HucMSC-Exo Induced N2 Polarization of Neutrophils: Implications for Angiogenesis and Tissue Restoration in Wound Healing.

Background: Neutrophils rapidly accumulate in large numbers at sites of tissue damage, exhibiting not only their well-known bactericidal capabilities but also playing crucial roles in angiogenesis and tissue repair. While exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exo) have emerged as a promising therapeutic tool, their exact mechanisms of action remain partly elusive. We hypothesize that HucMSC-Exo treatment may modulate neutrophil phenotypes, thereby significantly influencing wound healing outcomes. Methods: HucMSC-Exo were isolated via ultracentrifugation and subsequently administered through subcutaneous injection into full-thickness cutaneous wounds in mice. To determine the impact of host neutrophils on the healing effects of HucMSC-Exo in skin injuries, strategies including neutrophil depletion and adoptive transfer were employed. Flow cytometry was used to evaluate the proportion of N2 subtype neutrophils in both normal and diabetic wounds, and the effect of HucMSC-Exo on this proportion was assessed. Furthermore, the mitochondrial metabolic reprogramming driven by HucMSC-Exo during N2 polarization was investigated through JC1 staining, ATP quantification, fatty acid uptake assays, and assessment of FAO-related genes (Cpt1b, Acadm, and Acadl). Results: Depleting host neutrophils strikingly dampened prohealing effect of HucMSC-Exo on skin injury, while adoptive transfer of bone marrow neutrophils rescued this process. During normal healing process, some neutrophils expressed N2 markers, in contrast, diabetic wounds exhibited a reduced expression of N2 markers. After treatment with HucMSC-Exo, most neutrophils increased the phosphorylation of STAT6, leading to mitochondrial metabolic reprogramming and thus acquired an N2 phenotype. These N2 neutrophils, polarized by HucMSC-Exo, boosted the release of proangiogenic factors, particularly BV8, a myeloid cell-derived proangiogenic factor, and induced angiogenesis thereby favoring tissue restoration. Conclusion: This research uniquely demonstrates the identification of N2 neutrophils in skin injury and shows that HucMSC-Exo could skew neutrophils toward N2 phenotype, enhancing our insight into how cells react to HucMSC-Exo.

Integrated Bioinformatics Analysis Reveals Diagnostic Biomarkers and Immune Cell Infiltration Characteristics of Solar Lentigines.

Background: Solar lentigines (SLs), serving as a prevalent characteristic of skin photoaging, present as cutaneous aberrant pigmentation. However, the underlying pathogenesis remains unclear and there is a dearth of reliable diagnostic biomarkers. Objective: The aim of this study was to identify diagnostic biomarkers for SLs and reveal its immunological features. Methods: In this study, gene expression profiling datasets (GSE192564 and GSE192565) of SLs were obtained from the GEO database. The GSE192564 was used as the training group for screening of differentially expressed genes (DEGs) and subsequent depth analysis. Gene set enrichment analysis (GSEA) was employed to explore the biological states associated with SLs. The weighted gene co-expression network analysis (WGCNA) was employed to identify the significant modules and hub genes. Then, the feature genes were further screened by the overlapping of hub genes and up-regulated differential genes. Subsequently, an artificial neural network was constructed for identifying SLs samples. The GSE192565 was used as the test group for validation of feature genes expression level and the model's classification performance. Furthermore, we conducted immune cell infiltration analysis to reveal the immune infiltration landscape of SLs. Results: The 9 feature genes were identified as diagnostic biomarkers for SLs in this study. And an artificial neural network based on diagnostic biomarkers was successfully constructed for identification of SLs. GSEA highlighted potential role of immune system in pathogenesis of SLs. SLs samples had a higher proportion of several immune cells, including activated CD8 T cell, dendritic cell, myeloid-derived suppressor cell and so on. And diagnostic biomarkers exhibited a strong relationship with the infiltration of most immune cells. Conclusion: Our study identified diagnostic biomarkers for SLs and explored its immunological features, enhancing the comprehension of its pathogenesis.

Antifungal Activity and Potential Action Mechanism of Allicin against Trichosporon asahii.

