Oxidative stress-triggered pyroptosis mediates Candida albicans susceptibility in diabetic foot.

An accumulating trend of research demonstrates that diabetic patients are susceptible to skin infections with Candida albicans, but the mechanism still remains unclear. The intense oxidative stress (OS) responses were occurred in the lesion of diabetic mice footpads after C. albicans infection. Localised skin infections would lead to more severe complications while the severity of the condition worsens or the inadequate treatment. Notably, in this study, through the investigation of murine diabetic footpad C. albicans infection model and molecular biotechnology, including histopathological staining, immunofluorescence (IF) staining, quantitative real-time PCR (qPCR), western blot (WB), flow cytometry (FCM), sandwich enzyme-linked immunosorbent assay (ELISA) assays, we found that intense OS responses in the footpad tissue not only mediated the activation of NF-κB protein complex, but also triggered downstream pyroptosis and apoptosis through NLRP3 inflammasome, which is one of the potential reasons for the severe condition of infectious skin injuries in diabetic mice. Caspase-1, a classical signal pathway protein in pyroptosis, could promote pore formation on cell membranes and the release of the cytokine after NLRP3 inflammasome activation. With intense immune-inflammatory responses, the organism also stimulates immune organs such as the spleen and lymph nodes to produce negative feedback regulation and generate CD4+CD25+Foxp3+ Treg cells to rectify the process. Therefore, combined with the results of this work, it is possible to design and screen relevant drugs for NLRP3 inflammasomes as core targets to keep the OS response at a low level in the footpad tissues.

Effects of oestrogen on vulvovaginal candidosis.

As a frequently occurring infectious disease mainly caused by Candida albicans, vulvovaginal candidosis (VVC) affects more than 100 million women worldwide every year. Multiple factors that influence C. albicans colonisation have been linked to the incidence of VVC, including high levels of circulating oestrogen due to pregnancy, the use of oral contraceptives, and hormone replacement therapy. This review provides an overview of the current understanding of the mechanism(s) by which oestrogen contributes to VVC, which might provide meaningful guidance to the prevention and treatment of this disease.

Protection against peripheral artery disease injury by Ruan Jian Qing Mai formula via metabolic programming.

Ruan Jian Qing Mai formula (RJQM), a multicomponent herbal formula, has been widely used to treat peripheral arterial disease (PAD) in China. However, its active compounds and mechanisms of action are still unknown. First, RNA sequencing analysis of 15 healthy and 16 PAD samples showed that 524 PAD differential genes were significantly enriched in Go Ontology (ribonucleotide metabolic process, oxidoreductase complex, and electron transfer activity), Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA pathways (OXPHOS and TCA cycle), miRNA (MIR183), and kinase (PAK6). Fifty-three active ingredients in RJQM had similar structures to the seven drug molecules in CLUE. Then, network topology analysis of the 53 components-target-pathway-disease network yielded 10 active ingredients. Finally, computational toxicity estimations showed that the median lethal dose (LD50) of the 10 active ingredients was above 1000 mg/kg, and eight of them did not cause hepatotoxicity, mutagenicity, carcinogenicity, cytotoxicity, and immunotoxicity nor activate 12 toxic pathways. In conclusion, RJQM has a protection effect on PAD by regulating a complex molecular network. Part of the mechanism is associated with the regulation of OXPHOS by 10 active components, which may alleviate mitochondrial dysfunction and pathological metabolic programming.

Aneuploidy Enables Cross-Adaptation to Unrelated Drugs.

Aneuploidy is common both in tumor cells responding to chemotherapeutic agents and in fungal cells adapting to antifungal drugs. Because aneuploidy simultaneously affects many genes, it has the potential to confer multiple phenotypes to the same cells. Here, we analyzed the mechanisms by which Candida albicans, the most prevalent human fungal pathogen, acquires the ability to survive both chemotherapeutic agents and antifungal drugs. Strikingly, adaptation to both types of drugs was accompanied by the acquisition of specific whole-chromosome aneuploidies, with some aneuploid karyotypes recovered independently and repeatedly from very different drug conditions. Specifically, strains selected for survival in hydroxyurea, an anticancer drug, acquired cross-adaptation to caspofungin, a first-line antifungal drug, and both acquired traits were attributable to trisomy of the same chromosome: loss of trisomy was accompanied by loss of adaptation to both drugs. Mechanistically, aneuploidy simultaneously altered the copy number of most genes on chromosome 2, yet survival in hydroxyurea or caspofungin required different genes and stress response pathways. Similarly, chromosome 5 monosomy conferred increased tolerance to both fluconazole and to caspofungin, antifungals with different mechanisms of action. Thus, the potential for cross-adaptation is not a feature of aneuploidy per se; rather, it is dependent on specific genes harbored on given aneuploid chromosomes. Furthermore, pre-exposure to hydroxyurea increased the frequency of appearance of caspofungin survivors, and hydroxyurea-adapted C. albicans cells were refractory to antifungal drug treatment in a mouse model of systemic candidiasis. This highlights the potential clinical consequences for the management of cancer chemotherapy patients at risk of fungal infections.

