Intensification of therapy has been associated with early worsening of retinopathy prior to subsequent risk reduction. We sought to assess whether glycated hemoglobin (HbA1c) reduction, following flash monitoring, was associated with early worsening.An observational study in 541 individuals with type 1 diabetes and paired HbA1c and eye assessment prior to and following flash monitoring commencement.Change in HbA1c was −4 mmol/mol (IQR −9–1) (−0.4% (−0.8–0.1)) and 25% achieved a fall in HbA1c of ≥10 mmol/mol. The occurrence of the composite end point  was associated with baseline HbA1c >75 mmol/mol (9.0%) (HR 4.0 (95% CI 2.0 to 7.9), p<0.001) but not with fall in HbA1c of ≥10 mmol/mol (0.9%) (HR 1.6 (95% CI 0.8 to 3.2), p=0.203) over a follow-up period of 615 days (527–863). In multivariate analysis, diabetes duration (p=0.035) and prior retinopathy (p<0.001) were most predictive of the composite end point. Baseline HbA1c was the strongest predictor of worsening retinopathy (p=0.002) or new retinopathy (p=0.002) in multivariate analysis whereas change in HbA1c was not independently associated with either .Progression of eye disease is associated with baseline HbA1c, diabetes duration and previous retinopathy and such individuals should be monitored during intensification of glycemic therapy. Reassuringly, the extent of glucose lowering does not appear to be an independent risk factor for early worsening of eye disease in this context. Glycated hemoglobin (HbA1c) lowering has been associated with early worsening of diabetic retinopathy before risk reduction occurs.Baseline HbA1c and diabetes duration were associated with subsequent need for intervention for diabetic eye disease and with the development or worsening of diabetic eye disease.Change in HbA1c was not greater in those with subsequent panretinal photocoagulation, worsening of retinopathy or new development of retinopathy.When stratified based on HbA1c response, there were no differences in any eye outcomes when adjusted for baseline HbA1c.People with markedly elevated HbA1c, prior retinopathy and long diabetes duration require careful monitoring after commencement of flash monitoring, but these data offer reassurance that extent of HbA1c lowering may not be a major contributor to risk.Flash glucose monitoring provides users with an interstitial glucose value only on scanning a glucose sensor with a reader device or compatible mobile phone. In other respects, it is similar to conventional continuous glucose monitoring (CGM) in providing a 24 hours glucose trace and trend arrows.We conducted a prospective observational study of the first 589 individuals with type 1 diabetes commenced on National Health Service (NHS) funded flash monitoring  in a University hospital clinic  during February and March 2018. Glycemic outcomes for this cohort (including a group from another clinic within our center) have previously been reported, where the baseline characteristics are described in detail.10.1136/bmjdrc-2020-001668.supp1Supplementary dataThe primary outcome was an assessment of factors associated with the development of a composite end point comprising PRP, macular laser therapy or anti-VEGF therapy. Additional outcomes of interest were the individual components of the composite outcome, new onset of retinopathy, new onset of maculopathy and worsening of retinopathy. Retinopathy was classified as: none, mild background, intermediate  or proliferative. Worsening retinopathy was defined as at least one step up through these categories, with the most advanced category being recorded for individuals when a discrepancy existed between eyes. Retinopathy and maculopathy data were obtained from the national diabetic retinopathy screening programme,2 test or by Fisher’s exact test, when assumptions for χ2 test were not met. Logistic regression analysis was performed to identify associations with development or worsening of eye disease. Univariate analysis of the composite end point and need for PRP were assessed by log-rank test. Independent predictors of the composite end point and need for PRP were assessed by Cox proportional-hazard model. Significance was accepted at p<0.05. All analyses were performed using R Studio V.1.0.153.Data were largely non-normally distributed (as determined by Shapiro-Wilk test) and are presented as median and IQR. Paired data were analyzed by Wilcoxon signed-rank test and unpaired data by Mann-Whitney U test. Categorical data were analyzed by χClinical and demographic characteristics are summarized in At baseline, 31.4% had an HbA1c <58 mmol/mol (7.5%) which rose to 48.6% at next follow-up after commencing flash monitoring (p<0.001). Median change in HbA1c was −4 mmol/mol (−9–1) (−0.4% (−0.8–0.1)) at next follow-up (median 253 days (156–507), p<0.001) and −3 mmol/mol (−8–4) (−0.3% (−0.7–0.4)) at final follow-up (median 703 days (603–888), p<0.001). HbA1c reduction was more marked in individuals with HbA1c >75 mmol/mol (9.0%) at baseline (−11 mmol/mol (−23–−5), p<0.001) (−1.0% (−2.1–−0.5)). At baseline, 31.4% had an HbA1c <58 mmol/mol (7.5%), which rose to 48.6% at next follow-up after commencing flash monitoring (p<0.001); 47.5% achieved an HbA1c fall of 5 mmol/mol (0.5%) or greater , 25.0% achieved a fall of 10 mmol/mol (0.9%)  and 8.1% achieved a fall of 20 mmol/mol (1.8%) . Self-reported hypoglycemia measures, following commencement of flash monitoring, were not significantly different in those achieving an HbA1c reduction of 10 mmol/mol (0.9%) and those who did not .Median follow-up for occurrence of the composite end point  was 615 days (527–863) from commencement of flash monitoring. During this time, 34 of 541 individuals experienced an event at a median 218 days (112–469) from flash monitoring commencement, of whom 26 had a previous element of the composite end point prior to commencement . Twenty-seven individuals required PRP following flash monitoring commencement (median 187 days (100–382)), which was the first ever episode in 7/27 ), all of whom had maculopathy diagnosed prior to commencement and eight of whom had received previous laser treatment.At next follow-up, since commencement of flash monitoring (median 235 days (135–344)), 430 (79.5%) had no change in their retinopathy status . A deterioration was observed in 59 (10.9%) and improvement in 52 (9.6%). Next follow-up was within 6 months in 209/541 (47.3%) and within 12 months in 442 (81.7%). By final follow-up ), the corresponding figures were: no change in 279 (70.3%), deterioration in 72 (18.1%) and improvement in 46 (11.6%). For maculopathy, at first assessment, no change was observed in 496 (91.7%), deterioration in 28 (5.2%) and improvement in 17 (3.1%). The corresponding maculopathy figures at final assessment were 388 (97.7%), 3 (0.8%) and 6 (1.5%), respectively. Two hundred sixty-six individuals had no retinopathy at baseline of whom 48 (18.0%) had developed retinopathy at next follow-up .Younger age at diagnosis (13 years (8–21) vs 19 (12–31), p=0.010), longer diabetes duration (27 years (24–38) vs 23 (12–36), p=0.001), higher HbA1c at baseline (74 mmol/mol (57–81) vs 63 (55–71), p=0.006) (8.9% (7.4–9.6) vs 7.9 (7.2–8.6)) and the presence of previous eye disease or treatments  were allYounger age at diagnosis 13 years are all Worsening retinopathy status at next follow-up was associated with higher baseline HbA1c (69 mmol/mol (48–81) vs 63 (55–71), p=0.010) (8.5% (6.5–9.6) vs 7.9 (7.2–8.6)) and lower frequency of retinopathy at baseline  at first follow-up. It was not significantly associated with change in HbA1c following flash monitoring commencement . At finaNew onset of retinopathy  was associated with longer diabetes duration (24 years (14–33) vs 14 (8–30), p<0.001) and higher baseline HbA1c (65 mmol/mol (57–73) vs 61 (54–68), p=0.014) (8.1% (7.4–8.8) vs 7.7 (7.1–8.4)) but not change in HbA1c (−4 mmol/mol (−7–0) vs −3 (−9–−2), p=0.176) (−0.4 (−0.6–0) vs −0.3 (−0.8–−0.2)) Cox proportional-hazards analysis identified diabetes duration (HR 1.03 (95% CI 1.00 to 1.06) per year, p=0.035) and presence of baseline retinopathy (HR 6.58 (95% CI 2.26 to 19.20), p<0.001) but not baseline HbA1c (HR 1.03 (95% CI 1.00 to 1.05), p=0.075) or change in HbA1c (HR 0.99 (95% CI 0.96 to 1.03), p=0.702) as independently predictive of the composite end point.Cox proportional-hazards analysis identified baseline HbA1c (HR 1.03 per mmol/mol (95% CI 1.01 to 1.06), p=0.015) and presence of baseline retinopathy (HR 11.5 (95% CI 2.64 to 50.2), p=0.001) but not change in HbA1c (HR 1.01 per mmol/mol (95% CI 0.98 to 1.05), p=0.568) or diabetes duration (HR 1.02 per year (95% CI 0.99 to 1.05), p=0.153) as independently predictive of subsequent PRP.HbA1c at baseline (OR 1.04 per mmol/mol (95% CI 1.01 to 1.06), p=0.002) and diabetes duration (OR 1.03 per year (95% CI 1.01 to 1.05), p=0.005) but not change in HbA1c (OR 1.00 per mmol/mol (95% CI 0.97 to 1.04), p=0.830) were independently predictive of new development of retinopathy in logistic regression analysis. HbA1c at baseline (OR 1.03 per mmol/mol (95% CI 1.01 to 1.05), p=0.002) but not diabetes duration (OR 1.01 per year (95% CI 0.99 to 1.03), p=0.261) or change in HbA1c (OR 1.00 per mmol/mol (95% CI 0.97 to 1.03), p=0.930) were independently predictive of worsening retinopathy in logistic regression analysis.We have demonstrated that HbA1c lowering, in the context of flash monitoring commencement, is not independently predictive of clinically important changes in diabetic eye disease in the short-term. HbA1c prior to commencement of flash monitoring, as well as diabetes duration and pre-existing retinopathy were all independently associated with progression of eye disease. Intensification of glycemic management in type 1 diabetes is unequivocally associated with reduction in the development and progression of diabetic retinopathy,We hypothesized that advances in the management of type 1 diabetes since the early 1980s  and specific features of intensification relating to flash monitor use may have reduced the risk of early worsening of eye disease following HbA1c reduction. Randomization to intensive glycemic control in DCCT was associated with a threefold increase in severe hypoglycemia while we did not, as previously reported, observe an increase in severe hypoglycemia following commencement of flash monitoring.20The cohort described in our study is different from the DCCT cohort in a number of important respects: older age 46 vs 27 years), longer duration of diabetes (23 vs 6 years) and higher prevalence of prior diabetic eye disease intervention (12.9% vs none in DCCT). Baseline HbA1c (63 mmol/mol (7.9%) vs 76 mmol/mol (9.1%)) was lower in our study, although baseline HbA1c in those with a 10 mmol/mol (0.9%) or greater fall in HbA1c (72 mmol/mol (8.7%)) was similar to the DCCT cohort. Our cohort is broadly representative of typical clinical practice in the UK, although, as previously described, it is slightly skewed toward lower than average HbA1c, younger age, CSII use and lower socioeconomic deprivation than our total clinic population.7 years, The study is open to the usual criticisms of observational methodology particularly the potential influence of unmeasured confounders. As a ‘real-world’ assessment, the timing of HbA1c measurement and eye assessment was not uniform and reflected the expected variation in normal clinical practice. A minority of individuals did not have an eye assessment within 1 year of flash monitoring commencement, meaning some early worsening may have been missed. Lack of uniformly timed HbA1c and eye assessments limits our ability to comment on the potential influence of rate of change in HbA1c. However, we have no reason to suspect variation in follow-up intervals introduced any systematic bias. An advantage of this ‘real-world’ methodology is its likely generalizability, in the absence of stringent exclusion and inclusion criteria, to modern diabetes clinic populations in the UK and beyond. The DCCTElevated HbA1c, longer duration of diabetes and pre-existing retinopathy are all significant risk factors with respect to diabetic eye disease in people with type 1 diabetes commencing flash monitoring. However, in the short-term, subsequent change in HbA1c does not appear to independently predict the risk of retinopathy development or progression nor the need for interventions to treat advanced eye disease in a representative cohort of flash monitoring users. These single-centre observational findings, while offering a degree of reassurance, clearly require corroboration from larger national datasets and randomized controlled trials of novel glucose-lowering technologies. Understanding the risk of abrupt reduction in HbA1c, as well as the potential moderating influence of hypoglycemia and glucose variability, will become increasingly important as we approach the era of closed-loop insulin delivery.


As an independent, negative-prognostic biomarker for progression-free survival (PFS) and overall survival (OS), circulating tumor cells (CTCs) constitute a promising component for developing a liquid biopsy for patients with metastatic breast cancer (MBC). The effects of HER2-targeted therapy such as trastuzumab, pertuzumab, T-DM1, and lapatinib on CTC status and longitudinal enumeration were assessed in this trial.n = 28), patients with de novo HER2-positive MBC and no HER2-targeted therapy in the last 12 months prior to enrollment and start of HER2-targeted therapy (n = 15), and patients with HER2-nonamplified disease and no HER2-targeted therapy (n = 212).CTC status of 264 patients with MBC was analyzed prior to and after 4 weeks of a new line of palliative systemic therapy. CTCs were assessed using CellSearch®. Three groups were compared: patients with HER2-positive MBC receiving ongoing HER2-targeted therapy (p = 0.02) of patients, respectively. At least one CTC/7.5 ml was seen in 28.6, 53.3, and 67.0% (p < 0.001) of these patients. Furthermore, 3.6, 40.0, and 3.3% (p < 0.001) of the patients had at least one HER2-positive CTC. After 4 weeks of therapy 7.1, 0.0, and 31.1% (p = 0.001) of patients had still a positive CTC status (≥ 5 CTC/7.5 ml blood). At least one CTC/7.5 ml was still observed in 25.0, 20.0, and 50.5% (p = 0.004) of the patients. Furthermore, 7.1, 0.0, and 1.9% (p = 0.187) had at least one HER2-positive CTC. After 3 months of therapy, 35.7, 20.0, and 28.3% (p = 0.536) showed disease progression.Positive CTC status (≥ 5 CTC/7.5 ml blood) at enrollment was observed in the 3 groups for 17.9, 46.7, and 46.2% (HER2-targeted therapy seems to reduce the overall CTC count in patients with MBC. This should be taken into account when CTC status is used as an indicator for aggressive or indolent metastatic tumor disease. Worldwide, metastatic breast cancer (MBC) is a major cause of cancer-related death in women –3. MBC, Apart from solid metastases, a liquid biopsy as detector and surrogate of the systemic tumor burden is needed to take into account the heterogeneity of the disease . As an iAs CTCs reflect a subpopulation of the total tumor cell population, characterization and treatment of CTC might be a promising tool for optimizing therapy . Meng etRetrospective studies and xenograft models suggest that HER2-targeted monoclonal antibody therapies might even be able to target both HER2-nonamplified cancer cells and cancer stem cell populations via antibody-dependent, cell-mediated cytotoxicity (ADCC) . In a phTo further investigate these controversial findings, this retrospective study was conducted to demonstrate the immediate effect of HER2-targeted therapies on CTCs in the metastatic setting.n = 264). Exclusion criteria were: no blood draw after 4 weeks of therapy (n = 207), no blood draw at enrollment (n = 16), patients who were already included in the trial (n = 13), no follow-up information (n = 9), no metastatic disease (n = 3), or withdrawal of patient’s consent to participate in the study (n = 2). Furthermore, ≥ 5 CTCs per 7.5 ml peripheral blood was defined as CTC-positive ) after 4 weeks of HER2-targeted therapy  but the rate of positive CTC status (≥ 5 CTC) at enrollment was higher in the “New HER2 therapy” group (46.7 vs. 30.6%). After 4 weeks of therapy, ≥ 1 CTC were found in 20.0 vs. 38.8% of patients and a positive CTC status was observed in 0 vs. 22.4% of patients. PD rate was similar at 20.0 vs. 20.4%. PFS and OS were better in the “New HER2 therapy” group .As other retrospective studies and xenograft models have shown that HER2-targeted monoclonal antibody therapies might even be able to target HER2-nonamplified cancer cells and cancer stem cell populations including CTCs, the indication for HER2-targeted therapies might need to be extended , 26. AntThe clinical relevance of CTC status on prognosis, including HER2-positive patients, is undisputed . These fOne main limitation lies in the fact that this is a retrospective analysis. Another limitation is the small number of analyzed patients in the “HER2 Therapy” and “New HER2 therapy” groups. Indeed, the small number of patients with PD highlights the implicit difficulties in analyzing the rate of CTC-positive cases. The subgroup receiving anti-HER2 therapy can therefore only be reported descriptively and used to generate new hypotheses about therapy effects on CTC levels.First-line HER2-targeted therapy of metastatic breast cancer seems to reduce CTC levels greater than endocrine or chemotherapy. Ongoing anti-HER2 therapy seems to be associated with lower overall CTC levels.


Enterococcus faecium EBD1 significantly increased the ACE inhibitory ability to 75 ± 5%, while captopril showed an ACE inhibition of 92 ± 4%. An untargeted metabolomics approach using Ultra-high-performance liquid tandem chromatography quadrupole time of flight mass spectrometry revealed the abundance of vitamins, phenolic compounds, antioxidant peptides, DPP IV inhibitory peptides, and antihypertensive peptides in the fermented samples which may account for its strong ACE inhibition. Although fermented DP had decreased fatty acid levels, the amount of essential amino acid improved drastically compared to destarched rice. Our results show that fermenting Prozyme-treated destarched rice with Enterococcus faecium EBD1 generates abundant bioactive compounds necessary for developing antihypertensive functional foods.Enzyme treatment and fermentation of cereals are known processes that enhance the release of bound bioactive compounds to make them available for bioactivity. In this study, we tested the angiotensin converting enzyme (ACE) inhibitory ability of destarched rice, Prozyme 2000p treated destarched rice (DP), and fermented DP samples. Prozyme 2000p treatment increased the ACE inhibitory ability from 15 ± 5% to 45 ± 3%. Fermentation of the Prozyme 2000p treated samples with  Aliquots (1 mL) of the supernatants were filtered through 0.25 µm pore size Millex syringe filters  and transferred into LC-MS vials. LC-MS/MS analysis was carried out using a UHPLC  connected to a controller, a pump, a degasser, an autosampler, column oven, and a photodiode array detector (ExionLC) coupled to a quadrupole time-of-flight mass spectrometer (Q-TOF-MS) (X500R QTOF). The analytical column used was a 100 × 3 mm, Accucore C18 column . Solvent A consisted of water with 0.1% formic acid and solvent B was methanol. The chromatography was carried at a flow rate of 0.4 mL/min. A linear gradient was programmed for 25 min as follows: 0–3.81 min, 9% to 14% B; 3.81–4.85 min, 14% to 15% B; 4.85–5.89 min, 15% B; 5.89–8.32 min, 15% to 17% B; 8.32–9.71 min, 17% to 19% B; 9.71–10.40 min, 19% B; 10.40–12.48 min, 19% to 26% B; 12.48–13.17 min, 26% to 28% B; 13.17–14.21 min, 28% to 35% B; 14.21–15.95 min, 35% to 40% B; 15.95–16.64 min, 40% to 48% B; 16.64–18.37 min, 48% to 53% B; 18.37–22.53 min, 53% to 70% B; 22.53–22.88 min, 70% to 9% B; 22.88–25.00 min, 9% B. The injection volume was 5 μL. The Q-TOF-MS was set for the negative mode through a mass range of 100–1000 and a resolution of 5000. The capillary and cone voltages used to record full mass spectra were 1.45 kV and 30 V, respectively. The flow rate of Helium (the cone gas) was 45 L/h, while the flow rate of the desolvation gas (N2) was 900 L/h. The temperature of N2 was 250 °C, the ion source temperature was 120 °C, while the collision energies needed to obtain the MS/MS spectra were set at 15, 20, and 30 V.Each rice sample (1 g) was extracted with 20 mL of 50% methanol and placed on a mini rocker  overnight. The samples were mixed completely for 30 s using a vortex and subsequently centrifuged at 12,000× m/z) were ultimately imported into Microsoft Excel.All data were obtained and processed using SCIEX OS 1.0 software. A non-target algorithm was used for peak finding. Matrix and sample specific signals were separated from true contaminations using an automatic sample-control comparison algorithm. Compound identification was done by using empirical formula finding, MS/MS library searching, and online database searching. Compound names, peak area, retention time, similarity to metabolites in the database, and mass  statistical software system . http://biit.cs.ut.ee/clustvis/) was used in multivariate statistical analyses, including principal component analysis (PCA) and heat maps [ClustVis software  increased the ACE inhibitory ability of destarched rice from 15 ± 5% to 45 ± 3%  demonstrated a 92 ± 4% inhibitory ability.Prozyme treatment significantly  and essential amino acids. The fermented DP samples also contained known antihypertensive phenolic compounds as well as antihypertensive peptides which make the sample a promising material for developing cheap antihypertensive foods. Despite the antihypertensive potential of the fermented product, further studies regarding the ability of


An increase in PVP concentration to 20% w/v in both presence and absence of HA rendered fibers of approx. 1 µm. PVP 5%-HA 0.8% w/v fibers were loaded with 83.3 ± 14.0 µg FA per mg. After nanofibers crosslinking with ε-PL, blank and FA-loaded inserts showed a mean thickness of 270 ± 21 µm and 273 ± 41 µm, respectively. Blank and FA-loaded inserts completely released ε-PL within 30 min under sink conditions, whereas FA-loaded inserts released the antioxidant within 20 min. Both blank and FA-loaded inserts were challenged against Pseudomonas aeruginosa and Staphylococcus aureus, demonstrating their efficacy against relevant microbial species.Electrospun nanofibers are gaining interest as ocular drug delivery platforms that may adapt to the eye surface and provide sustained release. The aim of this work was to design an innovative ophthalmic insert composed of hyaluronan (HA) nanofibers for the dual delivery of an antioxidant  and an antimicrobial peptide . Polyvinylpyrrolidone (PVP) was added to facilitate the electrospinning process. Fibers with diameters of approx. 100 nm were obtained with PVP 5%-HA 0.8%  Electrospinning is becoming a popular technique due to the mild processing conditions and the versatility of substances that can be processed . It may Although less investigated, electrospun nanofibers of natural and synthetic polymers may also find applications as drug delivery platforms, particularly in those cases that thin devices with high specific surface area are required to facilitate the delivery of poorly soluble drugs ,10. DrugIn the ocular field, electrospun nanofibers are gaining interest as soft materials that can easily adapt to cornea and sclera surfaces and remain on the eye surface for a moderate period of time acting as sustained release platforms ,14,15. THyaluronic acid HA,  is a widPolyvinylpyrrolidone PVP, , anotherStreptomyces albulus with a variable number of L-lysine residues bound through amide linkage between ε-amino and α-carboxyl groups [Green or natural crosslinking agents have been proposed to prepare HA networks . ε-polyll groups ,35,36. TThe aim of this work was to design ocular inserts of HA-based fibers for ferulic acid FA,  and ε-PLEpsiliseen®-H  was purchased from Siveele B.V. . Trans-ferulic acid , Pluronic® F68 , polyvinylpyrrolidone , sodium hyaluronate  and Tryptan blue powder were from Sigma Aldrich . Polyethylene oxide  was purchased from Thermo Fisher Scientific . Ethanol absolute AnalaR Normapur®  was purchased from VWR Chemicals . Water was purified using reverse osmosis . Supragradient HPLC grade acetonitrile and acetic acid glacial (reagent grade) were from Scharlau Chemicals . NaCl was from Panreac Quimica S.L.U. .v/v) mixture overnight under gentle magnetic stirring at room temperature at different concentrations . HA was then added to PVP solutions and hydrated under magnetic stirring overnight at room temperature; 20%, 10%, 5% and 2.5% w/v of HA were added to 20%, 10%, 5% and 2.5% w/v PVP solutions, respectively. The pH of the solutions was close to neutrality.PVP was dissolved in EtOH:water 4:6 (w/v in EtOH:water 4:6 mixture (v/v) by vortexing before addition of PVP (5% w/v) and HA (0.8% w/v).For FA-loaded nanofibers, the antioxidant was solubilized in a concentration equal to 0.5% Rheological analysis of electrospinning dispersions was performed using a Rheolyst AR-1000N rheometer fitted with an AR2500 data analyzer, a Peltier plate, and a 6-cm in diameter and 2.1° cone geometry . Viscosity measurements were recorded at 20 °C. Experiments were performed using a single batch for each dispersion.w/v PVP solution. Voltages applied to each solution were 15 kV for solutions containing 5%–20% w/v PVP, 18 kV for 2.5% w/v PVP solution and 25 kV for 10% w/v PVP solution (solely or mixed with HA). FA-loaded nanofibers were prepared from FA dispersion  using 0.1 mL/h flow rate and 15 kV voltage. Electrospun fibers were deposited on a piece of aluminum foil above the aluminum collector plate. All experiments were carried out at room temperature and relative humidity of 30%–40%. Solutions (5 mL) were electrospun onto an aluminum grounded target placed at 25 cm from the needle tip. The solutions were placed in a 5 mL plastic syringe of polypropylene connected to a 22-needle gauge . The flow rate and high-voltage power source were controlled using a Yflow® Professional Electrospinning Machine . Flow rates were 0.1 mL/h for PVP 2.5%–HA 1% and PVP 5%–HA 0.8% dispersions, 0.5 mL/h for PVP 10%–HA 0.5%, and 0.6 mL/h for PVP 20%–HA 0.2% dispersions. In absence of HA, flow rate of 1 mL/h was used for electrospinning 20% Approx. 2 mg of FA-loaded nanofibers were immersed in 1 mL of MilliQ water for 5 min. Then, solutions were 20-fold diluted in MilliQ water and the absorbance recorded at 290 nm  for drug quantification. The experiments were carried out in triplicate.Scanning electron microscope (SEM) images of blank and drug-loaded nanofibers were obtained using a FESEM Ultra Plus  at various magnifications. Samples were put onto metal plates and sputter-coated with 10 nm thick iridium film  before viewing.2, layer thickness ≈ 0.5 mm) were placed on a Petri dish and 0.05 mL ε-PL 50 mg/mL were poured onto the nanofibers. Mixtures were then casted and dried at 37 °C overnight.Nanofibers-based inserts were prepared by crosslinking with ε-PL. Nanofibers  and the weight recorded. SEM images of blank and FA-loaded inserts were obtained as described in w/v of a 6 well plate at 35 °C under oscillation . Release medium (1 mL) was sampled every 10 min and immediately replaced with the same volume of NaCl 0.9%. FA and ε-PL amounts released were monitored up to 30 min, and samples filtered before drug quantification . Blank and FA-loaded inserts  were placed in wells containing 2 mL of NaCl 0.9% v/v) ratio, pumped at 1 mL/min. The injection volume was 90 μL and the absorbance was recorded at 290 nm. Retention time was 4.7 min. FA calibration curves were built in the range 5–25 µg/mL (R2 > 0.999).FA was quantified using a reverse-phase Symmetry® C18 cartridge , thermostated at 30 °C using a HPLC-UV system . The mobile phase was a mixture of acetonitrile: 2% acetic acid aqueous solution 19:81 (2 > 0.992).