Trichosporon asahii is an emerging opportunistic pathogen that causes potentially fatal disseminated trichosporonosis. The global prevalence of coronavirus disease 2019 (COVID-19) poses an increasing fungal infection burden caused by T. asahii. Allicin is the main biologically active component with broad-spectrum antimicrobial activity in garlic. In this study, we performed an in-depth analysis of the antifungal characteristics of allicin against T. asahii based on physiological, cytological, and transcriptomic assessments. In vitro, allicin inhibited the growth of T. asahii planktonic cells and biofilm cells significantly. In vivo, allicin improved the mean survival time of mice with systemic trichosporonosis and reduced tissue fungal burden. Electron microscopy observations clearly demonstrated damage to T. asahii cell morphology and ultrastructure caused by allicin. Furthermore, allicin increased intracellular reactive oxygen species (ROS) accumulation, leading to oxidative stress damage in T. asahii cells. Transcriptome analysis showed that allicin treatment disturbed the biosynthesis of cell membrane and cell wall, glucose catabolism, and oxidative stress. The overexpression of multiple antioxidant enzymes and transporters may also place an additional burden on cells, causing them to collapse. Our findings shed new light on the potential of allicin as an alternative treatment strategy for trichosporonosis. IMPORTANCE Systemic infection caused by T. asahii has recently been recognized as an important cause of mortality in hospitalized COVID-19 patients. Invasive trichosporonosis remains a significant challenge for clinicians, due to the limited therapeutic options. The present work suggests that allicin holds great potential as a therapeutic candidate for T. asahii infection. Allicin demonstrated potent in vitro antifungal activity and potential in vivo protective effects. In addition, transcriptome sequencing provided valuable insights into the antifungal effects of allicin.

Pathogenicity of phospholipase B1 of Trichosporon asahii in immunosuppressed mice.

BACKGROUND: Trichosporon asahii is an opportunistic pathogenic yeast-like fungus. Phospholipase B1 (PLB1) is an important virulence factor of pathogenic fungi such as Candida albicans and Cryptococcus neoformans, and there are few studies on the role of PLB1 in the pathogenicity of T. asahii. OBJECTIVES: To investigate the role of PLB1 in the pathogenicity of T. asahii. METHODS: A strain with low secretion of PLB1 (4848) was screened, a PLB1 overexpression strain (PLB1OX ) was constructed, and the differences in histopathology, fungal load of organ, survival time of mice, the levels of IL-6, IL-10, TNF-α, and GM-GSF in the serum and organs caused by the two strains were compared. RESULTS: Histopathology showed that spores and hyphae were observed in both groups, and PLB1OX led to more fungal invasion. The fungal loads in the kidney, lung, spleen and liver in the PLB1OX group were significantly higher than those in the 4848 group, and the survival time of mice was significantly lower than that in the 4848 group. The levels of TNF-α in the serum, liver, spleen, lung and kidney of the PLB1OX group were lower than those of the 4848 group, while the level of IL-10 in the serum was higher than that of the 4848 group. CONCLUSIONS: These results suggest that PLB1 can enhance the invasive function of T. asahii and affect the secretion of TNF-α and IL-10 which may affect the host antifungal immune response, providing evidence that PLB1 plays a role in the pathogenic infection of T. asahii.

Verifying the outcomes of artesunate plus 595-nm PDL in hypertrophic scars via determining BMP-7 and Fas level in model rabbits.

BACKGROUND AND OBJECTIVES: Single-use of artesunate (ART) or 595-nm pulsed-dye laser (PDL) has proven clinical efficacy in the treatment of hypertrophic scars (HSs), yet little research has been done on the combined use of ART and PDL. Bone morphogenetic protein-7 (BMP-7) and Fas are recognized to be two important proteins in reducing scar formation. This study was designed to observe the effect of ART combined with 595-nm PDL in the treatment of HS in rabbit models, and investigate the effect of such protocol on the expression of BMP-7 and Fas in rabbit models. STUDY DESIGN/MATERIALS AND METHODS: Twenty-four New Zealand white rabbits were randomly divided into the control group, ART group, PDL group, and combined treatment (ART + PDL) group. ART was respectively applied to the ART group and combined treatment group. Treatment was once every 2-week for a total of three sessions for both groups. Animals in the PDL group were simply treated with 595-nm PDL. Then, hematoxylin & eosin and Van Gieson straining, immunohistochemical study, enzyme-linked immunosorbent assay (ELISA), Cell counting kit-8 test, western blot assay, and real-time polymerase chain reaction (RT-PCR) were carried out to observe the development of HS samples and expression of BMP-7 and Fas proteins in the sample tissues. RESULTS: After treatment, the scar samples grew lower and flatter, which was particularly evident in the combined treatment group, with notably inhibited fibroblast and collagen compared to other groups (p < 0.001). Western blot assay and RT-PCR demonstrated that the expression of BMP-7 was most increased in scar samples treated by ART + PDL. BMP-7 level was correspondingly and notably upregulated in treatment groups, especially in the ART + PDL group. In addition, relevant expression of Fas was also higher after treatment, especially in the ART + PDL group compared to either ART or 595-nm PDL group. The difference was significant among groups (p < 0.001). CONCLUSIONS: Combined use of ART and 595-nm PDL can inhibit HSs in rabbit models via inhibiting extra fibroblast and collagens. The potential mechanism may be involved in enhanced BMP-7 and Fas expression. Our observations may create an alternative therapeutic strategy for HSs in the clinic.