Swiprosin-1 Promotes Mitochondria-Dependent Apoptosis of Glomerular Podocytes via P38 MAPK Pathway in Early-Stage Diabetic Nephropathy.

BACKGROUND/AIMS: Podocyte injury, especially podocyte apoptosis, plays a major role in early-stage diabetic nephropathy (DN). Swiprosin-1, also known as EF hand domain containing 2 (EFhd2), is a Ca2+-binding protein in different cell types. However, the function of swiprosin-1 in podocytes remains unknown. METHODS: The expression and distribution of swiprosin-1 were investigated in the mouse renal glomerulus and conditionally immortalized mouse podocyte cell line MPC-5. The expression of swiprosin-1 was also detected in streptozotocin (STZ)-treated mice and MPC-5 cells treated with high glucose (HG). Nephrin and podocin were detected by immunohistochemistry and immunofluroscence. Collagen IV, transforming growth factor-ß (TGF-ß) and fibronectin mRNA expressions were assayed by real-time PCR. Apoptotic proteins and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were detected by immunoblotting. RESULTS: Swiprosin-1 was found to be expressed in podocytes of the mouse glomerulus and MPC-5 cells. Swiprosin-1 expression was increased in STZ-treated mice and MPC-5 cells treated with HG. In Swiprosin-1-/- diabetic mice, kidney/ body weight, urinary albumin, podocyte foot process effacement and glomerular basement membrane thickening were attenuated; the downregulation of nephrin and podocin expression in the glomerulus was inhibited; and the upregulation of collagen IV, TGF-ß and fibronectin mRNA expression in the renal cortex was ameliorated as compared with those in diabetic swiprosin-1+/+ mice. In addition, the increased apoptosis of podocytes, proapoptotic protein expression and p38 phosphorylation in Swiprosin-1-/- diabetic mice were inhibited as compared with those in diabetic swiprosin-1+/+ mice. Knockdown of swiprosin-1 in MPC-5 cells reduced the apoptosis of podocytes, proapoptotic protein expression and p38 phosphorylation induced by HG. Targeted knockdown of p38 attenuated the increased apoptosis of MPC-5 cells over-expressing swiprosin-1. CONCLUSION: Swiprosin-1 expression in podocytes of the mouse glomerulus played a critical role in early-stage DN. Swiprosin-1 deficiency in early DN attenuated mitochondria-dependent podocyte apoptosis induced by hyperglycemia or HG via p38 MAPK signaling pathway.

Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling.

Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidy-independent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development.

The putative ABC transporter encoded by the orf19.4531 plays a role in the sensitivity of Candida albicans cells to azole antifungal drugs.

ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins in prokaryotic and eukaryotic cells. In the human fungal pathogen Candida albicans, there are 28 genes encoding ABC transporters and many of them have not been characterized so far. The orf19.4531 (also known as IPF7530) encodes a putative ABC transporter. In this study, we have demonstrated that disruption of orf19.4531 causes C. albicans cells to become tolerant to azoles, but not to polyene antifungals and terbinafine. Therefore, the protein encoded by orf19.4531 is involved in azole sensitivity and we name it as ROA1, the regulator of azole sensitivity 1 gene. Consistently, we show that the expression of ROA1 is responsive to treatment of either fluconazole or ketoconazole inC. albicans In addition, through a GFP tagging approach, Roa1 is localized in a small punctuate compartment adjacent to the vacuolar membrane. However, ROA1 is not essential for the in vitro filamentation of C. albicans cells.

Antifungal activity of Rubus chingii extract combined with fluconazole against fluconazole-resistant Candida albicans.

This study aimed to investigate the antifungal activity of Rubus chingii extract in combination with fluconazole (FLC) against FLC-resistant Candida albicans 100 in vitro. A R. chingii extract and FLC-resistant C. albicans fungus suspension were prepared. The minimum inhibitory concentration and fractional inhibitory concentration index of R. chingii extract combined with FLC against C. albicans were determined, after which growth curves for C. albicans treated with R. chingii extract, FLC alone and a combination of these preparations were constructed. Additionally, the mechanisms of drug combination against C. albicans were explored by flow cytometry, gas chromatographic mass spectrometry and drug efflux pump function detection. R. chingii extract combined with FLC showed significant synergy. Flow cytometry suggested that C. albicans cells mainly arrest in G1 and S phases when they have been treated with the drug combination. The drug combination resulted in a marked decrease in the ergosterol content of the cell membrane. Additionally, efflux of Rhodamine 6G decreased with increasing concentrations of R. chingii extract. R. chingii extract combined with FLC has antifungal activity against FLC-resistant C. albicans.

Adaptation of Candida albicans to growth on sorbose via monosomy of chromosome 5 accompanied by duplication of another chromosome carrying a gene responsible for sorbose utilization.

Candida albicans, a fungus that normally inhabits the digestive tract and other mucosal surfaces, can become a pathogen in immunocompromised individuals, causing severe or even fatal infection. Mechanisms by which C. albicans can evade commonly used antifungal agents are not fully understood. We are studying a model system involving growth of C. albicans on toxic sugar sorbose, which represses synthesis of cell wall glucan and, as a result, kills fungi in a manner similar to drugs from the echinocandins class. Adaptation to sorbose occurs predominantly due to reversible loss of one homolog of chromosome 5 (Ch5), which results in upregulation of the metabolic gene SOU1 (SOrbose Utilization) on Ch4. Here, we show that growth on sorbose due to Ch5 monosomy can involve a facultative trisomy of a hybrid Ch4/7 that serves to increase copy number of the SOU1 gene. This shows that control of expression of SOU1 can involve multiple mechanisms; in this case, negative regulation and increase in gene copy number operating simultaneously in cell.

Rapid on-site TLC-SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements.

A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC-SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS). The study showed that TLC-SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants.

Transposition of the Zorro2 retrotransposon is activated by miconazole in Candida albicans.

Zorro2 is a member of a non-long terminal repeat (LTR) retrotransposon family in Candida albicans, but as yet no clear evidence has been provided to establish either transcription or transposition activity for Zorro2. In this study, the relative expression changes of two open reading frames in Zorro2, ORF19.7274 and ORF19.7275, were examined in response to miconazole (MCZ), and were found to be increased by this treatment. As well, the copy number and the transcripts of Zorro2 in MCZ-induced resistant daughter strains were increased compared to the parental strain, indicating that transposition of Zorro2 occurred during long-term MCZ treatment. Intriguingly, the transcription activity of Zorro2 retrotransposons was significantly inhibited when the cells were treated with MCZ together with antioxidant N-acetyl-L-cysteine (NAC). As both the level of intracellular reactive oxygen species (ROS) and the expression of genes involving DNA repair activated by MCZ were reduced when combined with the treatment of NAC, we propose that the damage caused by accumulation of ROS under MCZ stress is a major reason for the transcription and transposition activation of the Zorro2 retrotransposon.

[Derivatization of berberine based on its synergistic antifungal activity with fluconazole against fluconazole-resistant Candida albicans].

Abstract: Our previous work revealed berberine can significantly enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans, which suggested that berberine has synergistic antifungal activity with fluconazole. Preliminary SAR of berberine needs to be studied for the possibility of investigating its target and SAR, improving its drug-likeness, and exploring new scaffold. In this work, 13-substitutited benzyl berberine derivatives and N-benzyl isoquinoline analogues were synthesized and characterized by 1H NMR and MS. Their synergetic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The 13-substitutited benzyl berberine derivatives 1a-1e exhibited comparable activity to berberine, which suggested that the introduction of functional groups to C-13 can maintain its activity. The N-benzyl isoquinolines, which were designed as analogues of berberine with its D ring opened, exhibited lower activity than berberine. However, compound 2b, 2c, and 4b showed moderate activity, which indicated that berberine may be deconstructed to new scaffold with synergistic antifungal activity with fluconazole. The results of our research may be helpful to the SAR studies on its other biological activities.

[Rapid detection of four antipertensive chemicals adulterated in traditional Chinese medicine for hypertension using TLC-SERS].

A novel facile method for on-site detection of antipertensive chemicals (e. g. nicardipine hydrochloride, doxazosin mesylate, propranolol hydrochloride, and hydrochlorothiazide) adulterated in traditional Chinese medicine for hypertension using thin layer chromatography (TLC) combined with surface enhanced Raman spectroscopy (SERS) was reported in the present paper. Analytes and pharmaceutical matrices was separated by TLC, then SERS method was used to complete qualitative identification of trace substances on TLC plate. By optimizing colloidal silver concentration and developing solvent, as well as exploring the optimal limits of detection (LOD), the initially established TLC-SERS method was used to detect real hypertension Chinese pharmaceuticals. The results showed that this method had good specificity for the four chemicals and high sensitivity with a limit of detection as lower as to 0.005 microg. Finally, two of the ten antipertensive drugs were detected to be adulterated with chemicals. This simple and fast method can realize rapid detection of chemicals illegally for doping in antipertensive Chinese pharmaceuticals, and would have good prospects in on-site detection of chemicals for doping in Chinese pharmaceuticals.

Effect of Additives on Tribological Performance of Magnetorheological Fluids.

In this study, nano-diamond (ND) and MoS2 powder are used as additives in a carbonyl iron-based magnetorheological fluid (MRF) to improve its tribological performance. MRFs are prepared by dispersing 35 wt.% of CI particles in silicone oil and adding different proportions (0, 1, 3, or 5 wt.%) of ND and MoS2 additives. Seven kinds of MRFs are made and tested using reciprocating friction and wear tester under different normal loads, and then the friction characteristics are evaluated by analyzing the experimental results. The morphological properties of MRFs and contacting surfaces before and after the tests are also observed using a scanning electron microscope and analyzed via energy-dispersive X-ray spectroscopy. The results show that the appropriate weight percentage of MoS2 additives may decrease the friction coefficient and wear zone. It is also demonstrated from detailed analyses of worn surfaces that the wear mechanism is influenced not only by additives, but also by the applied normal load and magnetic field strength.

A Review: Cytochrome P450 in Alcoholic and Non-Alcoholic Fatty Liver Disease.

Alcoholic fatty liver disease (FALD) and non-alcoholic fatty liver disease (NAFLD) have similar pathological spectra, both of which are associated with a series of symptoms, including steatosis, inflammation, and fibrosis. These clinical manifestations are caused by hepatic lipid synthesis and metabolism dysregulation and affect human health. Despite having been studied extensively, targeted therapies remain elusive. The Cytochrome P450 (CYP450) family is the most important drug-metabolising enzyme in the body, primarily in the liver. It is responsible for the metabolism of endogenous and exogenous compounds, completing biological transformation. This process is relevant to the occurrence and development of AFLD and NAFLD. In this review, the correlation between CYP450 and liver lipid metabolic diseases is summarised, providing new insights for the treatment of AFLD and NAFLD.

A mechanism study on the synergistic effects of rifapentine and fluconazole against fluconazole-resistant Candida albicans in vitro.

Candida albicans (C. albicans) is one of the most common clinical isolates of systemic fungal infection. Long-term and inappropriate use of antifungal drugs can cause fungal resistance, which poses a great challenge to the clinical treatment of fungal infections. The combination of antifungal drugs and non-antifungal drugs to overcome the problem of fungal resistance has become a research hotspot in recent years. Our previous study found that the combination of rifapentine (RFT) and fluconazole (FLC) has a significant synergistic against FLC-resistant C. albicans. The present study aimed to further verify the synergistic effect between FLC and RFT against the FLC-resistant C. albicans 100, and explore the underlying mechanism. The growth curve and spot assay test not only showed the synergistic effect of FLC and RFT on FLC-resistant C. albicans in vitro but exhibited a dose-dependent effect on RFT, indicating that RFT may play a principal role in the synergic effect of the two drugs. Flow cytometry showed that the combined use of RFT and FLC arrested cells in the G2/M phase, inhibiting the normal division and proliferation of FLC-resistant C. albicans. Transmission electron microscopy (TEM) demonstrated that FLC at a low concentration could still cause a certain degree of damage to the cell membrane in the FLC-resistant C. albicans, as represented by irregular morphologic changes and some defects observed in the cell membrane. When FLC was used in combination with RFT, the nuclear membrane was dissolved and the nucleus was condensed into a mass. Detection of the intracellular drug concentration of fungi revealed that the intracellular concentration of RFT was 31-195 fold that of RFT alone when it was concomitantly used with FLC. This indicated that FLC could significantly increase the concentration of RFT in cells, which may be due to the damage caused to the fungal cell membrane by FLC. In short, the present study revealed a synergistic mechanism in the combined use of RFT and FLC, which may provide a novel strategy for the clinical treatment of FLC-resistant C. albicans.

Sirtuins in macrophage immune metabolism: A novel target for cardiovascular disorders.

Sirtuins (SIRT1-SIRT7), as a family of NAD+-dependent protein modifying enzymes, have various catalytic functions, such as deacetylases, dealkalylases, and deribonucleases. The Sirtuins family is directly or indirectly involved in pathophysiological processes such as glucolipid metabolism, oxidative stress, DNA repair and inflammatory response through various pathways and assumes an important role in several cardiovascular diseases such as atherosclerosis, myocardial infarction, hypertension and heart failure. A growing number of studies supports that metabolic and bioenergetic reprogramming directs the sequential process of inflammation. Failure of homeostatic restoration leads to many inflammatory diseases, and that macrophages are the central cells involving the inflammatory response and are the main source of inflammatory cytokines. Regulation of cellular metabolism has emerged as a fundamental process controlling macrophage function, but its exact signaling mechanisms remain to be revealed. Understanding the precise molecular basis of metabolic control of macrophage inflammatory processes may provide new approaches for targeting immune metabolism and inflammation. Here, we provide an update of studies in cardiovascular disease on the function and role of sirtuins in macrophage inflammation and metabolism, as well as drug candidates that may interfere with sirtuins, pointing to future prospects in this field.

Aneuploidy underlies brefeldin A-induced antifungal drug resistance in Cryptococcus neoformans.

Cryptococcus neoformans is at the top of the list of "most wanted" human pathogens. Only three classes of antifungal drugs are available for the treatment of cryptococcosis. Studies on antifungal resistance mechanisms are limited to the investigation of how a particular antifungal drug induces resistance to a particular drug, and the impact of stresses other than antifungals on the development of antifungal resistance and even cross-resistance is largely unexplored. The endoplasmic reticulum (ER) is a ubiquitous subcellular organelle of eukaryotic cells. Brefeldin A (BFA) is a widely used chemical inducer of ER stress. Here, we found that both weak and strong selection by BFA caused aneuploidy formation in C. neoformans, mainly disomy of chromosome 1, chromosome 3, and chromosome 7. Disomy of chromosome 1 conferred cross-resistance to two classes of antifungal drugs: fluconazole and 5-flucytosine, as well as hypersensitivity to amphotericin B. However, drug resistance was unstable, due to the intrinsic instability of aneuploidy. We found overexpression of AFR1 on Chr1 and GEA2 on Chr3 phenocopied BFA resistance conferred by chromosome disomy. Overexpression of AFR1 also caused resistance to fluconazole and hypersensitivity to amphotericin B. Furthermore, a strain with a deletion of AFR1 failed to form chromosome 1 disomy upon BFA treatment. Transcriptome analysis indicated that chromosome 1 disomy simultaneously upregulated AFR1, ERG11, and other efflux and ERG genes. Thus, we posit that BFA has the potential to drive the rapid development of drug resistance and even cross-resistance in C. neoformans, with genome plasticity as the accomplice.

RIPK1 plays a crucial role in maintaining regulatory T-Cell homeostasis by inhibiting both RIPK3- and FADD-mediated cell death.

Regulatory T (Treg) cells play an essential role in maintaining immune balance across various physiological and pathological conditions. However, the mechanisms underlying Treg homeostasis remain incompletely understood. Here, we report that RIPK1 is crucial for Treg cell survival and homeostasis. We generated mice with Treg cell-specific ablation of Ripk1 and found that these mice developed fatal systemic autoimmunity due to a dramatic reduction in the Treg cell compartment caused by excessive cell death. Unlike conventional T cells, Treg cells with Ripk1 deficiency were only partially rescued from cell death by blocking FADD-dependent apoptosis. However, simultaneous removal of both Fadd and Ripk3 completely restored the homeostasis of Ripk1-deficient Treg cells by blocking two cell death pathways. Thus, our study highlights the critical role of RIPK1 in regulating Treg cell homeostasis by controlling both apoptosis and necroptosis, thereby providing novel insights into the mechanisms of Treg cell homeostasis.