Gasdermin E-mediated keratinocyte pyroptosis participates in the pathogenesis of psoriasis by promoting skin inflammation.

BACKGROUND: Psoriasis is a common chronic inflammatory disease with an unclear aetiology. Keratinocytes in psoriasis are susceptible to exogenous triggers that induce inflammatory cell death. OBJECTIVES: To investigate whether gasdermin E (GSDME)-mediated pyroptosis in keratinocytes contributes to the pathogenesis of psoriasis. METHODS: Skin samples from patients with psoriasis and from healthy controls were collected to evaluate the expression of GSDME, cleaved caspase-3 and inflammatory factors. We then analysed the data series GSE41662 to further compare the expression of GSDME between lesional and nonlesional skin samples in those with psoriasis. In vivo, a caspase-3 inhibitor and GSDME-deficient mice (Gsdme-/-) were used to block caspase-3/GSDME activation in an imiquimod-induced psoriasis model. Skin inflammation, disease severity and pyroptosis-related proteins were analysed. In vitro, tumour necrosis factor (TNF)-α-induced caspase-3/GSDME-mediated pyroptosis in the HACAT cell line was explored. RESULTS: Our analysis of the GSE41662 data series found that GSDME was upregulated in psoriasis lesions vs. normal skin. High levels of inflammatory cytokines such as interleukin (IL)-1ß, IL-6 and TNF-α were also found in psoriasis lesions. In mice in the Gsdme-/- and caspase-3 inhibitor groups, the severity of skin inflammation was attenuated and GSDME and cleaved caspase-3 levels decreased after imiquimod treatment. Similarly, IL-1ß, IL-6 and TNF-α expression was decreased in the Gsdme-/- and caspase-3 inhibitor groups. In vitro, TNF-α induced HACAT cell pyroptosis through caspase-3/GSDME pathway activation, which was suppressed by blocking caspase-3 or silencing Gsdme. CONCLUSIONS: Our study provides a novel explanation of TNF-α/caspase-3/GSDME-mediated keratinocyte pyroptosis in the initiation and -acceleration of skin inflammation and the progression of psoriasis.

Psoriasis is chronic and autoinflammatory common skin disease that affects 2­3% of the world's population. The disease is characterized by persistent inflammation in various body systems, including the skin and joints. However, the exact cause of the disease is unclear. In this study from China, we found that in people with psoriasis a protein called 'gasdermin E' (or 'GSDME') is increased in a type of skin cell called keratinocytes. In psoriasis, these keratinocytes are susceptible to a type of cell death called 'pyroptosis'. We aimed to find out whether pyroptosis caused by GSDME in keratinocytes contributes to the development of psoriasis. To do this, we looked at samples of skin from people with psoriasis and compared these to samples from healthy controls (those without psoriasis). Firstly, we investigated the levels of GSDME, another protein called caspase-3 and other inflammatory factors in the skin lesions from patients with psoriasis. Secondly, we analysed previously published data from 24 patients with psoriasis. Finally, we carried out a range of experiments to confirm our findings. We found that keratinocyte pyroptosis was mediated by the messenger proteins TNF-α/caspase-3, and that GSDME played a key role in the initiation and acceleration of skin inflammation and the progression of psoriasis. Targeting the GSDME pathway may be a novel strategy in treating psoriasis.

Penalized and constrained LAD estimation in fixed and high dimension.

Recently, many literatures have proved that prior information and structure in many application fields can be formulated as constraints on regression coefficients. Following these work, we propose a L 1 penalized LAD estimation with some linear constraints in this paper. Different from constrained lasso, our estimation performs well when heavy-tailed errors or outliers are found in the response. In theory, we show that the proposed estimation enjoys the Oracle property with adjusted normal variance when the dimension of the estimated coefficients p is fixed. And when p is much greater than the sample size n, the error bound of proposed estimation is sharper than k log ( p ) / n . It is worth noting the result is true for a wide range of noise distribution, even for the Cauchy distribution. In algorithm, we not only consider an typical linear programming to solve proposed estimation in fixed dimension , but also present an nested alternating direction method of multipliers (ADMM) in high dimension. Simulation and application to real data also confirm that proposed estimation is an effective alternative when constrained lasso is unreliable.

Potent in vitro synergism of fusidic acid (FA) and berberine chloride (BBR) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).

It was found in the present study that combined use of fusidic acid (FA) and berberine chloride (BBR) offered an in vitro synergistic action against 7 of the 30 clinical methicillin-resistant Staphylococcus aureus (MRSA) strains, with a fractional inhibitory concentration (FIC) index ranging from 0.5 to 0.19. This synergistic effect was most pronounced on MRSA 4806, an FA-resistant isolate, with a minimum inhibitory concentration (MIC) value of 1,024 µg/ml. The time-kill curve experiment showed that FA plus BBR yielded a 4.2 log10 c.f.u./ml reduction in the number of MRSA 4806 bacteria after 24-h incubation as compared with BBR alone. Viable count analysis showed that FA plus BBR produced a 3.0 log10 c.f.u./ml decrease in biofilm formation and a 1.5 log10 c.f.u./ml decrease in mature biofilm in viable cell density as compared with BBR alone. In addition, phase contrast micrographs confirmed that biofilm formation was significantly inhibited and mature biofilm was obviously destructed when FA was used in combination with BBR. These results provide evidence that combined use of FA and BBR may prove to be a promising clinical therapeutic strategy against MRSA.

Transcriptional response of Candida albicans biofilms following exposure to 2-amino-nonyl-6-methoxyl-tetralin muriate.

