bacteria to antibiotics and anti-bacterial compounds in plants, Allium jesdianum Boiss plant extract can be used in mouthwash compounds with its anti-microbial activity. Methods and Materials: The anti-bacterial and anti-fungal activity of A. jesdianum mouthwash was investigated on Streptococcus mutans, Streptococcus sanguis, S. salivarius and Candida albicans, and Candida tropicalis. To analyse the anti-microbial effect of this mouthwash, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution method. Results: The average MIC and MBC of A. jesdianum mouthwash for S. mutans were 1.56 and 3.12 (mg/ml), respectively, for S. salivarius, 0.25 and 0.65 (mg/ml), and for S. sanguis, respectively, 0.25 and 0.65 (mg/ml). The highest MIC and MBC values were for S. mutans, and the MIC and MBC values were equal for S. sanguis and S. salivarius. Average MIC and MBC were determined as 2.41 and 4.16 (mg/ml) for C. albicans and 2.34 and 5.72 (mg/ml) for C. tropicalis, respectively. MIC values of mouthwash were higher for C. albicans and MBC values for C. tropicalis. Conclusion: Our results showed a promising anti-fungal-anti-bacterial effect of A. jesdianum extract. A. jesdianum extract may be used as an alternative to chemical mouthwashes.

The discovery of an anti-Candida xanthone with selective inhibition of Candida albicans GAPDH.

OBJECTIVES: This study aimed to discover novel antifungals targeting Candida albicans glyceraldehyde-3-phosphate dehydrogenase (CaGAPDH), have an insight into inhibitory mode, and provide evidence supporting CaGAPDH as a target for new antifungals. METHODS: Virtual screening was utilized to discover inhibitors of CaGAPDH. The inhibitory effect on cellular GAPDH was evaluated by determining the levels of ATP, NAD, NADH, etc., as well as examining GAPDH mRNA and protein expression. The role of GAPDH inhibition in C. albicans was supported by drug affinity responsive target stability and overexpression experiments. The mechanism of CaGAPDH inhibition was elucidated by Michaelis-Menten enzyme kinetics and site-specific mutagenesis based on docking. Chemical synthesis was used to produce an improved candidate. Different sources of GAPDH were used to evaluate inhibitory selectivity across species. In vitro and in vivo antifungal tests, along with antibiofilm activity, were carried out to evaluate antifungal potential of GAPDH inhibitors. RESULTS: A natural xanthone was identified as the first competitive inhibitor of CaGAPDH. It demonstrated in vitro anti-C. albicans potential but also caused hemolysis. XP-W, a synthetic side-chain-optimized xanthone, demonstrated a better safety profile, exhibiting a 50-fold selectivity for CaGAPDH over human GAPDH. XP-W also exhibited potent antibiofilm activity and displayed broad-spectrum anti-Candida activities in vitro and in vivo, including multi-azole-resistant C. albicans. CONCLUSION: These results demonstrate for the first time that CaGAPDH is a valuable target for antifungal drug discovery, and XP-W provides a promising lead.

An immunocompetent host with blood-disseminated Aspergillus versicolor spondylitis: a case report and literature review.

Blood-disseminated Aspergillus spondylitis in immunocompetent individuals is rare. The clinical, imaging, and pathological manifestations of this condition are not specific. Therefore, this disease is prone to misdiagnosis and a missed diagnosis. Systemic antifungal therapy is the main treatment for Aspergillus spondylitis. We report a case of blood-disseminated Aspergillus versicolor spondylitis in a patient with normal immune function. The first antifungal treatment lasted for 4 months, but Aspergillus spondylitis recurred a few months later. A second antifungal treatment course was initiated for at least 1 year, and follow-up has been ongoing. Currently, there has been no recurrence.

Comprehensive risk factor predictions for 3-year survival among HIV-associated and disseminated cryptococcosis involving lungs and central nervous system.

BACKGROUND: The global mortality rate resulting from HIV-associated cryptococcal disease is remarkably elevated, particularly in severe cases with dissemination to the lungs and central nervous system (CNS). Regrettably, there is a dearth of predictive analysis regarding long-term survival, and few studies have conducted longitudinal follow-up assessments for comparing anti-HIV and antifungal treatments. METHODS: A cohort of 83 patients with HIV-related disseminated cryptococcosis involving the lung and CNS was studied for 3 years to examine survival. Comparative analysis of clinical and immunological parameters was performed between deceased and surviving individuals. Subsequently, multivariate Cox regression models were utilized to validate mortality predictions at 12, 24, and 36 months. RESULTS: Observed plasma cytokine levels before treatment were significantly lower for IL-1RA (p < 0.001) and MCP-1 (p < 0.05) when in the survivor group. Incorporating plasma levels of IL-1RA, IL-6, and high-risk CURB-65 score demonstrated the highest area under curve (AUC) value (0.96) for predicting 1-year mortality. For 1-, 2- and 3-year predictions, the single-factor model with IL-1RA demonstrated superior performance compared to all multiple-variate models (AUC = 0.95/0.78/0.78). CONCLUSIONS: IL-1RA is a biomarker for predicting 3-year survival. Further investigations to explore the pathogenetic role of IL-1RA in HIV-associated disseminated cryptococcosis and as a potential therapeutic target are warranted.

