A point mutation in the IAA14 promoter enhances callus formation and regeneration.

Callus formation induced by auxin accumulation is considered the first step of in vitro plant regeneration. In Arabidopsis, degradation of the Aux/IAA protein, IAA14, in response to auxin signaling, which activates the AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 along with a series of downstream transcription factors, also plays a critical role in this process. However, the specific mechanism by which auxin regulates callus formation remains unclear. By screening mutant library in the solitary root 1 (iaa14/slr) Arabidopsis background we obtained the callus formation related 2 (cfr2) mutant. The cfr2 mutant exhibited a stronger capacity for callus formation, as well as lateral root and adventitious root regeneration from leaf explants than wild type (WT) seedlings, but did not recover gravitropism capability. The auxin signal in cfr2 was significantly enhanced, and the expression of some downstream transcription factors was increased. Map-based cloning, whole genome resequencing, and phenotypic complementation experiments showed that the phenotypes observed in the cfr2 mutant were caused by a point mutation in the IAA14 promoter region. This mutation, which is predicted to disrupt the binding of LBD16, LBD19, and LBD30 to the IAA14 promoter, changed the expression pattern of IAA14 in cfr2. Taken together, our results identified a new mutation in the IAA14 promoter region, which affects the expression pattern of IAA14 and in turn its ability to control plant regeneration. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01493-y.

Glucosinolate O-methyltransferase mediated callus formation and affected ROS homeostasis in Arabidopsis thaliana.

Auxin-induced callus formation was largely dependent on the function of Lateral Organ Boundaries Domain (LBD) family transcription factors. We previously revealed that two IGMT (Indole glucosinolate oxy-methyl transferase) genes, IGMT2 and IGMT3, may be involved in the callus formation process as potential target genes of LBD29. Overexpression of the IGMT genes induces spontaneous callus formation. However, the details of the IGMT involvement in callus formation process were not well studied. IGMT1-4, but not IGMT5, are targeted and induced by LBD29 during the early stage of callus formation. Cell membrane and nucleus localized IGMT3 was mainly expressed in the elongation and maturation zones tissues of the primary root and lateral root, which could be further accumulated after CIM treatment. The igmts quadruple mutant, which obtained by CRISPR/Cas9 technology, exhibits a phenotype of attenuated callus formation. Enhanced indole glucosinolate anabolic pathway caused by IGMT1-4 overexpression promotes callus formation. In addition, the IGMT genes were involved in the reactive oxygen species homeostasis, which could be responsible for its role on callus formation. This study provides novel insights into the role of IGMTs gene-mediated callus formation. Activation of the Indole glucosinolate anabolic pathway is an inducing factor for plant callus initiation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01409-2.

Diffusion-weighted imaging diagnostic algorithm in patients with suspected pleural malignancy.

OBJECTIVES: The purpose of this study was to analyze the diagnostic performance and clinical application of diffusion-weighted imaging (DWI) in patients with suspected pleural malignancy (PM). METHODS: A retrospective review of patients with suspected PM was performed from March 2014 to August 2018 (NCT02320617). All patients underwent chest DWI and computed tomography (CT) with cytological or histopathological findings as reference standards. The diagnostic performance of DWI and CT was analyzed and compared. A DWI diagnostic algorithm with three sequential steps was established. RESULTS: Seventy patients (61.6 ± 13.6 years; 47 males and 23 females) were included. The sensitivity of DWI (94.2%, 49/52) for the diagnosis of PM was significantly higher compared with CT (67.3%, 35/52), with similar specificity (72.2% vs. 72.2%, respectively). The apparent diffusion coefficient of malignant lesions (1.15 ± 0.32 × 10-3 mm2/s) was lower compared with benign lesions (1.46 ± 0.68 × 10-3 mm2/s), but the cutoff value was difficult to define for overlap between groups. Approximately 62.5% (5/8) of invasive procedures were avoided when using the DWI diagnostic algorithm in patients with suspected PM without N3 lymph node or extra-thoracic metastasis. CONCLUSION: Including DWI into the diagnostic algorithm of suspected PM can effectively identify malignancy and avoid unnecessary invasive procedures, which may have some potential in clinical application. KEY POINTS: • Diffusion-weighted imaging can identify pleural malignancy much more efficiently than CT. • A diffusion-weighted imaging diagnostic algorithm helped to avoid unnecessary invasive procedures in patients without N3 lymph node or extra-thoracic lesions. • A hyperintense signal on DWI at a high b value (800 s/mm2) but not at a low b value (50 s/mm2) was a reliable signature of PM.