AIM: To identify changes in the gene expression profile of Candida albicans (C albicans) biofilms following exposed to 2-amino-nonyl-6-methoxyl-tetralin muriate(10b) and clarify the mechanism of 10b against C albicans biofilms. METHODS: Anti-biofilm activity of 10b was assessed by tetrazolium (XTT) reduction assay and the action mechanism against biofilms was investigated by cDNA microarray analysis and real-time RT-PCR assay. RESULTS: Ten differentially expressed genes were directly linked to biofilm formation and filamentous or hyphal growth (eg, NRG1, ECE1 and CSA1). Decreased gene expression was involved in glycolysis (eg, HXK2 and PFK1) and antioxidant defense (eg, SOD5), while increased gene expression was associated with enzymes that specifically hydrolyzed beta-1,3 glucan (XOG1), and with lipid, fatty acid and sterol metabolism (eg, SLD1, ERG6 and ERG2). Functional analysis indicated that addition of anti-oxidant ascorbic acid reduced inhibitory efficiency of 10b on mature biofilm. CONCLUSION: Inhibition of 10b on biofilm formation possibly depends on impairing the ability of C albicans to change its morphology via altering the expression of biofilm formation genes. Mitochondrial aerobic respiration shift and endogenous ROS augmentation might be a major contribution to reduce mature biofilm metabolic activity. The data may be useful for the development of new strategies to reduce the incidence of device-associated infections.

Proteomic analysis reveals a synergistic mechanism of fluconazole and berberine against fluconazole-resistant Candida albicans: endogenous ROS augmentation.

Our previous study showed that concomitant use of berberine (BBR) and fluconazole (FLC) provided a synergistic action against FLC-resistant Candida albicans (C. albicans) clinical strains in vitro. To clarify the mechanism underlying this action, we performed a comparative proteomic study in untreated control cells and cells treated with FLC and/or BBR in 2 clinical strains of C. albicans resistant to FLC. Our analyses identified 16 differentially expressed proteins, most of which were related to energy metabolisms (e.g., Gap1, Adh1, and Aco1). Functional analyses revealed that FLC + BBR treatment increased mitochondrial membrane potential, decreased intracellular ATP level, inhibited ATP-synthase activity, and increased generation of endogenous reactive oxygen species (ROS) in FLC-resistant strains. In addition, checkerboard microdilution assay showed that addition of antioxidant ascorbic acid or reduced glutathione reduced the synergistic antifungal activity of FLC + BBR significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the synergistic action of FLC + BBR against FLC-resistant C. albicans.

2-Amino-nonyl-6-methoxyl-tetralin muriate inhibits sterol C-14 reductase in the ergosterol biosynthetic pathway.

AIM: To investigate the action mechanism of a novel chemical structural aminotetralin derivate, 2-Amino-Nonyl-6-Methoxyl-Tetralin Muriate (10b), against Candida albicans (C albicans) in the ergosterol biosynthetic pathway. METHODS: Antifungal susceptibility test of 10b was carried out using broth microdilution method, the action mechanism of 10b against C albicans was investigated by GC-MS spectrometry and real-time RT-PCR assay, and cytotoxicity of 10b in vitro was assessed by MTS/PMS reduction assay. RESULTS: 10b reduced the ergosterol content markedly, and the 50% ergosterol content inhibitory concentration (ECIC(50) value) was 0.08 microg/mL. Although the sterol composition of 10b-grown cells was completely identical with that of erg24 strain, the content of ergosta-8,14,22-trienol in 10b-grown cells was much higher than that in erg24 strain. Real-time RT-PCR assay revealed a global upregulation of sterol metabolism genes. In addition, the 50% inhibitory concentration (IC(50) value) of 10b was 11.30 microg/mL for murine embryonic fibroblasts and 35.70 microg/mL for human normal liver cells. CONCLUSION: 10b possessed a mode of action different from that of azoles and morpholines, whose targets were sterol C-14 reductase (encoded by ERG24 gene) and sterol C-5 desaturase (encoded by ERG3) related enzyme. Although 10b seemed to reduce MTS/PMS reduction in a dose dependent manner, IC(50) value for mammalian cells was much higher than 50% minimum inhibitory concentration (MIC(50)) value for C albicans. This indicates that the formulation is preliminarily safe and warrants further study for possible human applications.

Synthesis and antifungal activities of novel 2-aminotetralin derivatives.

Novel 2-aminotetralin derivatives were synthesized as antifungal agents. The 2-aminotetralin scaffold was chemically designed to mimic the tetrahydroisoquinoline ring of the lead molecule described before. Their antifungal activities were evaluated in vitro by measuring the minimal inhibitory concentrations (MICs). Compounds 10a, 12a, 12c, 13b, and 13d are more potent than fluconazole against seven testing human fungal pathogens. Compound 10b exhibits much higher antifungal activities against all of the four fluconazole-resistant clinic Candida albicans strains than the control drugs including amphotericin B, terbinafine, ketoconazole, and itraconazole. The mode of action of some compounds to the potential receptor lanosterol 14alpha-demethylase (CYP51) was investigated by molecular docking. The studies presented here provide a new structural type for the development of novel antifungal compounds. Furthermore, 10b was evaluated in vivo by a rat vaginal candidiasis model, and it was found that 10b significantly decreases the number of fungal colony counts.

Hsp90 is involved in apoptosis of Candida albicans by regulating the calcineurin-caspase apoptotic pathway.

Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA) could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway.