Strong Synergism of Palmatine and Fluconazole/Itraconazole Against Planktonic and Biofilm Cells of Candida Species and Efflux-Associated Antifungal Mechanism

Fungal infections caused by Candida albicans and non-albicans Candida [NAC] species are becoming a growing threat in immunodeficient population, people with long-term antibiotic treatment and patients enduring kinds of catheter intervention. The resistance to one or more than one conventional antifungal agents contributes greatly to the widespread propagation of Candida infections. The severity of fungal infection requires the discovery of novel antimycotics and the extensive application of combination strategy. In this study, a group of Candida standard and clinical strains including C. albicans as well as several NAC species were employed to evaluate the antifungal potentials of palmatine (PAL) alone and in combination with fluconazole (FLC)/itraconazole (ITR) by microdilution method, checkerboard assay, gram staining, spot assay, and rhodamine 6G efflux test. Subsequently, the expressions of transporter-related genes, namely CDR1, CDR2, MDR1, and FLU1 for C. albicans, CDR1 and MDR1 for Candida tropicalis and Candida parapsilosis, ABC1 and ABC2 for Candida krusei, CDR1, CDR2, and SNQ2 for Candida glabrata were analyzed by qRT-PCR. The susceptibility test showed that PAL presented strong synergism with FLC and ITR with fractional inhibitory concentration index (FICI) in a range of 0.0049–0.75 for PAL+FLC and 0.0059–0.3125 for PAL+ITR in planktonic cells, 0.125–0.375 for PAL+FLC and 0.0938–0.3125 for PAL+ITR in biofilms. The susceptibility results were also confirmed by gram staining and spot assay. After combinations, a vast quantity of rhodamine 6G could not be pumped out as considerably intracellular red fluorescence was accumulated. Meanwhile, the expressions of efflux-associated genes were evaluated and presented varying degrees of inhibition. These results indicated that PAL was a decent antifungal synergist to promote the antifungal efficacy of azoles (such as FLC and ITR), and the underlying antifungal mechanism might be linked with the inhibition of efflux pumps and the elevation of intracellular drug content

Extraction of Extracellular Matrix in Static and Dynamic Candida Biofilms Using Cation Exchange Resin and Untargeted Analysis of Matrix Metabolites by Ultra-High-Performance Liquid Chromatography-Tandem Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS)

Fungal infections caused by Candida albicans poses a great threat to human health. The ability of biofilm formation is believed to be associated with resistance-related Candida infections. Currently, knowledge on extracellular matrix (EM) of C. albicans biofilm is limited. In this study, we introduced ion exchange resin, i.e., cation exchange resin (CER) and anion exchange resin (AER), in EM extraction of C. albicans biofilm as well as several non-albicans Candida (NAC) biofilms under static and dynamic states in combination with vortexing and ultrasonication (VU). The metabolites extracted from the dynamic C. albicans biofilm matrix using the CER-VU and VU were identified with ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) via untargeted filtration. Compared with other physical and chemical extraction methods, CER-VU was demonstrated to be an ideal approach with high-yield acquisitions of EM constituents including proteins, triglycerides and carbohydrates and low-level damages on fungal cell viability and integrity. The untargeted MS analysis further showed the high efficacy of CER-VU, as a large quantity of metabolites (217 versus 198) was matched comprising a great number of lipids, carbohydrates, amino acids, nucleic acids and their derivatives together with a high involvement of signaling pathways compared with the VU alone. However, combining the results from both the CER-VU and VU methods could generate more metabolites. In summary, the EM analysis of the dynamic C. albicans biofilm expands our understanding upon a comprehensive depiction of matrix components and provides another effective approach for EM extraction

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

High triglyceride-glucose (TyG) index is associated with poor prognosis of heart failure with preserved ejection fraction

Abstract Background The impact of insulin resistance on the prognosis of heart failure with preserved ejection fraction (HFpEF) remains unknown. This study aimed to investigate the association between the triglyceride-glucose (TyG) index, an easily calculated marker of insulin resistance, and the long-term prognosis of HFpEF. Methods A total of 823 patients with HFpEF were enrolled in the study. The TyG index was determined using the formula ln(fasting triglycerides [mg/dL] × fasting glucose [mg/dL]/2). The primary endpoint was all-cause death. The secondary endpoints were cardiovascular (CV) death and heart failure (HF) rehospitalization. Restricted cubic spline, multivariate Cox proportional hazard models, and competing risk models were used for analyses. Results During a median follow-up period of 3.16 years, 147 (17.8%) all-cause deaths, 139 (16.8%) CV deaths, and 222 (27.0%) HF rehospitalizations occurred. Restricted cubic spline analysis revealed a J-shaped association between the TyG index and the mortality and rehospitalization rates. In the multivariate Cox proportional hazard models, compared with those in the lowest TyG index tertile, patients in the highest tertile exhibited the greatest susceptibility to all-cause death (HR 1.53, 95% CI 1.19–1.98) and CV death (HR 1.52, 95% CI 1.19–1.96). In the competing risk model, a significant association between the TyG index and HF rehospitalization was observed (HR 1.31, 95% CI, 1.07–1.61). Conclusion A high TyG index is associated with an increased risk of mortality and rehospitalization in patients with HFpEF. The TyG index may serve as a promising prognostic marker for patients with HFpEF