Elaboration of a multimodal MRI-based radiomics signature for the preoperative prediction of the histological subtype in patients with non-small-cell lung cancer.

BACKGROUND: Non-invasive discrimination between lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) subtypes of non-small-cell lung cancer (NSCLC) could be very beneficial to the patients unfit for the invasive diagnostic procedures. The aim of this study was to investigate the feasibility of utilizing the multimodal magnetic resonance imaging (MRI) radiomics and clinical features in classifying NSCLC. This retrospective study involved 148 eligible patients with postoperative pathologically confirmed NSCLC. The study was conducted in three steps: (1) feature extraction was performed using the online freely available package with the multimodal MRI data; (2) feature selection was performed using the Student's t test and support vector machine (SVM)-based recursive feature elimination method with the training cohort (n = 100), and the performance of these selected features was evaluated using both the training and the validation cohorts (n = 48) with a non-linear SVM classifier; (3) a Radscore model was then generated using logistic regression algorithm; (4) Integrating the Radscore with the semantic clinical features, a radiomics-clinical nomogram was developed, and its overall performance was evaluated with both cohorts. RESULTS: Thirteen optimal features achieved favorable discrimination performance with both cohorts, with area under the curve (AUC) of 0.819 and 0.824, respectively. The radiomics-clinical nomogram integrating the Radscore with the independent clinical predictors exhibited more favorable discriminative power, with AUC improved to 0.901 and 0.872 in both cohorts, respectively. The Hosmer-Lemeshow test and decision curve analysis results furtherly showed good predictive precision and clinical usefulness of the nomogram. CONCLUSION: Non-invasive histological subtype stratification of NSCLC can be done favorably using multimodal MRI radiomics features. Integrating the radiomics features with the clinical features could further improve the performance of the histological subtype stratification in patients with NSCLC.

Effect of peptidases secreted by the opportunistic pathogen Scedosporium aurantiacum on human epithelial cells.

Peptidases secreted by a clinical high-virulence Scedosporium aurantiacum isolate (strain WM 06.482; CBS 136046) under normoxic and hypoxic conditions were separated via size-exclusion chromatography, and peptidase activities present in each fraction were determined using class-specific substrates. The fractions demonstrating peptidase activity were assessed for their effects on the attachment and viability of A549 human lung epithelial cells in vitro. Of the peptidases detected in the size-exclusion chromatography fractions, the elastase-like peptidase reduced cell viability, the chymotrypsin-like peptidase was associated with cell detachment, and the cysteine peptidases were able to abolish both cell attachment and viability. The loss of cell viability and attachment became more prominent with an increase in the peptidase activity and could also be specifically prevented by addition of class-specific peptidase inhibitors. Our findings indicate that peptidases secreted by S. aurantiacum can breach the human alveolar epithelial cell barrier and, thus, may have a role in the pathobiology of the organism.

Comparative Mitogenomic Analysis of Species Representing Six Subfamilies in the Family Tenebrionidae.

To better understand the architecture and evolution of the mitochondrial genome (mitogenome), mitogenomes of ten specimens representing six subfamilies in Tenebrionidae were selected, and comparative analysis of these mitogenomes was carried out in this study. Ten mitogenomes in this family share a similar gene composition, gene order, nucleotide composition, and codon usage. In addition, our results show that nucleotide bias was strongly influenced by the preference of codon usage for A/T rich codons which significantly correlated with the G + C content of protein coding genes (PCGs). Evolutionary rate analyses reveal that all PCGs have been subjected to a purifying selection, whereas 13 PCGs displayed different evolution rates, among which ATPase subunit 8 (ATP8) showed the highest evolutionary rate. We inferred the secondary structure for all RNA genes of Tenebrio molitor (Te2) and used this as the basis for comparison with the same genes from other Tenebrionidae mitogenomes. Some conserved helices (stems) and loops of RNA structures were found in different domains of ribosomal RNAs (rRNAs) and the cloverleaf structure of transfer RNAs (tRNAs). With regard to the AT-rich region, we analyzed tandem repeat sequences located in this region and identified some essential elements including T stretches, the consensus motif at the flanking regions of T stretch, and the secondary structure formed by the motif at the 3' end of T stretch in major strand, which are highly conserved in these species. Furthermore, phylogenetic analyses using mitogenomic data strongly support the relationships among six subfamilies: ((Tenebrionidae incertae sedis + (Diaperinae + Tenebrioninae)) + (Pimeliinae + Lagriinae)), which is consistent with phylogenetic results based on morphological traits.

[Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease].

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 µg/s and 17.57 µg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (µg/s) and 4.091 (µg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Research on the bearing behavior of single pile in self-weight collapsible loess areas.

