Azole resistance in Aspergillus fumigatus, a major cause of invasive aspergillosis, threatens public health. Known drivers include cyp51A/B mutations (e.g., TR34/L98H and TR46/Y121F/T289A), yet existing studies have largely focused on clinical isolates and known genetic determinants, leaving gaps in understanding broader genomic contributions. This study aimed to develop a machine learning–based framework to overcome limitations of traditional GWAS and identify novel resistance loci beyond cyp51A. A global collection of 590 A. fumigatus strains was analyzed, including whole-genome sequencing (WGS) data from 15 countries and resistance phenotypes using CLSI/EUCAST guidelines. Phylogenetic analysis revealed four clades without geographic clustering. Clade III harbored the highest proportion of resistant strains (ITR: 51.89%, POS: 50.48%, VOR: 38.68%), predominantly linked to cyp51A tandem repeats. In contrast, Clade IV strains frequently carried point mutations but showed lower resistance rates. GWAS was performed using PLINK and GAPIT frameworks, and 7,098 high confidence SNPs were selected for ML modeling. Ten classifiers were evaluated using repeated random 80:20 train-test splits, with five-fold cross-validation used for RFECV-based feature selection and model tuning where applicable. RF and XGBoost achieved superior performance, with mean AUCs > 95% and accuracy > 88% across all azoles. Penalized logistic regression outperformed SVM and AdaBoost. Decision trees exhibited the lowest accuracy. The SNP SCM000172.1_1781459 was identified as a key predictor for all three azoles. Cross-resistance analysis revealed significant overlap between ITR and POS resistance loci, whereas VOR-associated loci were distinct, suggesting divergent mechanisms. The findings provide actionable insights for resistance surveillance, antifungal development, and tailored treatment strategies.

Aspergillus fumigatus is a major opportunistic fungal pathogen, and increasing azole resistance poses a challenge for aspergillosis treatment. Squalene is an upstream precursor of ergosterol biosynthesis and may also be utilized by SHC-like triterpene cyclases, suggesting a potential link between squalene-associated metabolism, membrane adaptation, and azole response. However, the roles of SHC-like triterpene cyclase genes in A. fumigatus remain unclear. Here, we characterized three candidates, shc1, shc2, and shc3, using comparative bioinformatic analysis, gene deletion, phenotypic assays, azole susceptibility testing, transcriptomics, and host-interaction models. Sequence, genomic-context, phylogenetic, and structural analyses suggested divergence among the three candidates. Individual shc deletion caused limited effects on vegetative growth, whereas loss of shc1 mildly reduced susceptibility to voriconazole and posaconazole, as reflected by twofold MIC increases and lower inhibition rates. Transcriptomic analysis revealed distinct remodeling patterns, with Δshc3 showing the broadest transcriptional changes despite no detectable MIC shift. Targeted metabolite profiling and PI uptake analysis further supported an association between shc deletion, sterol/hopane-type triterpenoid balance, and membrane-associated properties. shc deletion also altered epithelial cell interaction phenotypes, while Δshc1 showed reduced lethality in Galleria mellonella. In clinical isolates, elevated shc transcription was associated with azole-resistant backgrounds. These findings suggest functional diversification among SHC-like triterpene cyclase genes and indicate that shc1 may contribute to azole-associated adaptation and virulence-related traits in A. fumigatus.

IntroductionAspergillus fumigatus is a major airborne fungal pathogen that causes invasive aspergillosis in immunocompromised individuals. This study aims to elucidate the autophagic mechanisms activated following the internalization of A. fumigatus conidia in human bronchial epithelial cells.MethodsSpecifically, we investigated the role of ULK1 and the autophagic processes in Beas2B cells upon A. fumigatus conidia internalization. The Beas2B cell line was used to assess the protein expression of ULK1, phosphorylated ULK1, and LC3-I/II via Western blotting. Autophagosome structures were examined using transmission electron microscopy. Gene silencing of ULK1 using siRNA and pharmacological inhibition with SBI-0206965 were performed. Secreted inflammatory cytokines were quantified using specific immunoassays.ResultsA. fumigatus conidia induced a time- and dose-dependent conversion of LC3-I to LC3-II, indicating autophagic activation resembling LC3-associated phagocytosis (LAP). ULK1 expression significantly increased post-infection, whereas genetic silencing of ULK1 reduced LC3-II conversion. Notably, common fungal polysaccharides and Dectin-1 did not influence this process, but the loss of complement receptor 3 (CR3) elevated both basal and conidia-induced autophagy, correlating with increased AMPK expression.DiscussionThis study reveals a novel ULK1-dependent autophagic response similar to LAP during A. fumigatus internalization, highlighting potential therapeutic targets for managing invasive aspergillosis in immunocompromised patients.