Effectiveness of artesunate combined with fractional CO2 laser in a hypertrophic scar model with underlying mechanism.

BACKGROUND AND OBJECTIVES: Both artesunate and fractional CO2 laser have been proved effective in the treatment of hypertrophic scars, yet little data are available for the efficacy of artesunate combined with fractional CO2 laser. In order to assess the pre-clinical significance and the underlying mechanism of this combined treatment profile, we attempted to observe the effectiveness of this therapy in rabbit models through determining the expression of BMP-7 and Fas. MATERIALS AND METHODS: Twenty-Four New Zealand white rabbits with established hypertrophic scar samples were randomly divided into control group and three treatment groups. Artesunate (20 µl/cm2) was injected into the rat's scar of artesunate and combination groups, while fractional CO2 laser (Combo mode, deep energy:10 mJ, super energy: 50 mJ) was applied to rats in fractional CO2 laser and combination groups at week 4 after model establishment. All rabbits underwent a total of 3 sessions of treatment once every 2 weeks. Histological and immunohistochemistry study, Western blot assay, cell viability, ELISA and RT-QPCR were performed at week 10 to observe the aspects of hypertrophic scar sample changes and expression of BMP-7 and Fas in the scar tissues. RESULTS: Compared with control group, hypertrophic scars and the collagen fibers were significantly inhibited after treatment, and higher inhibition was seen in the samples in combination group compared to that in artesunate and fractional CO2 laser groups (P < 0.01). Meanwhile, BMP-7 and Fas expressions were both notably increased in all treatment groups, and upregulation of the two proteins was dominant in combination group (P < 0.01). CONCLUSIONS: Artesunate combined with fractional CO2 laser is effective in hypertrophic scarring in this rabbit model. Our findings can serve as a potential alternative strategy to treatment of hypertrophic scar in clinical practice.

Proteomic analysis of serial isolates of Trichosporon asahii identifies host-specific adaptations using the TMT/MRM approach.

The opportunistic fungal pathogen Trichosporon asahii (T. asahii) is an important causal agent of mortality in immunocompromised patients and associated with frequent relapses, even with sufficient antifungal treatment. Investigating the proteomes of initial and recurrent isolates may help to identify within-host adaptive changes. In this study, using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) technology, we analyzed the proteomes of two T. asahii strains that were isolated 15 years apart from the same patient who suffered initial and recurrent episodes of systemic disseminated trichosporonosis. A total of 597 differentially expressed proteins were identified. Functional analysis showed that the increased proteins were primarily concentrated on peptide/protein/energy/drug metabolism and translation. Most of the results were determined to be consistent with the findings of phenotypic assays, such as tests for drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays. Moreover, we performed multiple reaction monitoring (MRM) mass spectrometry to verify 27 candidate proteins, and the results of this experiment were also highly consistent with the results of the TMT analysis. Therefore, to the best of our knowledge, these data provide the first molecular evidence of how the T. asahii proteome changes related to host-specific adaptation during human infection. SIGNIFICANCE: Systemic infection with Trichosporon asahii (T. asahii) has recently been recognized as an important causal agent of mortality in immunocompromised patients. Although triazole treatment usually works efficiently in the early phase of infection, many patients relapse. Hence, comparative analyses of the proteomics of initial and recurrent isolates may reveal evidence of adaptive changes within the host. Our study demonstrates that the recurrent strain has undergone proteomic changes using tandem mass tag (TMT)-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Moreover, the results of phenotypic assays, including drug susceptibility, temperature growth, biofilm formation, melanization and paromomycin assays, were highly consistent with the proteomic changes, and multiple reaction monitoring (MRM) verification also showed similar trends to the TMT results. In summary, our study is the first to investigate the adaptation of T. asahii under pressure from antifungal chemotherapy and host immune responses.