The negative skin frictional caused by loess collapse will decrease the bearing capacity of single pile, which is essential to the design of pile foundations in loess areas. In this study, a method for estimating the subsidence of soil layer at any depth is firstly proposed based on the total self-weight collapse value. Secondly, a new load transfer constitutive model for pile-soil interface is developed, which considers the nonlinear stress-strain relationship and the ultimate shear strength of soil. Then, a load transfer calculation model for pile foundation is established, which can calculate the pile axial force, the pile skin frictional, neutral point position and the settlement of a single pile. The calculation results are compared with the test data that obtained from a pile foundation on-site immersion test and the effectiveness of the calculation method is verified well. This calculation method may be useful for designing pile foundations in collapsible loess regions.

Dechlorination of wastewater from shell-based glucosamine processing by mangrove wetland-derived fungi.

Wastewater from processing crustacean shell features ultrahigh chloride content. Bioremediation of the wastewater is challenging due to the high chloride ion content, making it inhospitable for most microorganisms to survive and growth. In this study, mangrove wetland-derived fungi were first tested for their salt tolerance, and the highly tolerant isolates were cultured in shrimp processing wastewater and the chloride concentration was monitored. Notably, the filamentous fungal species Aspergillus piperis could remove over 70% of the chloride in the wastewater within 3 days, with the fastest biomass increase (2.01 times heavier) and chloride removal occurring between day one and two. The chloride ions were sequestered into the fungal cells. The genome of this fungal species contained Cl- conversion enzymes, which may have contributed to the ion removal. The fungal strain was found to be of low virulence in larval models and could serve as a starting point for further considerations in bioremediation of shell processing wastewater, promoting the development of green technology in the shell processing industry.

Physicochemical properties and inhibition effect on iron deficiency anemia of a novel polysaccharide-iron complex (LPPC).

Porphyran (P) was extracted from red algae Porphyra by boiling water. A novel polysaccharide-iron complex (LPPC) was prepared under the alkaline condition by adding a ferric chloride solution to the low molecular weight porphyran (LP) solution. Physicochemical properties and inhibition effect on iron deficiency anemia of this complex were studied. The content of iron(III) in the complex is 21.57% determined with iodometry. The results indicate that LPPC was product required. The complex can increase red blood cell count (RBC), hemoglobin (Hb), Serum iron (SI), spleen index, spleen mass and mass of mice with iron deficiency anemia (IDA). Although the structure and deeper mechanisms on hemolytic anemia of LPPC should be further studied, LPPC is hoped to be developed as a late-model iron supplement which has a synergism on anemia.

Deciphering the mitochondrial genome of Hemerocallis citrina (Asphodelaceae) using a combined assembly and comparative genomic strategy.

Hemerocallis citrina is a perennial herbaceous plant that is dedicated to mothers in Chinese culture and is widely distributed across the country. As a popular species with a long history of cultivation and utilization, it is renowned for its remarkable edible and medicinal value. In this study, we integrated Illumina short-read and Oxford Nanopore long-read sequencing to generate a complete mitochondrial genome (mitogenome) assembly of H. citrina. The H. citrina mitogenome has a multiple chromosomal structure consisting of three circular molecules that are 45,607 bp, 239,991 bp, and 182,864 bp long. We correspondingly annotated 66 genes, comprising 45 protein-coding genes (PCGs), 17 tRNA genes, and 4 rRNA genes. Comparative analysis of gene organization indicated that six syntenic gene clusters were conserved in the mitogenomes of the compared plants. The investigation of repeat content revealed repeat-rich nature of the H. citrina mitogenome, for which plentiful dispersed repeats were characterized to correlate with the size of the mitogenome. The codon usage behavior disclosed that Leucine (Leu) and Serine (Ser) were the most preferred amino acids in H. citrina, and nearly all of the codons with relative synonymous codon usage (RSCU) values greater than 1 showed the preference of A or T ending. Moreover, we inferred a total of 679 RNA editing sites in all mitochondrial PCGs, which presented perfect C-to-U types and tended to lead to the alteration of internal codons. Subsequent selective pressure analysis showed that the majority of the PCGs had undergone evolutionary negative selections, with atp9 in particular undergoing strong stabilizing selection, reflecting its indispensable function in mitogenomes. According to the phylogenetic analysis, H. citrina is close to the species Allium cepa (Amaryllidaceae) and Asparagus officinalis (Asparagaceae) in evolutionary terms. Overall, this project presents the first complete mitogenome of H. citrina, which could provide a reference genome for the comprehensive exploration of the Asphodelaceae family and can facilitate further genomic breeding and evolutionary research on this medicine-food homologous plant.