As a pivotal secondary metabolite of Aspergillus fumigatus, gliotoxin (GT) has many toxicological effects on mammalian cells; however, its function on LC3-associated phagocytosis (LAP) induced by A. fumigatus in macrophages is poorly understood. Here, it was found that pretreatment of macrophages with GT can significantly attenuate the conversion of LC3-II. In parallel, the expression of Rubicon, the putative indicator of LAP in macrophages, was dampened in a similar trend. Loss of ability to produce GT made the conidia of gliPΔ mutant of A. fumigatus induce more LC3-II conversion in THP1 cells, which could be inhibited by exogenous GT. Comparative transcriptomic analysis showed that GT can promote the expression of MAPK10, and the calcium-release regulatory pathway was enriched between the differentially expressed genes. Further, GT promotes the release of ROS at a concentration of 50 ng/mL, and inhibition by GT on LC3-II production in macrophages during A. fumigatus infection could be restored by pretreatment with calcium inhibitors but not affected by the inhibitors for JNK and siRNA for MAKP10. Collectively, our data demonstrated that GT exerts an inhibitory effect on LC3-associated phagocytosis in macrophages via a calcium-dependent mechanism, and MAPK10 exists downstream of LAP.

OBJECTIVES:To establish and validate a vitamin-related gene signature for predicting glioma prognosis and immune characteristics, and to explore the functional relevance of EEF2. METHODS:Survival-related vitamin-associated genes were identified from public glioma cohorts to build a prognostic signature. Its clinical relevance, immune associations, and predictive performance were validated. EEF2 function was tested in glioma cells, and its relationship with YPEL1 was explored. RESULTS:A six-gene signature (POSTN, IRX5, EEF2, RAB27A, MDM2, ENO1) independently predicted overall survival and distinguished glioma grade and molecular subtype across cohorts. High-risk gliomas showed elevated immune and stromal scores, reduced tumor purity, enriched immune-cell infiltration, C4 immune-subtype dominance, and pronounced immunosuppressive features comprising upregulated Cancer-Immunity Cycle inhibitors and immune checkpoints. A nomogram integrating the signature with clinicopathological factors accurately predicted 1-, 3-, and 5-year survival. Functionally, EEF2 knockdown promoted glioma cell proliferation, migration, and invasion, and positively regulated YPEL1, supporting its protective role. CONCLUSIONS:This vitamin-related signature links vitamin biology with glioma immunity and prognosis, and EEF2 represents a promising direction for future mechanistic and therapeutic research.

Introduction:Lung adenocarcinoma (LUAD), the most common subtype of non-small cell lung cancer, is a form of malignant pulmonary nodule that requires clinical differentiation from benign pulmonary nodules (BPN). The mechanisms underlying the development of LUAD are complex, and effective non-invasive methods for differentiating BPN from LUAD are lacking. This study aimed not only to distinguish BPN from LUAD using gut fungi and serum metabolites, but also to establish an integrated network of gut fungi-metabolite-cytokine interactions. Methods:Fecal and serum samples from individuals with BPN and patients with LUAD were subjected to internal transcribed spacer sequencing, ultra-performance liquid chromatography-tandem mass spectrometry, and multiplex Luminex assays to quantify gut fungi, metabolites, and cytokines, respectively. Results:A significant difference in gut fungal communities was observed between the BPN and LUAD groups. Multiple genera and species were more abundant in LUAD than in BPN. Docosapentaenoic acid n-6 (DPAn-6), indole-3-propionic acid (IPA), and interferon-γ-induced protein 10 (IP-10) were significantly elevated in the LUAD group. The integrated model established using a combination of gut fungi and metabolites demonstrated excellent performance in distinguishing BPN from LUAD. A network of interactions was established among differentially abundant gut fungi, serum metabolites, and cytokines. Conclusion:Our study identifies a novel panel of fungal and metabolite biomarkers for differentiating between BPN and LUAD, and constructs a multi-omics network that provides new insights into investigating the mechanistic role of gut mycobiota dysbiosis in LUAD.

Pterygium is a prevalent ocular disease characterized by abnormal conjunctival tissue proliferation, significantly impacting patients’ quality of life. However, the underlying molecular mechanisms driving pterygium pathogenesis remain inadequately understood. This study aimed to investigate gene expression changes following pterygium excision and their association with immune cell infiltration. Clinical samples of pterygium and adjacent relaxed conjunctival tissue were collected for transcriptomic analysis using RNA sequencing combined with bioinformatics approaches. Machine learning algorithms, including LASSO, SVM-RFE, and Random Forest, were employed to identify potential diagnostic biomarkers. GO, KEGG, GSEA, and GSVA were utilized for enrichment analysis. Single-sample GSEA was employed to analyze immune infiltration. The GSE2513 and GSE51995 datasets from the GEO database, along with clinical samples, were selected for validation analysis. Differentially expressed genes (DEGs) were identified from the PRJNA1147595 and GSE2513 datasets, revealing 2437 DEGs and 172 differentially regulated genes (DRGs), respectively. There were 52 co-DEGs shared by both datasets, and four candidate biomarkers (FN1, SPRR1B, SERPINB13, EGR2) with potential diagnostic value were identified through machine learning algorithms. Single-sample GSEA demonstrated increased Th2 cell infiltration and decreased CD8 + T cell presence in pterygium tissues, suggesting a crucial role of the immune microenvironment in pterygium pathogenesis. Analysis of the GSE51995 dataset and qPCR results revealed significantly higher expression levels of FN1 and SPRR1B in pterygium tissues compared to conjunctival tissues, but SERPINB13 and EGR2 expression levels were not statistically significant. Furthermore, we identified four candidate drugs targeting the two feature genes FN1 and SPRR1B. This study provides valuable insights into the molecular characteristics and immune microenvironment of pterygium. The identification of potential biomarkers FN1 and SPRR1B highlights their significance in pterygium pathogenesis and lays a foundation for further exploration aimed at integrating these findings into clinical practice.

Filamentous fungi present significant health hazards to immunocompromised individuals globally; however, the prompt and precise identification of them during infection remains challenging. In this study, a TaqMan probe-based multiplex real-time PCR (M-qPCR) assay was developed to detect simultaneously the target genes of four important pathogenic filamentous fungi: ANXC4 gene of Aspergillus fumigatus, EF1-α gene of Fusarium spp., mitochondrial rnl gene of Mucorales, and hcp100 gene of Histoplasma capsulatum. In this M-qPCR assay, the limit of detection (LoD) to all four kinds of fungi was 100 copies and the correlation coefficients (R2) were above 0.99. The specificity of this assay is 100%, and the minimum detection limit is 100 copies/reaction. In conclusion, an M-qPCR detection assay was well established with high specificity and sensitivity for rapid and simultaneous detection on four important filamentous fungi in the clinic. IMPORTANCE World Health Organization developed the first fungal priority pathogens list (WHO FPPL) in 2022. Aspergillus fumigatus, Mucorales, Fusarium spp., and Histoplasma spp. are the four types of pathogenic fungi with filamentous morphology in the critical priority group and high priority group of WHO FPPL. These four filamentous fungal infections have become more common and severe in immunocompromised patients with the increase in susceptible populations in recent decades, which resulted in a substantial burden on the public health system. However, prompt and precise identification of them during infection remains challenging. Our study established successfully a TaqMan probe-based multiplex real-time qPCR assay for four clinically important filamentous fungi, A. fumigatus, Fusarium spp., Mucorales, and Histoplasma capsulatum, with high sensitivity and specificity, which shows promising potential for prompt and precise diagnosis against fungal infection.

毛霉菌病是一类由毛霉目真菌感染引起的侵袭性疾病,常见于免疫功能低下患者,人群发病率每年可达到 1.2/100 万人,病死率为 40%～80%.自印度第二波新冠病毒感染疫情大流行起,世界范围内新冠相关毛霉菌病的发病率明显增加.人类主要通过吸入空气中的毛霉孢子而感染,偶尔通过摄入受污染的食物或创伤性创面接触而感染.增加毛霉菌病发病风险的重要因素包括血液系统恶性肿瘤、糖尿病、造血干细胞移植或实体器官移植受者等.临床类型主要分为鼻脑型、肺型、皮肤型、胃肠型和播散型.毛霉菌病诊断方法有影像学检查、组织病理学、真菌培养、聚合酶链式反应等.预防毛霉菌感染的主要措施包括治疗和管理、医院环境监测、个人安全维护方面,应定期监测血糖水平、避免可能造成外伤的活动、避免经空气和经皮肤传播毛霉孢子、保持个人卫生等.早期诊断、纠正潜在的诱发因素、感染组织的手术清创和适当的抗真菌治疗对于管理毛霉菌病至关重要.通过控制糖尿病、减少免疫抑制剂用量、纠正酸中毒、恢复中性粒细胞、早期手术干预以及两性霉素 B给药等措施,患者可能会有更好的预后.

Objective: Aspergillus fumigatus infection in the lungs is accompanied by the recruitment of innate immune cells, phagocytosis, and the release of inflammatory factors. Phospholipase D (PLD) is a key regulator of cell migration and phagocytosis, but the effect of PLD deficiency on antifungal infection in animals is unknown. This study aims to investigate the impact of PLD on the host immune response to A. fumigatus infection under either immunocompetent or immunosuppressed status. Methods: The invasive pulmonary aspergillosis mouse model was created using a modified protocol with immunosuppression by steroids. For collection of bronchoalveolar lavage fluid (BALF) from mice, the lungs were washed eight times with 0.5 ml of PBS. Total cell counts in BALF were determined using a Coulter Counter. The content of alveolar macrophages, neutrophils, and monocytes in BALF was examined by flow cytometry and analyzed by FlowJo V10 software. Multiplex immunoassays were used to determine the concentrations of inflammatory cytokines in BALF. Results: In immunocompetent mice, alveolar macrophages were the major cell population in BALF after A. fumigatus infection, and a number of neutrophils and monocytes were recruited in the alveoli. Loss of both pld1 and pld2 genes did not affect the content of alveolar macrophages, neutrophils, or monocytes in BALF. Under immunosuppression induced by hydrocortisone acetate, pld1-/-pld2-/- mice showed higher mortality after A. fumigatus infection and had a higher fungal burden and much lower number of prominent focal areas of dense inflammatory infiltrates in lung tissue than wild type mice. Moreover, interleukin (IL)-12p40 significantly decreased, and IL-10 markedly increased, in BALF from pld1 -/- pld2 -/- mice after infection. Conclusion: Our findings revealed that, during A. fumigatus infection, deficiency in both pld1 and pld2 in mice was not conducive to the infiltration of inflammatory cells into lung tissue but promoted the release of IL-10 and blocked the release of IL-12, thereby increasing fungal burden and mortality.

烟曲霉(Aspergillus fumigatus)是一种分布于世界各地的腐生真菌,属于人类临床常见的三大机会性致病真菌之一,是侵袭性曲霉菌病的主要病原菌.烟曲霉可以产生 DHN-黑色素(dihydroxynaphthalene melanin)和脓黑素(pyomelanin)这 2 种类型黑色素.本综述介绍烟曲霉黑色素产生的遗传代谢途径、功能以及与宿主免疫系统相互作用的最新认识,有助于更好地理解烟曲霉的病理生理特征,为烟曲霉感染快速诊断技术和新型抗真菌药物的研发提供理论依据.

Aspergillus fumigatus ( A. fumigatus ) is an important fungal pathogen and its conidia can be inhaled and interact with airway epithelial cells; however, the release of inflammatory factors from bronchial epithelial cells upon A. fumigatus infection and its regulation remained unclear. Here it was demonstrated that the release of IL-27, MCP-1 and TNF-α from BEAS-2B cells were upregulated upon stimulation by conidia, while mitogen-activated protein kinase signaling pathway was activated. Further, the inhibition of JNK, but not p38 and ERK, could inhibit inflammatory factors release and the LC3II formation in BEAS-2B cells induced by A. fumigatus conidia. In addition, an inhibitor of autophagy, bafilomycin A1 was able to significantly down-regulate the release of inflammatory factors in BEAS-2B cells upon A. fumigatus conidia, while rapamycin could reverse the effect of JNK inhibitor on IL-27 and TNF-α release. Taken together, these data demonstrated that JNK signal might play an important role in inflammatory factor release regulated by autophagy in bronchial epithelial cells against A. fumigatus infection.

Mucormycosis, an invasive fungal disease with severe consequences, poses a significant threat to immunocompromised individuals. However, the timely and accurate identification of Mucorales infection continues to present difficulties. In this study, novel detection techniques utilizing recombinase polymerase amplification (RPA) and quantitative real-time polymerase chain reaction (qPCR) were developed, specifically targeting the mitochondrial rnl gene, in order to address this challenge. The specificity of the RPA and qPCR assay was assessed by adding genomic DNAs extracted from 14 non-targeted strains, as well as human and mouse blood. No false-positive results were observed. Additionally, genomic DNAs from 13 species in five genera of order Mucorales were tested and yielded positive results in both methods. To further evaluate the sensitivity of the assays, DNAs from Rhizopus oryzae, Mucor racemosus, Absidia glauca, Rhizomucor miehei, and Cunninghamella bertholletiae were utilized, with concentrations ranging from 1 ng/μL to 1 fg/μL. The limit of detection (LoD) for the RPA assay was determined to be 1 pg., with the exception of Rhizomucor miehei which had a LoD of 1 ng. The LoD for the qPCR assay varied between 10 fg and 1 pg., depending on the specific species being tested. Sensitivity analysis conducted on simulated clinical samples revealed that the LoD for RPA and qPCR assays were capable of detecting DNA extracted from 103 and 101 colony forming units (CFU) conidia in 200 μL of blood and serum, respectively. Consequently, the real-time RPA and qPCR assays developed in this study exhibited favorable sensitivity and specificity for the diagnosis of mucormycosis.

烟曲霉是广泛存在于自然环境中的腐生真菌,其产生大量的分生孢子易被人体吸入肺中,在一定条件下可造成艾滋病患者和器官移植患者等免疫低下人群的感染.已有研究显示,临床中侵袭性曲霉感染致死率达 50％以上,而造成这种高致死率的原因与烟曲霉菌丝极性生长、压力应激和免疫逃逸等过程有关[1-2].此外,临床上也面临烟曲霉对抗真菌药物(如三唑类和棘白菌素类等)耐受水平不断升高的棘手难题,这其中重要的耐药分子机制包括药物靶点突变/靶点高表达、药物应激反应等[3-4].因此,阐明烟曲霉在药物耐受和致病力等方面的重要分子机制,有助于临床和科研工作中防治烟曲霉感染和开发新型的靶点药物.目前的证据显示,小GTPase蛋白可能是参与调控致病真菌耐药性和致病力的关键分子基础[5].该蛋白在真菌细胞中多以单体形式(21～30 kD)保守存在,可通过水解 GTP 实现开关模式转换,并以此参与细胞生长和分化、应激外界压力刺激等关键生命过程[5].小GTPase 蛋白根据分子特征和功能等分为Rab、Ras、Arf、Rho和Ran.

磷脂酰肌醇磷酸酶(phosphatidylinositol phosphatases,PIPases)Sac1是细胞内磷脂酰肌醇磷酸(phosphatidylinositol phosphates,PIPs)代谢途径中一类重要磷酸酶,可以使磷脂酰肌醇-4-磷酸去磷酸化,同时参与肌醇代谢、肌动蛋白细胞骨架重排、ATP转运等一系列细胞功能.该文对磷脂酰肌醇磷酸酶Sac1的结构、功能及其在哺乳动物细胞、酵母细胞和其他真核细胞中的功能研究最新发现进行了综述.

新型冠状病毒(2019-nCoV)引起医院感染流行情况严重,是新型冠状病毒肺炎(COVID-19)疫情传播的关键环节之一.医院感染防控是COVID-19疫情防控的关键环节.通过对国内外新闻及文献报道的2019-nCoV引起的各类医院感染流行及暴发情况,从流行特点和防控存在的主要问题进行梳理与分析,提出策略与建议.总结经验发现,领导重视程度不够,医务人员院感防控意识和培训不足,消毒及个人防护等防控措施落实不到位等是引起COVID-19院内感染的主要因素.建议将COVID-19院内感染融入疾控工作体系,建立健全第三方监督机制,加强多学科合作,切实提升我国医院感染防控能力.

目的 评价火神山医院隔离病区新型冠状病毒(SARS-CoV-2)污染情况,为新型冠状病毒肺炎(COVID-19)隔离病区制定合理的环境卫生管理策略提供科学依据.方法 2020年3月9日-3月29日在火神山医院随机选取2个普通隔离病区进行系统的样本采集工作.采集对象包括环境样本、医务人员个人防护用品(PPE)样本以及患者和医务人员手样本.采用棉签擦拭法采集样本,通过实时荧光定量聚合酶链反应检测法对样本进行SARS-CoV-2核酸检测,并对检测结果进行整理和分析.结果 共采集244份样本,经SARS-CoV-2核酸检测,171份环境样本中1份阳性,来自患者床栏,阳性率为0.58％;49份医务人员PPE样本均为阴性;患者和医务人员手样本共24份,其中1份患者手样本阳性,阳性率为4.17％.结论 合理规划隔离病区的布局和流程、严格落实环境消毒和手卫生制度能够有效降低隔离病区SARS-CoV-2污染程度,切断病毒传播途径,降低医务人员医院感染暴露风险.同时,COVID-19医院感染防控中还应加强患者个人卫生管理.

Aspergillus fumigatus causes a series of invasive diseases, including the high-mortality invasive aspergillosis, and has been a serious global health threat because of its increased resistance to the first-line clinical triazoles. We analyzed the whole-genome sequence of 15 A. fumigatus strains from China and found that long terminal repeat retrotransposons (LTR-RTs), including Afut1, Afut2, Afut3, and Afut4, are most common and have the largest total nucleotide length among all transposable elements in A. fumigatus. Deleting one of the most enriched Afut4(977-sac1) in azole-resistant strains decreased azole resistance and downregulated its nearby gene, sac1, but it did not significantly affect the expression of genes of the ergosterol synthesis pathway. We then discovered that 5'LTR of Afut4(977-sac1) had promoter activity and enhanced the adjacent sac1 gene expression. We found that sac1 is important to A. fumigatus, and the upregulated sac1 caused elevated resistance of A. fumigatus to azoles. Finally, we showed that Afut4(977- sac1) has an evolution pattern similar to that of the whole genome of azole-resistant strains due to azoles; phylogenetic analysis of both the whole genome and Afut4(977-sac1) suggests that the insertion of Afut4(977-sac1) might have preceded the emergence of azole-resistant strains. Taking these data together, we found that the Afut4(977-sac1) LTR-RT might be involved in the regulation of azole resistance of A. fumigatus by upregulating its nearby sac1 gene.

Coronavirus disease 2019 (COVID-19) pandemic has caused high number of infections and deaths of healthcare workers globally. Distribution and possible transmission route of SARS-CoV-2 in hospital environment should be clarified. We herein collected 431 environmental (391 surface and 40 air) samples in the intensive care unit (ICU) and general wards (GWs) of three hospitals in Wuhan, China from February 21 to March 4, 2020, and detected SARS-CoV-2 RNA by real-time quantitative PCR. The viral positive rate in the contaminated areas was 17.8% (28/157), whereas there was no virus detected in the clean areas. Higher positive rate (22/59, 37.3%) was found in ICU than that in GWs (3/63, 4.8%). The surfaces of computer keyboards and mouse in the ICU were the most contaminated (8/10, 80.0%), followed by the ground (6/9, 66.7%) and outer glove (2/5, 40.0%). From 17 air samples in the contaminated areas, only one sample collected at a distance of around 30 cm from the patient was positive. Enhanced surface disinfection and hand hygiene effectively decontaminated the virus from the environment. This finding might help understand the transmission route and contamination risk of SARS-CoV-2 and evaluate the effectiveness of infection prevention and control measures in healthcare facilities.