595-nm pulsed dye laser combined with fractional CO2 laser reduces hypertrophic scar through down-regulating TGFß1 and PCNA.

595-nm pulsed dye laser and fractional CO2 laser have been demonstrated effective to treat hypertrophic scar. The underlying mechanism may involve transforming growth factor-beta1 (TGFß1) and proliferating cell nuclear antigen (PCNA), but remains to be clarified. Our study was performed to investigate how 595-nm pulsed dye laser combined with fractional CO2 laser treats hypertrophic scars in a rabbit model through regulating the expression of TGFß1 and PCNA. Twenty-four New Zealand white rabbits were randomly divided into control group, pulsed dye laser group, fractional CO2 laser group, and pulsed dye laser + fractional CO2 laser (combination) group. Surgical wounds were made and allowed to grow into hypertrophic scars at day 28. Next, 595-nm pulsed dye laser (fluence: 15 J/cm2; square: 7 mm; pulse duration: 10 ms) was used in pulsed dye laser and combination group, while fractional CO2 laser (combo mode, deep energy: 12.5 mJ; super energy: 90 mJ) in fractional CO2 laser and combination groups, once every 4 weeks for 3 times. The appearance and thickness of hypertrophic scar samples were measured with hematoxylin-eosin and Van Gieson's straining. The expressions of TGFß1 and PCNA were evaluated by immunohistochemical and western blot analysis. A significant improvement was noted in the thickness, size, hardness, and histopathology of hypertrophic scar samples after laser treatment, especially in combination group. Scar Elevation Index (SEI), fiber density (NA), and collagen fiber content (AA) decreased most significantly in combination group (2.10 ± 0.14; 2506 ± 383.00; 22.98 ± 2.80%) compared to 595-nm pulsed dye laser group (3.35 ± 0.28; 4857 ± 209.40; 42.83 ± 1.71%) and fractional CO2 laser group (2.60 ± 0.25; 3995 ± 224.20; 38.33 ± 3.01%) (P < 0.001). Furthermore, TGFß1 and PCNA expressions were more suppressed in combination group (8.78 ± 1.03; 7.81 ± 1.51) than in 595-nm pulsed dye laser (14.91 ± 1.68; 15.73 ± 2.53) and fractional CO2 laser alone group (15.96 ± 1.56; 16.13 ± 1.72) (P < 0.001). The combination of 595-nm pulsed dye laser with fractional CO2 laser can improve the morphology and histology of hypertrophic scars in a rabbit model through inhibiting the expression of TGFß1 and PCNA protein. Our findings can pave the way for new clinical treatment strategies for hypertrophic scars.

Use of RNA Sequencing to Perform Comprehensive Analysis of Long Noncoding RNA Expression Profiles in Macrophages Infected with Trichosporon asahii.

Trichosporon asahii (T. asahii) is a clinically important opportunistic pathogenic fungus capable of causing systemic lethal infection in immunosuppressive and immunodeficient hosts. However, the mechanism of the host immune response upon T. asahii infection has not been elucidated. Recent evidence has shown that long noncoding RNAs (lncRNAs) play key roles in regulating the immune response to resist microbial infections. In this study, we analyzed the expression profiles of lncRNAs at 12 and 24 h post-infection (hpi) in THP-1 cells infected with T. asahii using RNA sequencing (RNA-Seq). A total of 64 and 160 lncRNAs displayed significant differentially expressed (DE) at 12 h and 24 hpi, respectively. Among these lncRNAs, 18 lncRNAs were continuous DE at two time points. The DE of eight candidate lncRNAs were verified by real time quantitative polymerase chain reaction (RT-qPCR). Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to analyze the cis-target genes of 18 DE lncRNAs. The results showed that they were enriched in signaling pathways related to the host immune response, indicating that these lncRNAs might play important roles in fungi-host interactions. Finally, we explored the function of lncRNA NEAT1 and found that the expression of TNF-α and IL-1ß declined after NEAT1 knockdown in T. asahii-infected THP-1 cells. To our knowledge, this is the first report of a expression analysis of lncRNAs in macrophages infected with T. asahii. Our study helps to elucidate the role of lncRNAs in the host immune response to early infection by T. asahii.