Aqua-{6,6'-dieth-oxy-2,2'-[ethane-1,2-diyl-bis(nitrilo-methanylyl-idene)]diphen-olato}zinc.

The mononuclear zinc title complex, [Zn(C(20)H(22)N(2)O(4))(H(2)O)], was obtained by the reaction of 3-eth-oxy-salicyl-aldehyde, ethane-1,2-diamine, and zinc acetate in methanol. The Zn atom is five-coordinated by two phenolate O and two imine N atoms of the tetradentate Schiff base ligand and by one water O atom, forming a square-pyramidal geometry. In the crystal, pairs of mol-ecules are linked via inter-molecular O-H⋯O hydrogen bonds, forming dimers.

Bis{4-chloro-2-[(2-hy-droxy-eth-yl)imino-meth-yl]phenolato}nickel(II) monohydrate.

The title mononuclear nickel(II) complex, [Ni(C(9)H(9)ClNO(2))(2)]·H(2)O, was obtained by the reaction of 5-chloro-salicyl-aldehyde, 2-amino-ethanol and nickel nitrate in methanol. The Ni atom is six-coordinated by two phenolate O, two imine N and two hy-droxy O atoms from two crystallographically different Schiff base ligands, forming an octa-hedral geometry. In the crystal, mol-ecules are linked through inter-molecular O-H⋯O and O-H⋯Cl hydrogen bonds.

The complete chloroplast genome sequence of the medicinal plant Persicaria filiformis.

Persicaria filiformis, known as a traditional Chinese herbal medicine, is a perennial plant of Polygonaceae wildly distributed in China. The complete chloroplast (cp) genome of P. filiformis was assembled and analyzed in this study. The length of the circular genome is 159,741 bp, with a rich GC content of 41.3%. The cp genome structure consists of a large single-copy region (LSC 84,432 bp), a small single-copy region (SSC 13,073 bp) and a pair of inverted repeat regions (IR 31,118 bp). The complete genome encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Phylogenetic analysis indicated that P. filiformis is most related to P. japonica.

Bioaccumulation of microcystin and its oxidative stress in the apple (Malus pumila).

The bioaccumulation and harmful effects of microcystins (MCs) and the activity of peroxidase (POD) and superoxide dismutase (SOD) were examined in the apple (Malus pumila) exposed in vitro with the crude extract of toxic cyanobacterial blooms from Dianchi Lake in southwestern China. The results showed that the growth and proliferation of M. pumila shoots in vitro decreased markedly after exposure to microcystins above 0.3 microg/ml. Recovered microcystins determined by enzyme-linked immunosorbent assay (ELISA) in M. pumila shoot cultures increased with exposure time and concentration. After 14 days exposure to the concentration of 3 microg/ml microcystins, M. pumila shoot cultures accumulated microcystins up to a concentration of 510.23 +/- 141.10 ng MC-LR equiv/g FW (fresh weight), equivalent to an accumulation rate of 36.45 ng/g day. POD activity was significantly increased after 7 days exposure to 3 microg/ml microcystins. After 14 days of exposure, microcystins caused POD to increase significantly at the concentration of 0.3 and 3 microg/ml. The activity of SOD was not affected by microcystins at concentrations up to 3 microg/ml on 7 days. After 14 days exposure to microcystins, SOD activity increased significantly at the concentration of 0.3 and 3 microg/ml in M. pumila shoot cultures.

Bis{2-eth-oxy-6-[2-(methyl-ammonio)ethyl-imino-meth-yl]phenolato}thio-cyanato-zinc(II) nitrate.

In the title compound, [Zn(NCS)(C(12)H(18)N(2)O(2))(2)]NO(3), the Zn(II) ion is chelated by the phenolate O and imine N atoms from two zwitterionic Schiff base ligands and is also coordinated by the N atom of a thio-cyanate ligand, giving a distorted trigonal-bipyramidal geometry. Intra-molecular N-H⋯O hydrogen bonds are observed in the complex cation. The nitrate anions are linked to the complex cations through N-H⋯O hydrogen bonds.

Fractionation and activity profiling of fruit bromelain from pineapples of Phuket variety growing in Thailand.

Bromelain is a type of protease found in both fruits and stems of pineapples. Stem bromelain has been extensively studied and is commercially available for applications in various industries. In contrast, studies of fruit bromelain are quite limited since most of pineapples have been consumed freshly, canned or juiced. Nowadays, the consumption of canned fruits, including canned pineapples has decreased greatly. Fruit bromelain could be a new growth point for pineapple industry. In this study, fruit bromelain was extracted from the pineapple juice of Phuket variety and some of its properties were studied. The enzyme was purified by precipitation using ammonium sulfate fractionation followed by ion-exchange and gel filtration chromatography. Consequently, the protease purification level was increased by 95.2 fold. The final specific activity was getting to 448,590 U/mg on average, dominated by cysteine proteases, with optimal activity at 45°C and pH ranging from 6 to 8. The study facilitates the molecular and application research of fruit bromelain. PRACTICAL APPLICATIONS: The research has been carried out at Funong Food Technology Co., Ltd., Guangdong, China, which produces primarily pineapple chunks and juice. As plenty of by-products, like peels and cores of pineapples, are produced, the techniques are employed to extract bromelain from the by-products. The techniques reported in this work are not new or advanced, however, they are applicable during the manufacturing process and the related equipment is easy to set up and operate. What's more, the practical application of the techniques is cost-effective for the manufactories. Take Funong Food Technology Co., Ltd. as an example, they was using 80% saturation ammonium sulfate to precipitate protein from pineapple juice and obtained a bromelain with activity of approximately 8,000 U/mg and yield of 1.7 kg per ton of juice. With the application of the techniques reported in this work, bromelain was first extracted by ammonium sulfate gradient precipitation, and then purified through ion-exchange and gel filtration chromatography. Each step of precipitation and purification generates a different level of activity and recovery of bromelain, ranging from around 2,506 to 448,590 U/mg, which allows for the production of bromelain according to the requirement of the market and brings more profits.

Growth and protease secretion of Scedosporium aurantiacum under conditions of hypoxia.

One of the micro-environmental stresses that fungal pathogens, such as Scedosporium aurantiacum, colonising human lungs encounter in vivo is hypoxia, or deficiency of oxygen. In this work, we studied the impacts of a hypoxic micro-environment (oxygen levels ≤1%) on the growth of a clinical S. aurantiacum isolate (WM 06.482; CBS 136046) and an environmental strain (S. aurantiacum WM 10.136; CBS 136049) on mucin-containing synthetic cystic fibrosis sputum medium. Additionally, profiles of secreted proteases were compared between the two isolates and protease activity was assessed using class-specific substrates and inhibitors. Overall, both isolates grew slower and produced less biomass under hypoxia compared to normoxic conditions. The pH of the medium decreased to 4.0 over the cultivation time, indicating that S. aurantiacum released acidic compounds into the medium. Accordingly, secreted proteases of the two isolates were dominated by acidic proteases, including aspartic and cysteine proteases, with optimal protease activity at pH 4.0 and 6.0 respectively. The clinical isolate produced higher aspartic and cysteine protease activities. Conversely, all serine proteases, including elastase-like, trypsin-like, chymotrypsin-like and subtilisin-like proteases had higher activities in the environmental isolate. Sequence similarities to 13 secreted proteases were identified by mass spectrometry (MS) by searching against other fungal proteases in the NCBI database. Results from MS analysis were consistent with those from activity assays. The clinical highly-virulent, and environmental low-virulence S. aurantiacum isolates responded differently to hypoxia in terms of the type of proteases secreted, which may reflect their different virulence properties.

FBW7 loss promotes epithelial-to-mesenchymal transition in non-small cell lung cancer through the stabilization of Snail protein.

The E3 ubiquitin ligase F-box and WD repeat domain containing 7 (FBW7α) functions as a putative tumor suppressor in non-small cell lung cancer (NSCLC) due to its regulation of a set of oncogenic proteins associated with cell proliferation and mitosis. Increasing efforts have been focused on the understanding of FBW7 in determining cell cycle progression and apoptosis induction, however, the correlation between FBW7 and tumor metastasis is not fully understood. In this study, we reported a potential anti-metastatic effect of FBW7 in non-small cell lung cancer (NSCLC). In this model, FBW7 inhibited cancer cell metastasis primarily by inducing ubiquitination and proteolysis of the transcriptional factor Snail, which suppressed E-cadherin cell tight junction protein expression. Loss of FBW7 would stabilize the Snail protein, thus, inhibit E-cadherin expression and promote metastasis in vitro and in vivo. Moreover, Snail ubiquitination and degradation were also achieved by pharmacological approach, in which the FBW7 agonist oridonin treatment led to Snail proteolysis. Furthermore, FBW7 silencing stabilized Snail protein and induced epithelial-to mesenchymal transition (EMT), and acquisition of migration and invasion properties in NSCLC. Overall, our study provides new insights into the FBW7-Snail axis in regulating cell migration and invasion, and suggests that targeting FBW7 may be a potent approach to inhibit metastasis in NSCLC.

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5-75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.