COVID-19-associated pulmonary aspergillosis (CAPA) in hematological patients: Could antifungal prophylaxis be necessary? A nationwide study.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) has emerged as a relatively common complication. Multiple studies described this relationship in critical patients, however its incidence and outcome in other risk groups such as immunosuppressed patients remains unknown. In this sense, we aimed to evaluate the rates and outcomes of CAPA in hematological patients and according to the different hematological malignances, comparing to invasive pulmonary aspergillosis (IPA) in non-COVID-19 ones. METHODS: Nationwide, population-based and retrospective observational cohort study including all adult patients with hematological malignancies admitted in Spain since March 1, 2020 to December 31, 2021. The main outcome variable was the diagnosis of IPA during hospitalization in hematological patients with or without COVID-19 at admission. The rate of CAPA compared to IPA in non-COVID-19 patients in each hematological malignancy was also performed, as well as survival curve analysis. FINDINGS: COVID-19 was diagnosed in 3.85 % (4367 out of 113,525) of the hematological adult inpatients. COVID-19 group developed more fungal infections (5.1 % vs. 3 %; p < 0.001). Candida spp. showed higher rate in non-COVID-19 (74.2 % vs. 66.8 %; p = 0.015), meanwhile Aspergillus spp. confirmed its predominance in COVID-19 hematological patients (35.4 % vs. 19.1 %; p < 0.001). IPA was diagnosed in 703 patients and 11.2 % (79 cases) were CAPA. The multivariate logistic regression analysis found that the diagnosis of COVID-19 disease at hospital admission increased more than two-fold IPA development [OR: 2.5, 95CI (1.9-3.1), p < 0.001]. B-cell malignancies - specifically B-cell non-Hodgkin lymphoma, multiple myeloma, chronic lymphocytic leukemia and acute lymphoblastic leukemia - showed between four- and six-fold higher CAPA development and 90-day mortality rates ranging between 50 % and 72 %. However, myeloid malignancies did not show higher CAPA rates compared to IPA in non-COVID-19 patients. CONCLUSION: COVID-19 constitutes an independent risk factor for developing aspergillosis in B-cell hematological malignancies and the use of antifungal prophylaxis during hospitalizations may be warranted.

The dynamic changes in olive fruit phenolic metabolism and its contribution to the activation of quiescent Colletotrichum infection.

Anthracnose, the most critical disease affecting olive fruits, is caused by Colletotrichum species. While developing olive fruits are immune to the pathogen regardless of the cultivar, the resistance level varies once the fruit ripens. The defense mechanisms responsible for this difference in resistance are not well understood. To explore this, we analyzed the phenolic metabolic pathways occurring in olive fruits and their susceptibility to the pathogen during ripening in two resistant cultivars ('Empeltre' and 'Frantoio') and two susceptible cultivars ('Hojiblanca' and 'Picudo'). Overall, resistant cultivars induced the synthesis of aldehydic and demethylated forms of phenols, which highly inhibited fungal spore germination. In contrast, susceptible cultivars promoted the synthesis of hydroxytyrosol 4-O-glucoside during ripening, a compound with no antifungal effect. This study showed that the distinct phenolic profiles between resistant and susceptible cultivars play a key role in determining olive fruit resistance to Colletotrichum species.

Influence of flavonoids from Sedum aizoon L. on mitochondrial function of Rhizopus nigricans in strawberry.

Rhizopus nigricans (R. nigricans), one of the fungi that grows the fastest, is frequently discovered in postharvest fruits, it's the main pathogen of strawberry root rot. Flavonoids in Sedum aizoon L. (FSAL) is a kind of green and safe natural substance extracted from Sedum aizoon L. which has antifungal activity. In this study, the minimum inhibitory concentration (MIC) of FSAL on R. nigricans and cell apoptosis tests were studied to explore the inhibitory effect of FSAL on R. nigricans. The effects of FSAL on mitochondria of R. nigricans were investigated through the changes of mitochondrial permeability transition pore(mPTP), mitochondrial membrane potential(MMP), Ca2+ content, H2O2 content, cytochrome c (Cyt c) content, the related enzyme activity and related genes of mitochondria. The results showed that the MIC of FSAL on R. nigricans was 1.800 mg/mL, with the addition of FSAL (1.800 mg/mL), the mPTP openness of R. nigricans increased and the MMP reduced. Resulting in an increase in Ca2+ content, accumulation of H2O2 content and decrease of Cyt c content, the activity of related enzymes was inhibited and related genes were up-regulated (VDAC1, ANT) or down-regulated (SDHA, NOX2). This suggests that FSAL may achieve the inhibitory effect of fungi by damaging mitochondria, thereby realizing the postharvest freshness preservation of strawberries. This lays the foundation for the development of a new plant-derived antimicrobial agent.

Synthesis and Antifungal Activity of Norbornene Carboxamide/sulfonamide Derivatives as Potential Fungicides Targeting Laccase.

To explore more potential fungicides with new scaffolds, thirty-seven norbornene carboxamide/sulfonamide derivatives were designed, synthesized, and assayed for inhibitory activity against six plant pathogenic fungi and oomycetes. The preliminary antifungal assay suggested that the title derivatives showed moderate to good antifungal activity against six plant pathogens. Especially, compound 6e presented excellent in vitro antifungal activity against Sclerotinia sclerotiorum (EC50 = 0.71 mg/L), which was substantially stronger than pydiflumetofen. In vivo antifungal assay indicated 6e displayed prominent protective and curative effects on rape leaves infected by S. sclerotiorum. The preliminary mechanism research displayed that 6e could damage the surface morphology and inhibit the sclerotia formation of S. sclerotiorum. In addition, the in vitro enzyme inhibition bioassay indicated that 6e displayed pronounced laccase inhibition activity (IC50 = 0.63 µM), much stronger than positive control cysteine. Molecular docking elucidated the binding modes between 6e and laccase. The bioassay results and mechanism investigation demonstrated that this class of norbornene carboxamide/sulfonamide derivatives could be promising laccase inhibitors for novel fungicide development.

Design, Synthesis, Antifungal Activity, and Molecular Docking Studies of Novel Chiral Isoxazoline-Benzofuran-Sulfonamide Derivatives.

Succinate dehydrogenase (SDH) is one of the most important molecular targets for the development of novel fungicides. With the emerging problem of resistance in plant fungal pathogens, novel compounds with high fungicidal activity need to be developed, but the study of chiral pesticides for the inhibition of highly destructive plant pathogens has been rarely reported in recent years. Therefore, a series of novel chiral isoxazoline-benzofuran-sulfonamide derivatives were designed to investigate potential novel antifungal molecules. The chiral target compound 3a was cultured as a single crystal and confirmed using X-ray diffraction. All the target compounds were tested for antifungal activity, and compounds 3c, 3i, 3s, and 3r were found to have significant antifungal effects against S. sclerotiorum with EC50 values of 0.42 mg/L, 0.33 mg/L, 0.37 mg/L, and 0.40 mg/L, respectively, which were superior to the commercial fungicide fluopyram (EC50 = 0.47 mg/L). The IC50 value of compound 3i against the SDH of S. sclerotiorum was 0.63 mg/mL, which was further demonstrated by enzyme activity assays. Scanning electron microscopy showed that 3i had a significant inhibitory effect on S. sclerotiorum. In addition, the fluorescence quenching analysis assay indicated that compound 3i had a similar effect with the positive control fluopyram. Molecular docking exhibited that target compounds with chiral configuration had better affinity than racemic configuration, and 3i possessed stronger action than fluopyram, which was in keeping with the in vitro test results. These results would provide a basis and reference for the development of novel chiral fungicides.

Deciphering antifungal and antibiofilm mechanisms of isobavachalcone against Cryptococcus neoformans through RNA-seq and functional analyses.

Cryptococcus neoformans has been designated as critical fungal pathogens by the World Health Organization, mainly due to limited treatment options and the prevalence of antifungal resistance. Consequently, the utilization of novel antifungal agents is crucial for the effective treatment of C. neoformans infections. This study exposed that the minimum inhibitory concentration (MIC) of isobavachalcone (IBC) against C. neoformans H99 was 8 µg/mL, and IBC dispersed 48-h mature biofilms by affecting cell viability at 16 µg/mL. The antifungal efficacy of IBC was further validated through microscopic observations using specific dyes and in vitro assays, which confirmed the disruption of cell wall/membrane integrity. RNA-Seq analysis was employed to decipher the effect of IBC on the C. neoformans H99 transcriptomic profiles. Real-time quantitative reverse transcription PCR (RT-qPCR) analysis was performed to validate the transcriptomic data and identify the differentially expressed genes. The results showed that IBC exhibited various mechanisms to impede the growth, biofilm formation, and virulence of C. neoformans H99 by modulating multiple dysregulated pathways related to cell wall/membrane, drug resistance, apoptosis, and mitochondrial homeostasis. The transcriptomic findings were corroborated by the antioxidant analyses, antifungal drug sensitivity, molecular docking, capsule, and melanin assays. In vivo antifungal activity analysis demonstrated that IBC extended the lifespan of C. neoformans-infected Caenorhabditis elegans. Overall, the current study unveiled that IBC targeted multiple pathways simultaneously to inhibit growth significantly, biofilm formation, and virulence, as well as to disperse mature biofilms of C. neoformans H99 and induce cell death.