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Acetohydroxyacid synthase (AHAS, EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, is the target for more than 50 commercially available herbicides, and is a promising target for antimicrobial drug discovery. Herein, we have expressed and purified AHAS from Candida auris, a newly identified human invasive fungal pathogen. Thirteen AHAS inhibiting herbicides have K i values of <2 μM for this enzyme, with the most potent having K i values of <32 nM. Six of these compounds exhibited MIC50 values of <1 μM against C. auris (CBS10913 strain) grown in culture, with bensulfuron methyl (BSM) being fungicidal and the most potent (MIC50 of 0.090 μM) in defined minimal media. The MIC50 value increases to 0.90 μM in media enriched by the addition of branched-chain amino acids at the expected concentration in the blood serum. The sessile MIC50 for BSM is 0.6 μM. Thus, it is also an excellent inhibitor of the growth of C. auris biofilms. BSM is nontoxic in HEK-293 cells at concentrations >100 μM and thus possesses a therapeutic index of >100. These data suggest that targeting AHAS is a viable strategy for treating C. auris infections.

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Fungal infections have become a major concern in the medical community, especially those caused by Candida spp. Within this species, Candida albicans stands out for being an opportunistic commensal fungus that can cause superficial and invasive infections. Current antifungal therapy involves the local and/or systemic use of drugs such as azoles, polyenes, and echinocandins. These antifungals are based on highly specific target sites, and the development of resistance may occur with changes in the enzymatic pathways that serve as the drug targets. Thus, the development of new antifungal drugs is highly recommended to prevent drug resistance. The present investigation evaluated the antifungal activity of silver-containing microcrystals such as silver vanadate (α-AgVO3), silver tungstate (α-Ag2WO4), and silver molybdate (β-Ag2MoO4). In addition to having antimicrobial activity, such compounds should not cause damage to underlying tissues. Thus, to better assess the biocompatibility of new compounds, a new three-dimensional (3D) coculture model involving three cell lines was developed. The validation of the model was based on fluorescent markers and confocal laser microscopy. The biocompatibility of silver-containing microcrystals was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 3D coculture was infected with C. albicans biofilm and challenged with α-AgVO3, α-Ag2WO4, and β-Ag2MoO4. The action of microcrystals on C. albicans biofilm was evaluated by colony-forming units (CFU/ml) and LIVE/DEAD staining. In addition, production of proinflammatory cytokines interleukin 6 (IL-6), IL-8, IL-1β, and tumor necrosis factor α (TNF-α) was measured by cytometric bead array kit using flow cytometry. The 3D coculture model described here proved to be adequate to assess both the biocompatibility of the new materials and the infectious processes. Regarding the biocompatibility of the microcrystals, only α-AgVO3 (15.62 µg/ml) showed a decrease in cell viability. The antibiofilm activity of α-Ag2WO4 was similar to that of the standard drug (fluconazole). Although α-Ag2WO4 was able to induce the production of IL-6, IL-8, and IL-1β, no differences in cytokine production were observed between noninfected and infected models treated with this microcrystal. β-Ag2MoO4 inhibits the production of TNF-α in the infected model; however, it showed no antibiofilm activity. Based on the biocompatibility and antifungal findings, α-Ag2WO4 is a promising material for treating C. albicans infection.

In the current COVID-19 pandemic, the next generation of innovative materials with enhanced anti-SARS-CoV-2 activity is urgently needed to prevent the spread of this virus within the community. Herein, we report the synthesis of chitosan/α-Ag2WO4 composites synthetized by femtosecond laser irradiation. The antimicrobial activity against Escherichia coli, Methicilin-susceptible Staphylococcus aureus (MSSA), and Candida albicans was determined by estimating the minimum inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC). To assess the biocompatibility of chitosan/α-Ag2WO4 composites in a range involving MIC and MBC/MFC on keratinocytes cells (NOK-si), an alamarBlue™ assay and an MTT assay were carried out. The SARS-CoV-2 virucidal effects was analyzed in Vero E6 cells through viral titer quantified in cell culture supernatant by PFU/mL assay. Our results showed a very similar antimicrobial activity of chitosan/α-Ag2WO4 3.3 and 6.6, with the last one demonstrating a slightly better action against MSSA. The chitosan/α-Ag2WO4 9.9 showed a wide range of antimicrobial activity (0.49–31.25 µg/mL). The cytotoxicity outcomes by alamarBlue™ revealed that the concentrations of interest (MIC and MBC/MFC) were considered non-cytotoxic to all composites after 72 h of exposure. The Chitosan/α-Ag2WO4 (CS6.6/α-Ag2WO4) composite reduced the SARS-CoV-2 viral titer quantification up to 80% of the controls. Then, our results suggest that these composites are highly efficient materials to kill bacteria (Escherichia coli, Methicillin-susceptible Staphylococcus aureus, and the yeast strain Candida albicans), in addition to inactivating SARS-CoV-2 by contact, through ROS production.

This study aimed to evaluate the accuracy of panoramic radiographs obtained by digital system based on charge coupled device (CCD) and a phosphor storage plate (PSP) system with two different file formats, JPEG irreversible type and BMP. Linear measurements of artificial bony defects were made in dry mandibles with a digital caliper. Measures of digital panoramic radiographs were taken in ImageJ(r) software. Each measurement was performed twice by an experienced examiner. The intra-rater agreement was considered very strong (Pearson's correlation coefficient of 0.97). There was no significant difference between the linear measurements of dry mandibles, considered the gold standard, in relation to measurements obtained from digital radiographs (p=0.47). The error of these measures for the CCD was 1.04 mm (9.97%) for JPEG images and 1.03 mm (9.99%) for those with BMP format. For the PSP images, these values were 1.48 mm (14.94%) and 1.43 mm (14.43%), respectively. Although there was no statistical difference between the images with JPEG and BMP format, for both digital systems (p=1.00 for CCD and p=0.98 for PSP system), errors made on radiographs obtained by PSP system were significantly higher than those of CCD (p<0.05). The size of the files saved in JPEG was also significantly lower (p=0.005) compared with the files saved in BMP. It may be concluded that the digital radiographs, both saved in JPEG and BMP formats, are accurate, regardless the used digital system, and JPEG images are more suitable for teleradiology.

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