ε-PL quantification was performed by using a colorimetric method . BrieflyHen’s Egg Test Chorioallantoic Membrane (HET-CAM) assay  was carr2 flasks using complete culture medium ) until 80% confluence was reached. Then, cells were trypsinized using TripLE solution  and seeded in wells of 96 well plates (5000 cells/well). After 24 h, culture medium was removed and 100 μL of the sample solutions (4 mg/mL) and 100 μL of fresh culture medium were added to each well. Culture medium was used as negative control. Cells were again incubated at 37 °C for 24 or 48 h. At each timepoint, the medium was discarded and the cells were washed with PBS. Immediately after, freshly prepared CCK8 working solution (100 μL) consisting of 90 μL of culture medium and 10 μL of CCK-8 reagent  was added to each well and then the cells incubated for 2 h at 37 °C. Finally, absorbance (450 nm) was recorded using a microplate reader .Fibers and inserts were weighed (4 mg) and added in separate to 1 mL of culture medium. The tubes were vortexed until specimen dissolution and kept at 37 °C. BalB/3T3 cells  were cultured at 37 °C in 75 cmPseudomonas aeruginosa  or Streptococcus aureus . Blank and FA-loaded inserts were placed on the inoculated agar plates and the zones of inhibition were measured after incubation at 35 °C for 24 h. All experiments were carried out in duplicate.The antibacterial activity was evaluated on Müller–Hinton agar plates seeded with p < 0.05.All data are reported as mean value ± sd. Differences were analyzed using ANOVA and multiple range test . Differences were considered statistically significant when The present study was aimed at designing an ophthalmic platform able to increase FA residence time on the ocular surface, assuring in the meantime ε-PL release to the eye. Ophthalmic inserts could offer an increased ocular residence, controlled drug release and accurate dosing. The improved residence time of the drug in the conjunctival sac may enhance drug ocular availability with less frequent administration and fewer side effects than eyedrops .HA based nanofibrous membranes have been previously investigated as biomimetic tissue engineering scaffolds, wound healing materials, and drug delivery systems . Howeverv/v during the preparation of HA solutions  did not ameliorate the electrospinning process and the Taylor cone was not stable.High MW sodium hyaluronan (MW 600–1100 kDa) was chosen for preparing electrospinning solutions at low concentrations. Preliminarily, electrospinning of HA aqueous dispersion was investigated. In accordance to literature , fibers w/v Pluronic F68 to 0.2% w/v HA in 20% EtOH: 80% water and 40% EtOH: 60% water mixtures (v/v) did not lead to stable Taylor cone either. Similar results were observed by using 0.5% w/v HA concentration under the same conditions. The addition of PEO 200 (12% w/v) to 0.2%, 0.3%, 0.5%, 0.8% w/v HA aqueous solution did not improve the process either.Incorporation of other chemical compounds was also investigated to overcome unfavorable physicochemical properties of HA solutions. Ionic surfactants may increase the net charge density and the instability of the charged jet leading to finer fibers . The addNext attempt was carried out with high molecular weight PVP (360 kDa) due to its already demonstrated capability to render electrospun nanofibers . HA:PVP v/v are shown in w/v dispersions (not shown). Fibers with diameters of approx. 100 nm were obtained for 5% and 10% w/v PVP concentration; namely PVP 5%–HA 0.8% w/v and PVP 10%–HA 0.5% w/v had average diameters of 116 ± 48 and 48 ± 13 nm. An increase in PVP concentration to 20% w/v in both presence and absence of HA rendered fibers of approx. 1 µm; namely, the average diameters of PVP 20% w/v and PVP 20%–HA 0.2% w/v were 1224 ± 429 and 1166 ± 346 nm. The fibers showed homogenous surface indicating that all the process parameters were well controlled.SEM micrographs of electrospun fibers prepared varying PVP and HA concentrations in EtOH:water 4:6 w/v and PVP 20%–HA 0.2% w/v .2. Conversely, crosslinked FA-loaded inserts appeared light yellow showing a mean thickness of 273 ± 41 µm and a weight of 46.8 ± 8.8 µg/cm2. SEM images of blank cross-linked inserts revealed the presence of nanofibers structures even after electrostatic crosslinking, whereas these structures seemed not to be conserved on inserts surface in case of FA- loaded inserts  released 91% ± 6% in 10 min and 100% of the loaded drug in 20 min. Concerning ε-PL loading, FA presence did not affect the peptide concentration into the inserts (12.2% ± 2.8% and 17.6% ± 3.1% w/w for blank and FA-loaded inserts) neither the peptide release. Blank inserts released 78% ± 20%, 77% ± 15% and 100% of ε-PL in 10, 20 and 30 min, respectively. Similarly, FA-loaded inserts released 70% ± 26%, 93% ± 9% and 100% of peptide in 10, 20 and 30 min, respectively. It should be noticed that under physiological conditions the volume of tears is limited and thus the insert could hydrate, adhere to the mucus on the ocular surface and release the drug more slowly [Then, the inserts were tested regarding their capability to release both FA and ε-PL. Although no specific methods are reported in Pharmacopoeias for studying drug release from ophthalmic dosage forms, in vitro studies under sink conditions in NaCl 0.9% medium may be useful as first step for studying the behavior of ophthalmic drug delivery systems . The weie slowly ,52. Stroe slowly , which cPseudomonas aeruginosa and Staphylococcus aureus and the growth inhibition zones  may contribute to forecast the suitability of the crosslinked inserts for ocular application.


Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, with the exception of clade III. Azoffluxin also inhibits efflux in highly azole-resistant strains of Candida albicans, another human fungal pathogen, increasing their susceptibility to fluconazole. Furthermore, azoffluxin enhances fluconazole activity in mice infected with C. auris, reducing fungal burden. Our findings suggest that pharmacologically targeting Cdr1 in combination with azoles may be an effective strategy to control infection caused by azole-resistant isolates of C. auris. Candida auris is resistant to multiple drugs including the common antifungal fluconazole. Here, Iyer et al. identify a compound that potentiates fluconazole activity against C. auris in vitro and in vivo by inhibiting a major efflux pump and thus increasing intracellular fluconazole accumulation.The fungal pathogen  Although the focus has primarily been on pan-resistant bacteria, there is growing concern over the multidrug-resistant fungal pathogen, Candida auris. This emerging pathogen has galvanized researchers, health care workers, and the media due to its high rates of drug resistance and transmissibility3. In its most recent report, the U.S. Centers for Disease Control and Prevention classified C. auris as one of only five pathogens that are the most urgent threat to public health4. Thus, the emergence of C. auris highlights the need for more therapeutic options to combat drug-resistant fungal infections.The rise in antimicrobial resistance has become a major threat to public healthC. auris has an interesting history. Since it was first identified in 2009 in Japan5, genomic analyses have revealed the near simultaneous emergence of distinct lineages across six continents, encompassing over 30 countries within the past ~400 years7. Currently, the majority of C. auris isolates fall into four major geographical clades: South Asian (I), East Asian (II), African (III), and South American (IV)7. This species has a remarkable ability to persist on human skin and other surfaces for extended periods of time, which facilitates hospital transmission amongst patients who are already vulnerable to infection9. Additionally, the prevalence of drug resistance amongst C. auris isolates is widespread as recent studies show that over 80% of clinical isolates are resistant to the azole antifungal fluconazole7. Resistance levels vary significantly between clades, with some isolates exhibiting resistance to all three major antifungal drug classes available to treat systemic infections7.C. auris isolates is challenging from a clinical perspective as fluconazole is the most widely administered antifungal. This is due to its oral bioavailability, broad spectrum of activity, and favorable safety profile10. Fluconazole inhibits the biosynthesis of ergosterol, the major sterol in fungal cell membranes, through inhibition of lanosterol demethylase, which is encoded by ERG1111. Inhibition leads to an increase in the Erg11 substrate lanosterol, and the production of aberrant sterol intermediates, including 14-α-methyl-3,6-diol13. Mechanisms of fluconazole resistance amongst C. auris isolates are highly variable and often clade specific, the nuances of which are still being elucidated. One major mechanism of fluconazole resistance involves point mutations in hot spot regions in its target gene ERG11, which are known to confer resistance in other fungi16. In addition to ERG11 mutations that are shared across all clades7, the most common substitutions found in clade I and IV are Erg11Y132F or Erg11K143R, whilst clade III isolates commonly have an Erg11F126L substitution. Notably, strains from clade II generally have no specific ERG11 mutations and include the most sensitive isolates16. In addition to target alteration, C. auris encodes an array of multidrug transporters, several of which are strongly induced under various conditions, including fluconazole treatment20. Finally, C. auris isolates possess other genetic alterations that could confer fluconazole resistance, such as gene duplication leading to a higher copy number of ERG1118, or transcriptional upregulation of efflux pumps through mutations in TAC1B21. Overall, the diversity of C. auris resistance mechanisms is extensive, and the prevalence of fluconazole resistance threatens to render this important therapeutic obsolete in treatment of the rising number of C. auris infections world-wide.The prevalence of fluconazole resistance amongst 10. By identifying agents that re-sensitize pathogens to existing therapeutics, the lifespan of existing antifungals could be extended. In vitro data suggests combining existing antifungals can be effective against C. auris23. An excellent example of the potential value of combination therapy is provided by iKIX1, a novel compound that inhibits interaction of the transcription factor Pdr1 with the Mediator complex in the fungal pathogen Candida glabrata, thus preventing upregulation of the multidrug transporter Pdr524. Combination treatment with iKIX1 and fluconazole abrogated intrinsic azole resistance and improved survival in a murine model of C. glabrata infection24. Clearly, the inclusion of agents capable of impairing the most common, readily anticipated modes of antifungal resistance provides a rational, readily implemented strategy in the development of more efficacious combination treatment regimens.A well-established strategy to thwart drug resistance and restore antimicrobial efficacy is the use of combination therapy, which has been successfully implemented for many difficult to treat infections, including HIV-AIDS, tuberculosis, and malariaC. auris to identify molecules that specifically enhanced the activity of fluconazole. We identified azoffluxin as a compound that synergized with fluconazole by increasing intracellular fluconazole accumulation through inhibition of the major multidrug efflux transporter Cdr1. Using azoffluxin as a chemical probe, we established that efflux is a major mechanism of resistance in isolates belonging to three of the four major C. auris clades. Notably, clade III isolates carrying specific mutations in ERG11, in addition to upregulating the multi-drug transporter Mdr1, remained resistant to fluconazole in the presence of azoffluxin, despite the compound blocking efflux of Nile red and fluconazole in these isolates. Azoffluxin showed cross-species activity by potentiating fluconazole activity against a resistant isolate of Candida albicans, the most common human fungal pathogen25. In culture, azoffluxin transformed fluconazole from ineffective to highly active in rescuing mammalian cells infected with drug-resistant C. auris. In mice infected with drug-resistant C. auris, azoffluxin not only enhanced fluconazole activity but also reduced fungal burden by ~1000-fold as a single agent. Collectively, our findings demonstrate pharmacological inhibition of Cdr1 function may be an effective way to control infection with azole-resistant isolates of C. auris.In this study, we applied a combinatorial approach to screening of a chemically diverse library against an azole-resistant strain of C. auris, we screened a diversity-oriented, synthetic library created by Boston University’s Center for Molecular Discovery (BU-CMD). This library of 2454 molecules, many natural product-inspired, has been curated to encompass greater structural complexity than conventional chemical libraries26, which is a feature that increases the likelihood of identifying compounds with bioactivity against microorganisms28. The BU-CMD library was screened at 50 μM in the absence or presence of a concentration of fluconazole that inhibited growth of the fluconazole-resistant clade I C. auris strain VPCI 673/P/12 by ~20%. Through Sanger sequencing we confirmed this strain harbored both an Erg11K143R substitution and Tac1bA640V substitution. Compounds that reduced growth after 48 h compared to the control by 7-median absolute deviations from the median alone were classified as single agent antifungals; their mechanism of action has been described elsewhere29. Compounds for which antifungal activity was only observed in combination with fluconazole were classified as fluconazole potentiators . The fractional inhibitory concentration index (FICI) calculated for the combination was 0.25, with values <0.5 indicating a synergistic interaction  reduced the fluconazole minimum inhibitory concentration (MIC) >8-fold, from >256 μg/mL to 32 μg/mL on YPD agar , C. glabrata (BG2), and Saccharomyces cerevisiae (BY4741), to represent diverse fungi. Interestingly, azoffluxin did not enhance the activity of fluconazole against any of these species even in the presence of the highest concentration of fluconazole that did not impair growth in each species on its own  in the cdr1Δ strain . Examining whole genome sequences of 304 isolates representing each of the four major clades7, identified a unique non-synonymous substitution, N647T, in the transcription factor domain of Mrr1 (B9J08_004061) in 49 of 51 clade III isolates. In C. albicans, Mrr1 is a transcription factor that controls the expression of the major facilitator superfamily (MFS) transporter Mdr1, which is involved in fluconazole efflux41. Indeed, when expression of MDR1 (B9J08_003981) was assessed in the clade III isolates B11221 and B11222, we observed a >6-fold increase in expression under nearly all conditions tested compared to the clade I screening strain Ci6684, suggesting that MDR1 is constitutively upregulated in clade III isolates  that does not contain the Erg11 substitutions or the Mrr1 substitution shared by most members of this clade7. While more sensitive to fluconazole than other clade members at baseline, this strain showed increased Nile red accumulation upon treatment with azoffluxin  cells when infected with fungusnal Fig. . Howevernal Fig. . Notablytro Fig. .Fig. 7PrC. auris (stained pink) in wells exposed to azoffluxin alone or low fluconazole alone, comparable to the untreated co-culture. With combination treatment, we observed an intact human cell monolayer which was similar to the no fungus control, and scant fungal burden. In the high fluconazole condition, some disruption of the human cell monolayer was evident and fungal cells were readily apparent, suggesting that high fluconazole was effective at reducing fungal toxicity to the monolayer, but less effective than our combination treatment in arresting fungal proliferation  staining of polysaccharides to visualize effects of the various treatments on both the fungal and human elements within the co-cultures. Results supported findings obtained using the quantitative assay. We observed extensive damage to the human cell monolayer  and sloughing in conjunction with the presence of abundant ion Fig. .48, and caspofungin, a stable clinical antifungal  as a new compound that enhances the susceptibility of resistant fungal pathogens to diverse intracellularly acting antifungals. This compound inhibits the activity of multidrug efflux transporters, most notably the ABC transporter Cdr1. Enhanced multidrug efflux is a frequently encountered and problematic mechanism of antimicrobial resistance54. 3,3-diarylated oxindoles are a subset of the medicinally “privileged” 3,3-disubstituted oxindole class, which have a rich history of reported biological activities. For example, the diphenolic oxindole oxyphenisatin and other 3,3-diarylated oxindoles have been widely reported to inhibit the growth of diverse cancer cell lines, in many cases the activity being ascribed to inhibition of eIF2α-mediated translation initiation56. Another 3,3-diarylated oxindole BHPI, has been reported to act as a non-classical agonist of human estrogen receptor α and was recently shown to deplete intracellular ATP in estrogen receptor-positive cancer cells, thereby disrupting ATP-dependent ABC transporter-mediated drug efflux57. Whether BHPI might directly inhibit human or fungal ABC transporters is unknown. Other 3,3-bisaryl oxindoles have been reported as mineralocorticoid receptor antagonists58, as well as antioxidants59. Although azoffluxin falls into the general class of 3,3-disubstituted oxindoles, its specific substituents impart structurally distinct features to the compound. In addition to differences at the level of chemical structure, divergence in the processes reported to be impacted by other class members between mammalian and fungal cells, and azoffluxin’s lack of cytotoxicity to human cells and good tolerability in mice make it unlikely that azoffluxin operates through the mechanisms previously described for other 3,3-disubstituted oxindoles.The 3,3-diarylated oxindole we named azoffluxin was identified as an unexpected side product in a Lewis-acid mediated Friedel-Crafts/Prins reaction process intended to generate spirocyclic oxindolesC. auris strains belonging to clades I, II, and IV were sensitized to fluconazole, implicating efflux as a major factor contributing to their high azole-resistance. Indeed, inhibition of efflux in these strains markedly reduced their fluconazole resistance, despite the presence of Erg11 alterations, most notably Erg11Y132F and Erg11K143R that have been reported to cause a >4-fold increase in azole MIC16. Furthermore, azoffluxin reduced azole resistance of a C. albicans strain harboring numerous resistance-conferring mutations in genes such as ERG11, TAC1, MDR1, and UPC245. Deletion of CDR1 in specific strains of C. auris and C. albicans abolished azoffluxin-mediated potentiation. In contrast, we did not observe azoffluxin-mediated fluconazole potentiation in the more divergent fungal species C. glabrata and S. cerevisiae. One explanation would be that Pdr5, the homologous efflux pump in these species, is not inhibited by azoffluxin, which is plausible given the relatively low 56% sequence identity between Cdr1 and Pdr560. Alternatively, efflux may not be a driving factor in determining the fluconazole sensitivity of the strains used as representatives of these species.Using azoffluxin as a chemical probe, we found that C. auris, we found that isolates from clade III were distinct in their lack of susceptibility to the fluconazole-sensitizing effect of azoffluxin. Clade III isolates often express an Erg11 variant with V125A/F126L substitutions and harbor a unique substitution in Mrr1 (N647T), which likely leads to upregulation of the MFS pump Mdr17. Unlike clade I and IV, this clade does not possess candidate drug-resistance substitutions in Tac1b21. While we confirmed upregulation of MDR1 and CDR1 in clade III isolates compared to clade I, azoffluxin treatment of these strains still increased accumulation of fluconazole, and Nile red which is a substrate of both Mdr1 and Cdr1. The mechanism(s) underlying this observation is unclear but include the possibility that azoffluxin inhibits Mdr1 as well as Cdr1. Alternatively, inhibition of Cdr1 in these strains might be sufficient to increase compound accumulation despite Mdr1 upregulation, or azoffluxin is able to inhibit the function of yet other efflux pumps for which these compounds are also a substrate34. Irrespective of their mechanism, our findings and the observation that azoffluxin synergized with fluconazole against an atypical clade III isolate that did not carry the Erg11V125A/F126L or Mrr1N647T substitutions suggest that one or more of these resistance-conferring mutations is responsible for the inability of azoffluxin to potentiate fluconazole activity against most clade III isolates. In light of a previous report that Mdr1 does not play a role in fluconazole resistance of clade I isolates20 and our finding that azoffluxin enhanced fluconazole accumulation in clade III isolates, it is likely that the impact of Mdr1 upregulation on azole resistance in clade III strains is negligible. Rather, the specific Erg11V125A/F126L mutant target protein in most clade III strains is likely the dominant determinant of their fluconazole resistance and would explain the inability of an efflux pump inhibitor such as azoffluxin to restore azole sensitivity.Within S. cerevisiae genetic interaction networks show that perturbation through deletion of specific ABC transporter genes can paradoxically lead to an increase in azole resistance61. This response was mediated, at least in part by compensatory upregulation of PDR5 expression, as deletion of PDR5 restored fluconazole sensitivity61. We find that expression of efflux genes such as CDR1 and CDR4-1 were upregulated in C. auris in response to azoffluxin, while genes such as SNQ2-2 were downregulated. This pattern highlights the complex connectivity of the efflux network as well as the potential contribution of general stress responses to efflux pump expression. Regardless, this upregulation did not preclude the ability to sensitize cells to fluconazole. The robust activity of azoffluxin despite the compensatory upregulation of efflux genes reflects the strong dependence of high level fluconazole-resistance on Cdr162. Consistent with this dependence, the highly resistant C. albicans clinical isolate CaCi-17 was susceptible to azoffluxin-fluconazole combination treatment. In contrast, azoffluxin had no impact on azole sensitivity of the earlier clinical isolate CaCi-2, which possesses no bona fide mutations and is far more susceptible to fluconazole. Such insights underscore the value of azoffluxin as a chemical probe to discern the relative role of efflux in mediating antifungal drug resistance across diverse fungal pathogens.Efflux is regulated by complex and highly interconnected genetic circuitry. Recent analyses of 66. In the case of efflux inhibitors, not only does this strategy enhance the efficacy of the other compound, but if applied early in the course of intervention, it can also reduce the rate at which resistance emerges67. Despite the conceptual appeal, no efflux inhibitor combination therapies have proven effective in patients52. This failure in clinical translation has largely been due to host toxicity, off target effects, and/or the poor pharmacokinetics that have plagued current efflux inhibitors such as verapamil, cyclosporin A, and valspodar66. In this study, we found azoffluxin to be very well tolerated both in culture and in mice. As well, azoffluxin significantly enhanced fluconazole activity in an immunocompromised mouse model of invasive C. auris infection, and reduced fungal burden by ~1000-fold even as a single agent, consistent with the role of Cdr1 in virulence of C. glabrata50.From a therapeutic perspective, utilizing a chemical combination in which one compound targets an essential process and the other disables a major resistance mechanism provides an attractive strategy that has been explored for both antimicrobial and cancer treatment52. Encouragingly, azoffluxin remains able to sensitize azole-resistant Candida strains despite the presence of target-based resistance mechanisms. While overall results are encouraging, they are clearly discovery-phase in nature and will require further development for translation to clinical application. As the number of drug-resistant infections continues to rise, there remains a need to understand the relative contribution of different resistance mechanisms to the diminishing efficacy of our limited antifungal armamentarium and to design new resistance-evasive treatment strategies.An additional distinct challenge to the efficacy of efflux inhibitors is the emergence of target-based and other resistance mechanisms that can render efflux a less important factor contributing to the overall resistance level of a fungal pathogenAll strains, plasmids, and oligonucleotides used in this study are listed in C. auris strain with CDR4-1 (B9J08_000479) deleted was constructed using homologous recombination and an electroporation transformation approach35. Approximately 1 kb of sequence homology upstream of CDR4-1 was amplified using primers oLC8164/oLC8165 and ~1 kb of homology downstream of CDR4-1 was amplified with oLC8166/oLC8167. The interior primer of each set contained 40 bp homology to a nourseothricin (NAT) resistance marker from pLC1049, which was amplified with primers oLC6296/oLC6304. Using fusion PCR with nested primers oLC8168/oLC8169, the NAT cassette and CDR4-1 homology regions were combined into a single DNA fragment. This PCR product was ethanol precipitated. C. auris CaLC5083 cells were prepared by growing in 50 mL YPD medium to an OD600 of 1.6–2.2. Cells were pelleted for 5 min at 3000×g, then subcultured for 1 h in 10 mL 10 mM Tris-HCl (Bioshop), and 1 mM EDTA (Bioshop) in ddH2O (1x TE buffer) and 0.1 M Lithium Acetate (Sigma). In all, 250 µL of 1 M DTT was added to the culture for 30 min. Cells were washed twice in cold ddH2O followed by a wash in cold 1 M sorbitol (Bioshop). In all, 3 μg of DNA was electroporated into 40 µL CaLC5083 cell suspension in a 2-mm electroporation cuvette (VWR) with the following settings on a BTX ECM830 electroporator: 1.8 kV, 200 Ω, 25 µF and outgrown in YPD medium for 4 h at 30 °C. Transformants were plated on YPD plates containing 150 μg/mL NAT. Colonies were patched and genotyped for integration of the deletion construct (oLC8164/oLC6308 and oLC274/oLC8167) and for the absence of the wild-type allele (oLC8052/oLC8053).The CDR1 (C3_05220W) were deleted by a transient CRISPR method68. The guide construct was made of two components from pLC1081: the SNR52 promoter amplified with the universal primer oLC6929 and guide specific primer oLC6966, and the guide scaffold and terminator amplified with the guide specific primer oLC6967 and universal primer oLC6927. The fusion construct was PCR amplified with the universal nested primers oLC6928/olC692969. Repair template was digested from pLC1083 by ApaI (NEB) and SacI (NEB). Gene deletion was verified by the absence of the CDR1 specific amplicon with oLC6968/oCL696969.Both alleles of TAC1 back into the tac1△/tac1△ deletion mutant CaLC425570 to match gain-of-function strains below. oLC1096 was digested by BamHI (NEB) and SacII (NEB) and used to transform CaLC4255. NAT resistant transformants was further triaged by a histidine auxotrophy. Re-introduction of the TAC1 ORF was verified by the presence of the oLC7041 and oLC7042 amplicon. Subsequently, pLC1092 was digested by BamHI and SacII and used in a second transformation. Transformants prototrophic for histidine were further selected for NAT resistance.Stepwise insertion of wild-type TAC1A2029-G2031△  into the tac1△/ tac1△ deletion mutant CaLC4255. oLC1097 was digested by BamHI and SacII and used to transform CaLC4255. NAT resistant transformants was further triaged by a histidine auxotrophy. Re-introduction of the TAC1A2029-G2031△ ORF was verified by the presence of the oLC7041 and oLC7042 amplicon. Subsequently, pLC1093 was digested by BamHI and SacII and used in a second transformation. Transformants prototrophic for histidine were further selected for NAT resistance.Stepwise insertion of a TAC1A2914G  into the tac1△/ tac1△ deletion mutant CaLC4255. oLC1098 was digested by BamHI and SacII and used to transform CaLC4255. NAT resistant transformants was further triaged by a histidine auxotrophy. Re-introduction of the TAC1A2914G ORF was verified by the presence of the oLC7041 and oLC7042 amplicon. Subsequently, pLC1094 was digested by BamHI and SacII and used in a second transformation. Transformants prototrophic for histidine were further selected for NAT resistance.Stepwise insertion of a TAC1A2929G  into the tac1△/ tac1△ deletion mutant CaLC4255. oLC1099 was digested by BamHI and SacII and used to transform CaLC4255. NAT resistant transformants was further triaged by a histidine auxotrophy. Re-introduction of the TAC1A2929G ORF was verified by the presence of the oLC7041 and oLC7042 amplicon. Subsequently, pLC1095 was digested by BamHI and SacII and used in a second transformation.Stepwise insertion of a Transformants prototrophic for histidine were further selected for NAT resistance.CDR1 (B9J08_000164) in C. auris B12037 a homologous recombination and electroporation transformation approach was used35. Approximately 1 kb of sequence homology upstream of CDR1 was amplified using primers oLC6020/oLC6305 and ~1 kb of homology downstream was amplified with oLC6306/oLC6025. The interior primer of each set contained 40 bp homology to a nourseothricin (NAT) resistance marker from pLC1049, which was amplified with primers oLC6296/oLC6304. Using fusion PCR with nested primers oLC6024/oLC6307, the NAT cassette and CDR1 homology regions were combined into a single DNA fragment. This PCR product was ethanol precipitated, 5 μg of DNA was electroporated into CauLC6554, and transformants were plated on YPD plates containing 150 μg/mL NAT. Colonies were patched and genotyped for integration of the deletion construct (oLC6221/oLC6308 and oLC274/oLC6023) and for the absence of the wild-type allele (oLC6231/oLC6169).To delete t test in Microsoft Excel (version 16.41) unless otherwise stated.All data presented in this study are derived from two biological experiments in which both results agreed, and data shown in the figures are from technical replicates from a single biological replicate, representative of both, unless otherwise stated. All statistical analysis was performed by a two-sided Student’s All fungal strains were stored in 25% glycerol in YPD medium  and maintained at −80 °C. Strains were grown in either YPD or RPMI medium . The mammalian cell line of human embryonic kidney 293T cells was stored in glycerol and cultured in DMEM medium (Sigma) with 10% fetal bovine serum .C. auris. All compounds were dissolved in DMSO  at 5 mM. RPMI medium alone or containing 128 μg/mL fluconazole  was inoculated with ~1 × 103 cells/mL of C. auris (VPCI 673/P/12) from a saturated overnight culture. Both types of media were dispensed at 100 μL per well into 96-well, flat bottom, microtiter plates (Sarstedt). In total, 1 μL of DMSO-solubilized compound from the library was added into each well to a final concentration of 50 μM. Cells were incubated for 48 h at 30 °C and OD600 was read (Molecular Devices SpectraMax Plus 384). After the initial screen, all secondary chemical susceptibility assays were performed on