Aspergillus fumigatus, an opportunistic and allergenic pathogenic fungus, is responsible for a range of clinical disorders in humans, including invasive aspergillosis (IA), which can lead to severe infections in immunocompromised individuals. Unfortunately, the emergence of azole resistance has become a significant challenge in combating IA, necessitating further investigations into the underlying mechanisms of resistance. In this study, we conducted an integrated proteomic and phosphoproteomic analysis of biofilm proteins from both azole-resistant and wildtype strains of A. fumigatus under voriconazole pressure. Our proteomic analysis identified 148 upregulated and 146 downregulated proteins in the azole-resistant strains, while phosphoproteomic analysis revealed 316 upregulated phosphopeptides and 109 downregulated phosphopeptides, suggesting extensive phosphorylation modifications associated with azole resistance. Upon excluding the impact of protein changes, we identified 133 proteins with differential expression solely at the phosphorylation level, comprising 104 upregulated and 29 downregulated proteins. Functional annotation and analysis highlighted the significance of these differentially expressed phosphoproteins in cell wall integrity, filamentous growth, and high-osmolarity stress response, with 33 MAPK pathway-associated proteins displaying phosphopeptide level regulation. These findings provide valuable insights into the mechanisms behind azole resistance in A. fumigatus and offer potential new drug targets for combating this pathogenic fungus in humans.

Objective:To summarize clinical characteristics of patients with Aspergillus fumigatus infection in a hospital in Nanjing, to preliminarily assess azole resistance in clinical isolates of Aspergillus fumigatus, and to investigate risk factors for the emergence of azole-resistant Aspergillus fumigatus. Methods:Clinical isolates of Aspergillus fumigatus were collected from inpatients in Department of Laboratory, Nanjing Drum Tower Hospital from March 2017 to February 2021. Clinical data on these infected patients were analyzed, azole sensitivity testing and mutation analysis of the cyp51A gene and its promoter region were performed for these Aspergillus fumigatus isolates. Results:A total of 201 strains of Aspergillus fumigatus were collected, and mainly isolated from sputum specimens. Among the infected patients, there were 131 males and 70 females, and their age were 64.2 ± 15.8 years. The patients were mainly collected from department of respiratory medicine (79 cases), department of intensive medicine (34 cases), department of rheumatology (19 cases), etc. Among these patients, common underlying diseases included interstitial pneumonia (32 cases), malignant tumors (18 cases), pneumonia (13 cases), trauma (12 cases), systemic lupus erythematosus (8 cases), etc. Drug susceptibility testing showed that 6 (2.99%) strains of Aspergillus fumigatus were resistant to itraconazole and posaconazole, and 3 patients infected with azole-resistant Aspergillus fumigatus had used antifungal drugs before testing. Sequencing was performed on the cyp51A gene and its promoter region in the 6 strains of azole-resistant Aspergillus fumigatus, and showed TR34/L98H/S297T/F495I mutation in 5 strains and TR34/L98H mutation in 1 strain. Conclusion:Compared with previously published data about azole resistance in China during 2010 -2015, the resistance of Aspergillus fumigatus to azoles in Nanjing Drum Tower Hospital did not increase from 2017 to 2021, and the mechanism of azole resistance was mostly associated with TR34/L98H/S297T/F495I mutation in the cyp51A gene and its promoter region.

Azole-resistant Aspergillus fumigatus makes a major challenge to the chemotherapy for invasive aspergillosis, whereas cyp51A gene mutation is the most dominant mechanism for azole resistance. Moreover, biofilm contributes to drug resistance for A. fumigatus, and extracellular matrix (ECM) is essential to protect live cells from antifungal drugs. Therefore, we performed a comparative proteomic study on the biofilm ECM of both the wild-type and azole-resistant strains of A. fumigatus under azole pressure. In total, 2377 proteins were identified, of which 480 and 604 proteins with differential expression were obtained from the wild-type and azole-resistant A. fumigatus in exposure to itraconazole respectively (fold change > 2 or < 0.5, P-value < 0.05). We found that a high proportion of regulated proteins were located in cytoplasm, nucleus, and mitochondria. Meanwhile, GO and KEGG analyses revealed that metabolic process and ribosome pathway were significantly enriched. Particularly, differentially expressed proteins in response to azole pressure of both the wild-type and resistant strains were further analyzed. Our results indicated that these changes in biofilm ECM proteins were related to ergosterol synthesis, oxidative stress, efflux pumps, DNA repair, DNA replication, and transcription.

目的 构建我国烟曲霉cyp51A基因序列BLAST数据库.方法 对本课题组收集的143株耐药与非耐药临床烟曲霉进行cyp51A基因测序,使用BLAST工具包汇总测序结果,构建本地数据库.结果 此BLAST数据库包含143株耐药与非耐药烟曲霉cyp51A基因序列,以1株耐药与1株非耐药待测烟曲霉标本的cyp51A序列对此数据库进行检索和比对可以确定烟曲霉cyp51A基因型.结论 本数据库有望用于cyp51A基因的检索与比对,鉴定烟曲霉cyp51A耐药基因型.

生物膜的形成对临床感染的发生发展产生重要影响[1].真菌生物膜是由真菌细胞和菌丝以及胞外基质包裹形成的复杂群落,能够表现出比游离真菌更强的致病性和耐药性[2].随着激素、免疫抑制剂等的广泛应用和各种人工植入物的使用,真菌感染的发病率显著上升,生物膜相关的感染及其耐药带来的治疗困难已成为严重的临床问题[3].几乎所有致病真菌均能够形成生物膜和胞外基质,尤其在侵袭性真菌病中更为常见,目前关于生物膜的研究大多集中在白念珠菌和其他念珠菌属以及烟曲霉等致病真菌[2].蛋白质组学技术在大规模水平上分离和鉴定蛋白质,近年来随着技术不断进步发展,已经在医学领域得到广泛应用,成为重要的研究手段之一[4].目前在生物膜研究中,蛋白质组学不仅分离鉴定出了相关特征性蛋白,还证明了真菌生物膜耐药性可能与其生长过程中的某些与能量和代谢相关酶的表达上调或下调有关,例如增强了对表面相关蛋白的调节,活性氧的积累,氧化应激反应的上调等,但具体机制尚未完全阐明[5-6].蛋白质组学技术通过提供真菌生物膜蛋白质全面和系统的描述,为研究其耐药相关蛋白提供了新的思路.本文将综述蛋白质组学技术的研究进展及其在真菌生物膜中的应用.

It is uncertain whether Bmi-1 deficiency could lead to skin aging by redox imbalance and DNA damage. In this study, we first confirmed that Bmi-1 had a relatively high expression level in the skin and Bmi-1 expression levels gradually decreased with age. Then, we studied the role of Bmi-1 in the skin using a Bmi-1-/- mouse model. Bmi-1-/- mice were supplemented with or without pyrroloquinoline quinone (PQQ) for 5 weeks, and their skin phenotypes were compared with Bmi1-/- and wild-type littermates. Our results showed that Bmi-1-/- mice displayed decreased vertical thickness of skin, sparse hair follicles, and thinner and more irregular collagen bundles. Mechanistically, increased oxidative stress with reducing antioxidant capacity and induced DNA damage occurred in Bmi-1-/- mice. Subsequently, this would lead to reduced cell proliferation, increased cell senescence and matrix metalloproteinases (MMPs), and the degradation of fibroblast function and further reduce collagen synthesis. All pathological alterations in the skin of Bmi-1-/- mice were alleviated by PQQ supplementation. These results demonstrated that Bmi-1 might play a key role in protection from skin aging by maintaining redox balance and inhibiting DNA damage response and will be a novel and potential target for preventing skin aging.

Aspergillus fumigatus (A. fumigatus) is the most common airborne opportunistic fungal pathogen. Biofilm formation is one of the main pathogenic mechanisms of A. fumigatus. During the past decades, A. fumigatus azole resistance has become prevalent due to the medical and agricultural use of antifungal drugs and fungicides. Until now, the role of fungal biofilms in azole resistance of A. fumigatus remains unclear. In the present study, we compared biofilm drug susceptibility and biofilm formation under itraconazole of azole-resistant strains, sensitive strains, and standard strains, separately. The biofilm viability and matrix thickness at the early and the late stage were measured by XTT assay and Calcofluor white. Our results showed that the sessile minimum inhibitory concentration of itraconazole, which describing the inhibition of drugs on fungi sessile with biofilm, was much higher than the traditional minimal inhibitory concentration of itraconazole. Additionally, low concentrations of itraconazole inhibited biofilm formation of A. fumigatus strains. Notably, biofilm formation by azole-resistant strains could not be inhibited by high concentrations of itraconazole but could be effectively restrained by low concentrations of micafungin, revealing the efficacy of a cell-wall inhibitor to disrupt A. fumigatus biofilm formation. However, late-stage biofilms of both azole-resistant strains and standard strains were hard to disrupt using itraconazole. We found that itraconazole was effective to prevent A. fumigatus biofilm formation at the early stage. For the treatment of A. fumigatus biofilm, our findings suggest that an early-stage preventive strategy is preferred and micafungin is effective to control the azole-resistant strain infection.

Introduction::Pterygium inversum unguis (PIU) refers to a disorder that distal portion of the nail bed adheres to ventral surface of the nail plate, leading to the absence of the distal groove. This is not a common condition in existing literature, thus its pathogenesis has not been elucidated. The occurrence can be congenital, idiopathic, or secondary. Here we present a case of PIU associated with systemic lupus erythematosus (SLE).Case presentation::A 33-year-old woman presented with distal nail bed of the bilateral fingers (excluding the thumbs) adheres to the ventral surface of the nail plate, associated with a malar rash, photosensitivity, and arthritis, whose further laboratory examinations confirmed immunologic disorders. All these clinical characteristics and laboratory results point to the diagnosis of PIU secondary to SLE. Treatment of SLE and topical application of tretinoin 0.025% were performed and resulted in the improvement of most discomforts but PIU. There has no aggravation or alleviation of the PIU during a year of follow-up.Discussion::Acquired form of PIU is reportedly associated with connective tissue diseases or other conditions, which may be caused by abnormal distal circulation or exposure to certain chemical stimuli. Some therapies may be available, while the most effective strategy is to treat potential disorder. Clinicians should be vigilant to find out the underlying causes of PIU, so as to obtain better therapeutic efficacy.Conclusion::We observed a rare disorder of PIU associated with SLE. Hence when identifying a patient of PIU, comprehensive evaluations and long-term follow-up are imperative to to detect the development of connective tissus diseases, such as SLE.

The opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in cytochromeP450 enzyme Cyp51. In this study, we indicated that in vitro azole situation results in emergence of azole-resistant mutations. There are previously identified azole-resistant cyp51A mutations (M220K, M220I, M220R, G54E and G54W mutations) and we successfully identified in this study two new mutations (N248K/V436A, Y433N substitution) conferring azole resistance among 18 independent stable azole-resistant isolates. The Galleria mellonella model of A. fumigatus infection experiment verified that Cyp51A mutations N248K/V436A and Y433N reduce efficacy of azole therapy. In addition, a predicted Cyp51A 3D structural model suggested that Y433N mutation causes the reduced affinities between drug target Cyp51A and azole antifungals. This study suggests that drug selection pressure make it possible to isolate unidentified cyp51A mutations conferring azole resistance in A. fumigatus.

Itraconazole is an antagonist of the component Smoothened of Hedgehog pathway, which can inhibit the growth of medulloblastoma, basal cell carcinoma, and melanoma, etc. To research the binding mechanism of the Smoothened and triazoles, we used docking and molecular dynamics simulations on the Smoothened crystal structure and six triazoles. Unlike vismodegib, itraconazole can effectively bind into the pocket in the C-terminal domain of the Smoothened crystal structure instead of the N-terminal domain. The binding of itraconazole can change the conformation of the N-terminal domain even although itraconazole only had limited area contacting with N-terminal domain of the Smoothened. Besides, the binding of Itraconazole will not affect the binding of vismodegib. The strong binding affinity could be demonstrated between itraconazole and the Smoothened. Posaconazole and ketoconazole also had the strong binding affinity and the similar binding mode with the Smoothened crystal structure.

Malignant melanoma is the deadliest form of all skin cancers. Itraconazole, a commonly used systemic antifungal drug, has been tested for its anti-tumor effects on basal cell carcinoma, prostate cancer, and non-small cell lung cancer. Whether itraconazole has any specific anti-tumor effect on melanoma remains unknown. However, the goal of this study is to investigate the effect of itraconazole on melanoma and to reveal some details of its underlying mechanism. In the in vivo xenograft mouse model, we find that itraconazole can inhibit melanoma growth and extend the survival of melanoma xenograft mice, compared to non-itraconazole-treated mice. Also, itraconazole can significantly inhibit cell proliferation, as demonstrated by Ki-67 staining in itraconazole-treated tumor tissues. In in vitro, we show that itraconazole inhibits the proliferation and colony formation of both SK-MEL-28 and A375 human melanoma cells. Moreover, we demonstrate that itraconazole significantly down-regulates Gli-1, Gli-2, Wnt3A, beta-catenin and cyclin D1, while it up-regulates Gli-3 and Axin-1, indicating potent inhibitory effects of itraconazole on Hedgehog (Hh) and Wnt signaling pathways. Furthermore, itraconazole significantly suppresses the PI3K/mTOR signaling pathway - indicated by the down-regulated phosphorylation of p70S6K, 4E-BP1 and AKT - but has no effect on the phosphorylation of MEK or ERK. Our data suggest that itraconazole inhibits melanoma growth through an interacting regulatory network that includes Hh, Wnt, and PI3K/mTOR signaling pathways. These results suggest that this agent has several potent anti-melanoma features and may be useful in the synergesis of other anti-cancer drugs via blockage of the Hh, Wnt and PI3K/mTOR signaling pathways.

Mutations of CYP51A protein (Cytochrome P450 14-α Sterol demethylase) play a central role in the azole resistance of Aspergillus fumigatus The available structural models of CYP51A protein ofA. fumigatus are built based on that of Homo sapiens and that of Mycobacterium tuberculosis, of which the amino acid homology is only 38% and 29% compared with CYP51A protein ofA. fumigatus, respectively. In the present study, we constructed a new 3D structural model ofA. fumigatus CYP51A protein based on a recently resolved crystal structure of the homologous protein in the fungus S. cerevisiae, which shares 50% amino acid homology with A. fumigatus CYP51A protein. Three azole molecules, itraconazole, voriconazole, and posaconazole, were docked to the wild-type and the mutant A. fumigatus CYP51A protein models, respectively, to illustrate the impact of cyp51A mutations to azole-resistance. We found the mutations that occurred at L98, M220, and Y431 positions would decrease the binding affinity of azoles to the CYP51A protein and therefore would reduce their inhibitory effects. Additionally, the mutations of L98 and G432 would reduce the stability of the protein, which might lead to conformational change of its binding pocket and eventually the resistance to azoles.

ABSTRACT Seventy-two A. fumigatus clinical isolates from China were investigated for azole resistance based on mutations of cyp51A . We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.

患者女,43岁,全身皮肤黏膜水疱、红斑3年余,加重伴肩关节痛2个月.3年前,患者双手背出现米粒至花生大小的水疱,伴瘙痒,逐渐累及头皮、面颈、躯干、四肢和口腔黏膜,口腔内水疱破溃后形成溃疡,可自愈,皮肤水疱痊愈后遗留色素减退或色素沉着斑,皮疹反复发生.近2个月皮损加重,伴双侧肩关节疼痛.病程中轻度多饮、多食、多尿,近1年体重减轻9 kg.既往史:8年前面部出现红斑,外院诊断红斑狼疮,应用糖皮质激素治疗1年,皮疹消退后停药,未定期复查.体检:各系统检查无异常.皮肤科检查:下颌、颈前可见不规则淡红斑,其上密集水疱,躯干及四肢散在水疱,水疱粟粒至花生大小,疱壁紧张,疱液淡黄色,Nikolsky征阴性(图1);全身皮肤散在大小不一色素减退及色素沉着斑;口腔内颊黏膜见2处指甲大小淡红斑.双侧肩关节运动时疼痛,无压痛及叩痛,双上肢外展上举不能至肩水平面.实验室检查:血常规正常.

Mitochondrial genome is a genetic marker for the identification and classification of fungi,and is also of great value to phylogenetic analysis in fungi.Canadian researchers have introduced the term of Fungal Mitochondrial Genome Project (FMGP) recently.Fungal mitochondrial genomes,which are closed circuar DNA ranging from 10 to 80 kb,usually include respiratory chain subunit gene,adenosine triphosphate (ATP) synthase complex subunit gene,ribosomal RNA and tRNA genes.Further more,fungal mitochondrial genomes are pleiomorphic and can serve as the DNA fingerprint of different fungal species in fungal discrimination,as well as in the classification and phylogenetic analysis of pathogenic fungi.

Objective To investigate the impact of mutations in the V2 domain of HIV-1 envelop glycoprotein (gp) 120 gene on the recognition of neutralizing antibodies (NAbs) specific to the other domains of gp120.Methods HIV-1 pseudoviruses (JR-FL) containing wild type or V2-mutant gp120 monomers were constructed,and the neutralization of CD4-binding site-specific and CD4-induced NAbs to the HIV-1 pseudoviruses was observed.Enzyme linked immunosorbent assay (ELISA) was performed to evaluate the binding affinity of CD4-binding site-specific and CD4-induced NAbs to wild type or V2-mutant gp120.Results Neither CD4-binding site-specific nor CD4-induced NAbs could neutralize the wild type JR-FL pseudoviruses,but both of them could neutralize pseudoviruses containg the gp120 V2 mutant at a low concentration.There was no significant difference in the binding affinity to CD4-binding site-specific NAbs between the wild type and mutant gp120,while the ELISA binding curves of wild type and mutant gp120 against CD4-induced NAbs were separate,and the affinity of CD4-induced NAbs to the mutant gp120 (L175P) was notably higher than that to the wild type gp120.Conclusion The mutations in the V2 domain of HIV-1 gp120 may affect the antiviral activity of NAbs.

目的:构建我国8种重要病原真菌DNA条形码数据库,评价rDNA-ITS区在系统发育学中的价值.方法:进行8种病原真菌的ITS区序列测定和序列分析,使用NJ法和BI法构建种系发育树.结果:ITS序列在2株须癣毛癣菌和2株红色毛癣菌之间完全一致；而在不同物种的真菌间,该序列存在数十到数百个核苷酸替代差异.依据ITS区构建的医学真菌系统发育树与传统分类学一致.结论:ITS区适合作为我国重要医学真菌物种鉴别的DNA条形码.

Objective To study the impact of V2 mutations on neutralizing ability of HIV-1-specific neutralizing antibodies. Methods We tested the influence of L175P mutation to the neutralizing ability of V3-specific antibodies by pseudotype virus and the binding affinity of those V3-spesific antibodies to gpl20 monomer by ELISA. Results We found L175P mutation changed the neutralizing ability of V3-specific antibodies. However, L175P mutation showed no effects on the binding affinity of these antibodies to gpl20 monomer. Conclusion Our results revealed the L175P mutation at V2 loop changed the natural trimmer structure of gp120 and enhanced the neutralizing ability of V3-specific antibodies.