Comprehensive circRNA-microRNA-mRNA network analysis revealed the novel regulatory mechanism of Trichosporon asahii infection.

BACKGROUND: Invasive Trichosporon asahii (T. asahii) infection frequently occurs with a high mortality in immunodeficient hosts, but the pathogenesis of T. asahii infection remains elusive. Circular RNAs (circRNAs) are a type of endogenous noncoding RNA that participate in various disease processes. However, the mechanism of circRNAs in T. asahii infection remains completely unknown. METHODS: RNA sequencing (RNA-seq) was performed to analyze the expression profiles of circRNAs, microRNAs (miRNAs), and mRNAs in THP-1 cells infected with T. asahii or uninfected samples. Some of the RNA-seq results were verified by RT-qPCR. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to analyze the differentially expressed mRNAs. A circRNA-miRNA-mRNA network was constructed and verified by dual-luciferase reporter assay and overexpression experiments. RESULTS: A total of 46 circRNAs, 412 mRNAs and 47 miRNAs were differentially expressed at 12 h after T. asahii infection. GO and KEGG analyses showed that the differentially expressed mRNAs were primarily linked to the leukocyte migration involved in the inflammatory response, the Toll-like receptor signaling pathway, and the TNF signaling pathway. A competing endogenous RNA (ceRNA) network was constructed with 5 differentially expressed circRNAs, 5 differentially expressed miRNAs and 42 differentially expressed mRNAs. Among them, hsa_circ_0065336 was found to indirectly regulate PTPN11 expression by sponging miR-505-3p. CONCLUSIONS: These data revealed a comprehensive circRNA-associated ceRNA network during T. asahii infection, thus providing new insights into the pathogenesis of the T. asahii-host interactions.

Effect of Artesunate Combined With Fractional CO2 Laser on the Hypertrophic Scar in a Rabbit Model.

BACKGROUND AND OBJECTIVES: Hypertrophic scar (HS), a common complication in wound healing, is characterized by the disarrangement of collagen, fibers, and extracellular matrix. Artesunate (ART) can inhibit the abnormal formation of fibroblasts and collagens. Fractional CO2 laser (FCO2 L) can facilitate tissue remodeling and the absorption of drugs into ablative microthermal columns in HS. So far, no research has investigated the efficacy of ART combined with an FCO2 L in treating HS. To investigate the theoretical basis and clinical significance of this combination, we established a rabbit model of HS to observe the change in the expression of transforming growth factor ß1 (TGF-ß1) and proliferating cell nuclear antigen (PCNA). STUDY DESIGN/MATERIALS AND METHODS: Forty New Zealand white rabbits were randomly divided into four groups: control group, ART group, FCO2 L group, and ART + FCO2 L (combination) group. Four wounds were surgically established in the ear of each rabbit and allowed to develop into HS. ART (20 µL/cm2 ) was injected in ART and combination groups, and FCO2 L (combo mode, deep energy:10m J, super energy: 50 mJ) in FCO2 L and combination groups on the 28th day after HS occurred. Three rounds of treatment were applied (once every 14 days). HS samples were measured by hematoxylin and eosin staining, Van Gieson staining, immunohistochemistry, and Western blot analysis on the 70th day. RESULTS: The morphological and histopathological changes in HS were significant. HSs were smoother and smaller and the collagen fibers were thinner and less disordered in the combination group than those in ART and FCO2 L groups. Meanwhile, the hypertrophic index (HI), fiber density (NA), and collagen fiber content (AA) were lower in the combination group (1.54 ± 0.15, 3.30 ± 0.22, 30.37 ± 1.41%) than in the ART group (2.51 ± 0.22, 4.69 ± 0.16, 44.68 ± 2.30%) and FCO2 L group (1.99 ± 0.14, 4.13 ± 0.12, 37.74 ± 1.38%) (P < 0.01). Additionally, the expressions of TGF-ß1 and PCNA protein were suppressed in the ART group (0.30 ± 0.03, 0.25 ± 0.03) and FCO2 L group (0.35 ± 0.03, 0.32 ± 0.05), and the suppression was more significant in the combination group(0.07 ± 0.02, 0.07 ± 0.02) (P < 0.01). CONCLUSIONS: The combination of ART and FCO2 L can effectively reduce HS in the rabbit model. This is the first report about this combination in the treatment of HS. A novel treatment is expected to be based on our findings. Lasers Surg. Med. © 2021 Wiley Periodicals LLC.

Specific microRNA/mRNA expression profiles and novel immune regulation mechanisms are induced in THP-1 macrophages by in vitro exposure to Trichosporon asahii.

BACKGROUND: Trichosporon asahii is considered the most prominent species associated with invasive trichosporonosis, but little is known about the pathogenesis of T. asahii infection in the host. MicroRNAs (miRNAs) are a class of noncoding endogenous small RNAs that play vital roles by manipulating immune responses against pathogenic microorganisms. Nevertheless, the exact functions of miRNAs in T. asahii infection are still unknown. OBJECTIVE: To investigate the interactions involved in the miRNA immune response in THP-1 macrophages following in vitro exposure to T. asahii. METHODS: We utilized next-generation sequencing to detect differentially expressed (DE) miRNAs and mRNAs in THP-1 cells after 24 h of in vitro exposure to T. asahii. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to verify the sequencing results. The miRNA-mRNA regulatory network was constructed with the DE miRNAs and DE mRNAs. We performed Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of the predicted targeting mRNAs in the miRNA-mRNA network. A dual-luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) were utilized to demonstrate the reliability of the miR-342-3p/Dectin-1 pair. RESULTS: A total of 120 DE miRNAs and 588 DE mRNAs were identified after 24 h of in vitro exposure to T. asahii. The miRNA-mRNA regulatory network was constructed with 39 DE miRNAs and 228 DE mRNAs. KEGG pathway analysis revealed that the up-regulated DE mRNAs in the complex interaction network were mainly involved in immune-related pathways. In addition, we verified the target relationship between miR-342-3p and Dectin-1 and found that miR-342-3p could promote the expression of TNF-α and IL-6 by negatively regulating Dectin-1. CONCLUSIONS: This study evaluated the expression profiles of miRNA/mRNA and revealed the immunological consequences of THP-1 macrophages in response to T. asahii exposure. Moreover, our data suggest that miR-342-3p can indirectly promote inflammatory responses and may be a potential therapeutic target against trichosporonosis.

Genomic and transcriptome identification of fluconazole-resistant genes for Trichosporon asahii.

Trichosporon asahii infection is difficult to control clinically. This study identified a case with over 15 years of T. asahii infection-related systemic dissemination disease and conducted genome and transcriptome sequencing to identify fluconazole-resistant genes in fluconazole-resistant versus susceptible strains isolated from this patient's facial skin lesions. The data revealed mutations of the ergosterol biosynthetic pathway-related genes in the T. asahii genome of the fluconazole-resistant strain, that is, there were 36 novel mutations of the ERG11 gene, three point mutations (V458L, D457V, and D334S) in the ERG3, and a missense mutation (E349D) in ERG5 in the fluconazole-resistant strain of the T. asahii genome. To ensure that ERG11 is responsible for the fluconazole resistance, we thus simultaneously cultured the strains in vitro and cloned the ERG11 CDS sequences of both fluconazole-susceptible and -resistant strains into the Saccharomyces cerevisiae. These experiments confirmed that these mutations of ERG11 gene affected fluconazole resistance (> 64 µg/ml vs. <8 µg/ml of the MIC value between fluconazole-resistant and -susceptible strains) in Saccharomyces cerevisiae. In addition, expression of ergosterol biosynthesis pathway genes and drug transporter was upregulated in the fluconazole-resistant strain of T. asahii. Collectively, the fluconazole resistance in this female patient was associated with mutations of ERG11, ERG3, and ERG5 and the differential expression of drug transporter and fatty acid metabolic genes.

Rapid and Simple Detection of Trichosporon asahii by Optimized Colony PCR.

Trichosporon asahii is the major pathogen causing invasive trichosporonosis. Conventional methods of its detection are time-consuming or costly and often require complex DNA extraction and purification steps, which hinders rapid clinical diagnosis. In this study, we evaluated colony PCR, which directly uses colonies or trace clinical samples as the template for amplification, for rapid detection of T. asahii infection. Four methods, namely, direct colony, freeze-thaw, glass beads, and enzymolysis, were compared to select the best DNA extraction strategy. We subsequently designed and screened species-specific primers targeting the intergenic spacer 1 (IGS1) of the ribosomal DNA of T. asahii and used them to detect mock infection clinical samples. The species-specific colony PCR based on glass beads proved advantageous, with short procedure time (154.8 ± 0.6 min), good sensitivity (detection limit, 102 CFU/mL), and specificity for T. asahii, indicating that this method can be used for the rapid and simple identification of clinical samples of T. asahii infection.

Adipose tissue-derived stem cells ameliorates dermal fibrosis in a mouse model of scleroderma.

OBJECTIVE: To investigate the therapeutic potential of adipose-derived stem cells (ADSCs) for limited cutaneous scleroderma (LS) in mouse models. METHODS: ADSCs were isolated from pathogen-free female C57BL/6 mice and LS was induced in wild type (WT) C57BL/6 mice via daily injection of bleomycin (0.1 mL × 300 µg/mL) for 4 weeks; then the ADSCs were subcutaneously injected into the dorsal area in the model treatment group, and 100 µL of phosphate-buffered saline (PBS) solution was injected into the same site in the model control group. Green fluorescent protein (GFP) was used to track the cells using an in vivo imaging system on days 7, 14, 21, and 28 after transplantation. All mice were sacrificed and histologic analyses were performed after 4 weeks, and the skin thickness, collagen deposition and the total content of hydroxyproline were evaluated. Additionally, immunohistochemistry were performed to compare the tissue expression and distribution of TGF-ß1 and VEGF between the ADSCs treatment group and the treatment control group. RESULTS: WT C57BL/6 LS mouse model were successfully established and GFP in vivo fluorescence imaging showed that the translated ADSCs survived at the local for at least 4 weeks. Compared with the control group, the ADSCs treatment group significantly attenuated bleomycin-induced dermal fibrosis, reduced the skin thickness and the total content of hydroxyproline (P < 0.05). The ADSCs treatment group displayed significantly lower levels of TGF-ß1 and higher levels of VEGF than the control group (P < 0.05). CONCLUSIONS: ADSCs may provide a feasible and practical treatment for autoimmune diseases such as LS and ameliorate dermal fibrosis.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

Abstract The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64.

The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

The antifungal effect of silver nanoparticles on Trichosporon asahii.

BACKGROUND/PURPOSE: Silver nanoparticles are receiving increasing attention in biomedical applications. This study aims at evaluating the antifungal properties of silver nanoparticles against the pathogenic fungus Trichosporon asahii. METHODS: The growth of T. asahii on potato dextrose agar medium containing different concentrations of silver nanoparticles was examined and the antifungal effect was evaluated using minimum inhibitory concentration. Scanning and transmission electron microscopy were also used to investigate the antifungal effect of silver nanoparticles on T. asahii. RESULTS: Silver nanoparticles had a significant inhibitory effect on the growth of T. asahii. The minimum inhibitory concentration of silver nanoparticles against T. asahii was 0.5 µg/mL, which was lower than amphotericin B, 5-flucytosine, caspofungin, terbinafine, fluconazole, and itraconazole and higher than voriconazole. Silver nanoparticles obviously damaged the cell wall, cell membrane, mitochondria, chromatin, and ribosome. CONCLUSION: Our results demonstrate that silver nanoparticles have good antifungal activity against T. asahii. Based on our electron microscopy observations, silver nanoparticles may inhibit the growth of T. asahii by permeating the fungal cell and damaging the cell wall and cellular components.

Angiotensin II stimulates melanogenesis via the protein kinase C pathway.

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which serve a crucial function in hyperpigmentation. The aim of the present study was to determine the effects of angiotensin II (Ang II) on melanogenesis and to elucidate the molecular events of Ang II-induced melanogenesis. Experiments were performed on human melanocytes to elucidate the pigmenting effect of Ang II and the underlying mechanisms. The elements involved in melanogenesis, including melanin content, tyrosinase (TYR) activity, and microphthalmia-associated transcription factor (MITF) and TYR expression at the mRNA and protein levels were evaluated. Melanin content and TYR activity increased in response to Ang II treatment in a concentration-dependent manner. MITF and TYR mRNA and protein expression levels were increased significantly in response to Ang II in a concentration-dependent manner. The Ang II-induced increase in melanin synthesis was reduced significantly in response to co-treatment with Ro-32-0432, a protein kinase C (PKC) inhibitor, whereas co-treatment with H-89, a PKA inhibitor, did not attenuate the Ang II-induced increase in melanin levels. These results suggest that PKC is required for Ang II-induced pigmentation in human melanocytes and that the mechanism involves the PKC pathway and MITF upregulation.