fresh sample aliquots that were first assessed for purity by UPLC-MS-ELSD analysis.A set of 2,454 compounds from the Boston University Center for Molecular Discovery (BU-CMD) library were used to identify compounds that enhance fluconazole activity against 71. Plates were incubated at 30 °C for the indicated time period. Growth was quantified by measuring OD600 and corrected for medium background. All strains were assessed in biological duplicate experiments with technical duplicates. Growth was normalized to untreated controls and plotted as a heat map using Java TreeView (version 1.1.6r4). For dose-response matrixes fractional inhibitory concentration index at 90% growth inhibition (FICI90) was calculated using the formula: 30. The fluconazole Etest susceptibility assay was performed by plating 200 μL of 5 × 106 cells/mL on YPD agar (1%) plates with either 50 μM azoffluxin or containing DMSO35. Etest strips (bioMérieux) were placed on top after drying and plates were incubated for 24 h at 30 °C and imaged. Etest susceptibility assays were performed in biological duplicate.Compound potency was assessed alone by dose-response assays or in combination with another compound by dose-response matrixes in 96-well plates, or 384-well, flat bottom, microtiter plates (Corning)2O or DMSO.BU-CMD hit compounds were newly supplied and dissolved in DMSO. Gepinacin , cerulenin , cycloheximide (BioShop), caspofungin (generously provided by Merck), and amphotericin B (Sigma) were dissolved in DMSO. Fluconazole was dissolved in sterile ddH1H NMR spectra were recorded at 400 or 500 MHz at ambient temperature unless otherwise stated. 13C NMR spectra were recorded at 100 or 125 MHz at ambient temperature unless otherwise stated. Chemical shifts are reported in parts per million. Data for 1H NMR are reported as follows: chemical shift, multiplicity , coupling constants, and integration. All 13C NMR spectra were recorded with complete proton decoupling. Analytical thin layer chromatography was performed using 0.25 mm silica gel 60-F plates. Flash chromatography was performed using 200–400 mesh silica gel  or prepack column  by Interchim. Isolated yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Analytical LC-MS experiments were performed using a Waters Acquity UPLC (ultra-performance liquid chromatography) with a binary solvent manager, SQ mass spectrometer, Waters 2996 PDA (photodiode array) detector and evaporative light scattering detector (ELSD).R)-1--4-methylpent-4-en-2-ol.All compounds tested in biological assays were determined to be >95% pure by UPLC-MS-ELSD analysis. For validation, the screening hit CMLD012336 was resynthesized via Lewis-acid mediated condensation of 6-fluoro-3,3-dimethoxyindolin-2-one and , trimethylorthoformate , and methanol (30 mL). p-Toluenesulfonic acid monohydrate  was added and the reaction was heated to reflux for 5.5 h. After cooling to ambient temperature, the reaction was diluted with diethyl ether and neutralized with a saturated solution of sodium bicarbonate. The organic layer was separated, and the aqueous layer was extracted twice with diethyl ether. The combined organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was taken up in diethyl ether, filtered over a pad of celite/Na2SO4, and concentrated to give a yellow solid. The crude yellow solid was purified flash column chromatography  to give product 6-fluoro-3,3-dimethoxyindolin-2-one . LCMS m/z [M-OMe]+ 180. 1H NMR  δ 9.57 , 7.44 , 6.79 , 6.72 , 3.48 . 13C NMR  δ 173.1, 166.4, 164.5, 145.0, 144.9, 128.3, 109.3, 109.1, 100.1, 99.9, 51.2. HRMS [M+Na]+ calcd. for C10H10FNO3Na 234.0545, found 234.0542.To a flame-dried 100 mL round bottomed flask equipped with a reflux condenser under an atmosphere of N2 was stirred a suspension of copper iodide  in THF (2 mL) cooled to −40 °C using an acetonitrile/CO2 bath. To this suspension was added isopropenylmagnesium bromide . The reaction was stirred at −40 °C for 35 min. Next, a solution of 5-[[(2 R)-oxiran-2-yl]methoxy]-1,3-benzodioxole  in THF was added dropwise. The reaction was stirred at −40 °C for 150 min. The brown colored mixture was quenched at −40 °C by dropwise addition of saturated aqueous ammonium chloride (0.4 mL). The product mixture was then filtered over a pad of Celite/SiO2/Na2SO4. This pad was eluted with 60% ethyl acetate in hexanes (100 mL) and ethyl acetate (60 mL) to afford the desired product (493 mg) as a colorless oil in quantitative yield. 1H NMR  δ 6.71 , 6.53 , 6.35 , 5.93 , 4.91 , 4.85 , 4.14 , 3.92 , 3.83 , 2.32 , 1.81 . 13C NMR  δ 154.1, 148.3, 141.7, 113.7, 107.9, 105.8, 101.2, 98.2, 72.8, 67.8, 41.9, 22.5. HRMS [M+Na]+ calcd. for C13H16O4Na 259.0946, found 259.0944.In a flame-dried 50 mL round bottom flask under an atmosphere N2 was stirred 6-fluoro-3,3-dimethoxy-indolin-2-one  and (R)-1--4-methylpent-4-en-2-ol  in dichloromethane (4.2 mL). To this reaction was added magnesium sulfate . The reaction was cooled to 0 °C in an ice bath and scandium(III) triflate  was added. The reaction was allowed to slowly warm to room temperature. After stirring at room temperature overnight, the reaction was filtered through a pad of Celite eluting with dichloromethane. After concentration in vacuo, the crude residue was purified by flash column chromatography  to afford azoffluxin . LCMS m/z [M+H]+ 620. At ambient temperature, azoffluxin exhibits multiple sets of broadened 1H and 13C NMR peaks due to rotamers by restricted rotation of the biaryl system. NMR peaks were found to coalesce upon heating to 150 °C. NMR chemical shifts at both temperatures are reported.In a two dram vial under N1H NMR  δ 10.60 , 7.35-7.17 , 6.77-6.72 , 6.67-6.55 , 6.48 , 6.14 , 5.95-5.90 , 5.88-5.84 , 4.67-4.63 , 4.57-4.46 , 3.78-3.38 , 3.30-3.33 , 1.95-1.72 , 1.65-1.55 ; 13C NMR  δ 180.0, 179.8, 163.5, 163.3, 161.0, 160.9, 153.0, 152.5, 151.9, 151.6, 147.7, 147.5, 147.23, 147.17, 143.2, 143.0, 142.95, 141.2, 141.0, 140.8, 130.8, 130.3, 127.2, 127.1, 126.8, 126.6, 121.1, 120.7, 112.6, 112.3, 109.2, 108.25, 108.2, 108.0, 107.8, 107.6, 101.7, 101.6, 97.9, 97.6, 97.2, 97.0, 96.95, 96.8, 74.0, 73.5, 73.1, 72.9, 67.5, 67.4, 67.3, 67.0, 59.3, 59.0, 42.2, 42.0, 41.8, 41.6, 23.13, 23.08, 23.05, 22.97. 1H NMR  δ 9.94 , 7.21 , 6.69-6.58 , 6.41 , 5.88 , 4.75-4.71 , 4.68 , 4.65 , 3.76-3.57 , 2.07-1.83 , 1.70 , 1.68 ; 13C NMR  δ 179.5, 162.6 , 152.7, 147.6, 143.5, 143.4, 143.1, 142.0, 130.7, 127.2, 127.1, 121.8, 112.1, 112.0, 108.8, 107.8, 107.5, 101.5, 97.8, 97.6, 97.2, 74.2, 68.3, 68.1, 59.6, 42.2, 41.4, 22.90, 22.85. HRMS [M+H]+ calcd. for C34H35FNO9 620.2296, found 620.2294.C. auris the targeted metabolomics profiling protocol established by Hoepfner et al.13 in S. cerevisiae was used. Cells were subcultured to an OD600 of 0.1 in 10 mL of RPMI supplemented with the indicated compound concentration for 18 h with agitation. After incubation growth (OD600) was normalized and cell pellets washed and resuspended in 100 µL of PBS. Cell suspension was treated with 1 mL methanol/CHCl3 (2:1 v/v) supplemented with 0.01% w/v butylated hydroxytoluene. Acid washed glass beads were added to each sample and they were vortexed for 10 min. Samples were pelleted by centrifugation for 5 min at 16,000×g. Transferring the supernatant to a new vial, 400 μL 50 mM citric acid in H2O, and 600 μL CHCl3 was added and vortexed for 10 min. Samples were again centrifuged for 5 min at 16,000×g. The organic phase was collected and dried. For LC-MS analysis, samples were resuspended in ethanol with cholesterol included as an internal standard. Samples were separated on Acquity UPLC BEH C18 column  using the Acquity UPLC I-Class coupled to a Xevo G2-S QToF equipped with an APCI source (Waters). Chromatographic methods were adopted from Hoepfner et al. as well as the selective reaction monitoring mass transitions specific for each sterol in subsequent quantification steps. TargetLynx  was used for peak finding, smoothing and area calculations. All samples were run in biological duplicate and technical triplicate, and a representative replicate was plotted in GraphPad Prism (version 8.4.2).To quantify the abundance of sterols in C. auris was subcultured from overnight cultures at a starting OD600 of 0.4 in 5 mL of YPD in the presence of the indicated compound concentration for 1 h with agitation. Cells were then transferred to falcon tubes and pelleted at 3000×g for 5 min at 4 °C. Media was removed, and cells were washed with 5 mL of cold PBS three times with centrifugation of 2000×g for 5 min in between. Cells were resuspended in 1 mL cold PBS, flash frozen in liquid nitrogen, and stored at −80 °C overnight. The following day, cells were thawed on ice, 25 μL of 6 N NaOH was added to each falcon tube, and samples were vortexed for 15 s. In all, 500 μL of 10 mM sodium phosphate (pH 6.0) was added to each sample followed by vortexing for 15 s. Compounds were extracted with 5 mL of CH2Cl2 and vortexed for 5 min, followed by centrifugation for 10 min at 4000×g at 4 °C. The organic phase was collected and dried. Before subsequent LC-MS analysis, samples were resuspended in 50 μL MeCN:H2O. The resuspended cell extracts (10 µl) were separated on an Acquity UPLC BEH C18 column  using the Acquity UPLC I-Class coupled to a Xevo G2-S QToF equipped with an electrospray ionization (ESI) source. Chromatography followed a gradient method (A: water + 0.1% (v/v) formic acid, B: MeCN + 0.1% (v/v) formic acid | 0–9 min: 10% B to 95% B at 0.125 µL min−1). Both fluconazole and azoffluxin were detected using selected reaction monitoring mass transitions 307.110 [M+H]+ → 220.0685 and 602.227 [M-H2O+H]+ → 286.0515, respectively. TargetLynx (Waters) was used for peak finding, smoothing and area calculations. All samples were run in biological duplicate and technical triplicate and a representative replicate was plotted in GraphPad Prism.600 0.1 in YPD for 3 h in the presence of compound as indicated. Cells were then pelleted at 3000×g at 4 °C, washed with cold PBS, flash frozen with liquid nitrogen, and stored at −80 °C. Cells were lysed by bead beating 4x for 30 s with 1 min on ice in between. RNA was extracted from lysed cells using the QIAGEN RNeasy kit and DNase treated using the QIAGEN RNase free DNAase Set. Complementary DNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative real-time-PCR (RT-qPCR) was performed using in a 384-well plate, with a 10 μL reaction volume using Fast SYBR Green Master Mix (Applied Biosystems) and the BioRad CFX-384 Real Time System with the following cycling conditions: 95 °C for 3 min, then 95 °C for 10 s and 60 °C for 30 s, for 40 cycles. The melt curve was completed with the following cycle conditions: 95 °C for 10 s and 65 °C for 10 s with an increase of 0.5 °C per cycle up to 95 °C. Reactions were performed in technical triplicate using the primer pairs: CDR1 (oLC6125/oLC6126), MDR1 (oLC8050/oLC8051), CDR4-1 (oLC8052/oLC8053), CDR4-2 (oLC8054/oLC8055), SNQ2-1 (oLC8060/oLC8057), SNQ2-2 (oLC8058/oLC8059), and normalized to the house keeping genes ACT1 (oLC5727/oLC5728) and GPD1 (oLC5729/oLC5730). Primer sequences are included in To determine changes in efflux gene expression, strains were subcultured from a saturated overnight culture at an OD34. Cells were subcultured from an OD600 0.1 in YPD for 4 h until exponential phase was reached. For cells treated with azoffluxin, 50 μM was added for 10 min prior to a 20-min incubation of all stained cells with 7 μM (3.5 mM in DMSO stock) of Nile red (Sigma). Cells were then pelleted for 1 min at 3000×g and resuspended in PBS. To quantify fluorescence, a CytoFlex Flow Cytometer (Beckman Coulter) was used. Cells were added to flat bottom, transparent, 96-well plate (Beckman Coulter). Each sample was run using the CytExpert Software (version 2.4) until ~20,000 events had been recorded. Populations were gated to exclude debris and doublets, and the median PE value was taken for each sample  to each well, incubating for 10 min, and reading luminescence on a Tecan Infinite 200 Pro. All experiments were performed in technical quadruplicate and biological duplicate and plotted in GraphPad Prism.To assess the ability of azoffluxin to rescue mammalian cell growth in co-culture experiments, 20 μL of 293 T cells were seeded at 1 × 104 cells/well for 24 h in six-well plates (Corning). In all, 2.5 × 103 exponential phase C. auris cells were added to mammalian cells followed by indicated drug or solvent concentrations. Plates were incubated for 48 h at 37 °C. Cultures were fixed with 4% formaldehyde (BioShop) in medium overnight, fixative was removed, and the plate was dried. Fixed cells were then hydrated with 1 mL ddH2O and this was removed from each well. In total, 1 mL PAS solution was added and cells were incubated for 5 min, followed by removal and 2x washes with ddH2O by pipetting. In all, 1 mL Schiff’s reagent was added, and plates were incubated for 15 min. The cells were then thoroughly rinsed for 5 min with ddH2O. In all, 1 mL hematoxylin was applied for 3 min and cell were rinsed again. Cells were allowed to dry and were then imaged. Experiments were performed in biological duplicate, with one representative image being shown.The cellular glycoproteins were stained in co-culture experiments using a periodic-acid Schiff (PAS) staining kit (Sigma) as per manufacturer’s instructions. Mammalian 293 T cells were seeded at 5 × 10C. auris Ci6684 to a final concentration of ~1 × 103 cells/mL in a 96-well plate. Plates were incubated at 30 °C and growth was measured after 48 h by OD600. All experiments were performed in technical triplicate and biological duplicate.To assess the ability of azoffluxin to retain activity after exposure to mouse serum, a 10x concentration of azoffluxin was incubated for 1 h in 100% mouse plasma in sodium citrate buffer . This was incubated along with the control compounds gepinacin, which is known to be metabolized by serum, and caspofungin, which is not metabolized by serum. Compounds were incubated at either at 37 °C or on ice, or in the absence of serum in a YPD medium control at 37 °C. 10 μL of the mixture was then added to 90 μL of YPD inoculated with 2O. At the indicated times post dose (n = 3 per time point), mice were bled via a submandibular site and whole blood was collected in lavender top K2EDTA tubes. Plasma was processed from whole blood by centrifugation at 9600×g for 10 min. In total, 50 µL plasma was either used straight or was diluted in commercial K2EDTA CD-1 plasma (BioIVT) and mixed with 100 µL of methanol containing 0.15% formic acid, 3 mM Ammonium Acetate, and 37.5 ng/mL N-benzylbenzamide internal standard. The mixture was vortexed, incubated for 10 min at room temperature, and then centrifuged at 16,100 g for 5 min. Supernatant was collected and recentrifuged. The final supernatant was analyzed by LC-MS/MS. Analytical standards and quality control samples were prepared in a similar fashion by spiking commercial K2EDTA CD-1 mouse plasma with known quantities of azoffluxin. Chromatography conditions were as follows: an Agilent (Santa Clara) C18 XDB, 5 µm packing, 50 × 4.6 mm size column was used for reverse phase chromatography. Buffer A consisted of dH2O + 2 mM NH4 acetate and 0.1% formic acid and Buffer B consisted of methanol + 2 mM NH4 acetate and 0.1% formic acid. Gradient conditions utilized were: 0.01–0.5 min 3% B, 0.5–1.5 min gradient to 100% B,1.5–2.5 min 100% B, 2.5–2.6 min gradient to 3% B, 2.6–3.6 min 3% B. A 619.9 to 286.1 transition was used to quantitate azoffluxin while the 619.9 to 384.2 transition was used as the qualifier ion pair. N-benzylbenzamide (212.1 to 91.1 transition) was used as an internal standard. Data were analyzed using Analyst software  A value 3x above the signal obtained in the blank plasma was designated as the limit of detection (LOD). The limit of quantitation (LOQ) was defined as the lowest concentration on the standard curve at which back calculation yielded a concentration within 20% of the theoretical value and above the LOD signal. The LOQ for azoffluxin was 0.1 ng/ml. Pharmacokinetic properties were evaluated using the noncompartmental analysis tool in WinNonlin . Sparse sampling was used for data analysis. Terminal half-life was calculated as the ln(2)/λz where λz is a first order rate constant associated with the terminal (log-linear) portion of the curve. It is estimated by linear regression of time vs. log concentration by the software for three or more of the final nonzero data points. Tmax  and Cmax  were determined by visual inspection. Area under the concentration time curve (AUClast) from time 0 to the last observed concentration was determined by linear trapezoidal analysis. Apparent Volume of Distribution (Vz_F) is based on the terminal phase and is calculated as Dose/λz*AUCinf_obs while Apparent Clearance (CL_F) is calculated as Dose/AUCinf_obs. Calculation of Absolute Vz and CL requires knowledge of IP bioavailability (F) which is calculated as AUCIP/AUCiv X Doseiv/DoseIP but was not determined here because an iv PK was not performed. Mean residence time (MRTlast) is the average time a molecule of drug spends in the system before before the last measurable concentration and is calculated by dividing the area under the curve formed by time and the product of concentration and time (AUMC) divided by the AUC.The pharmacokinetics of azoffluxin in mice were measured by the Preclinical Pharmacology Core at UT Southwestern Medical School. All animal work was approved and conducted under the oversight of the UT Southwestern Institutional Animal Care and Use Committee, which uses the Guide for the Care and Use of Laboratory Animals when establishing animal research standards. Mice were maintained at 20.5–22.2 °C, at 48–52% humidity, with light/dark alternating every 12 h. 21 6-week-old female CD-1 (Charles River) mice were dosed intraperitoneally (IP) with 10 mg/kg azoffluxin. Azoffluxin was formulated at 1 mg/mL in 25% PEG-400 (Sigma); 10% DMSO; 0.1% Tween-80 (Sigma), and 65% ddHn = 5). Mice were monitored twice daily for signs of distress such as hunching, ruffled fur, weight loss, difficulty moving, or reduced drinking or eating to prevent and minimize unnecessary pain for 21 days.The in vivo tolerability of azoffluxin was determined in outbred ICR (CD-1) mice (Envigo). Animal toxicity studies were approved by the Institutional Animal Care and Use Committee of The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center (#31413), according to the NIH guidelines for animal housing and care. Mice were maintained at 20.5–22.2 °C, at 30–70% humidity, with light/dark alternating every 12 h. Mice weighing ~ 25 g were rendered neutropenic by administration of two doses of cyclophosphamide  given IP and cortisone acetate  given subcutaneously on day −2 and +3, relative to treatment. To prevent bacterial infection, mice received antibacterial prophylaxis consisting of 50 μg/mL enrofloxacin (Bayer) in the drinking water starting the same day of immunosuppression. Mice were treated with 10 mg/kg of azoffluxin formulated as described above twice daily for 4 days or left untreated , The Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals, and Public Health Service Policy. Mice were maintained at 22.2 °C, at 45% humidity, with light/dark alternating every 12 h. Specific-pathogen-free, six-week-old female ICR (CD-1) mice weighing 23 to 27 g were used (Envigo). Mice were rendered neutropenic by cyclophosphamide  subcutaneous injection 4 days (150 mg/kg) and 1 day (100 mg/kg) before infection and 2 days after infection (100 mg/kg). C. auris isolate B11801was subcultured on SDA 24 h prior to infection. The inoculum was prepared by placing three to five colonies into 5 mL of sterile pyrogen-free 0.9% saline that had been warmed to 35 °C. The final inoculum was adjusted to a 0.6 transmittance at 530 nm. Final inoculum was determined to be 5.97 ± 0.03 log10 CFU/mL. Disseminated infection was achieved by injection of 0.1 mL of inoculum via the lateral tail vein 2 h prior to the start of drug therapy. Mice were treated with either saline as a control, fluconazole 32 mg/kg administered subcutaneously every 12 h, azoffluxin 10 mg/kg administered IP every 6 h, or fluconazole 32 mg/kg administered subcutaneously every 12 h and azoffluxin 10 mg/kg administered IP every 6 h. Animals were monitored every 6 h for signs of distress. Mice displaying hunching, ruffled fur, difficulty moving, or reduced drinking or eating were immediately euthanized. Mice were treated for 96 h, then sacrificed by CO2 asphyxiation. After sacrifice, the kidneys of each mouse were removed and placed in sterile 0.9% saline at 4 °C. Kidney homogenates were prepared and serially diluted 1:10. Aliquots were plated on SDA for viable fungal colony counts after incubation for 24 h at 35 °C. The lower limit of detection was 100 CFU/mL. Results were expressed as the mean number of CFU per kidney for three mice. Controls were sampled at the start and end of the 96-hour treatment period. These experiments were performed in two replicates.The antifungal activity of azoffluxin was assessed using a well-characterized modelFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary