Pathogenic Aspergillus Strains Identification and Antifungal Susceptibility Analysis of 452 Cases with Otomycosis in Jingzhou, China.

OBJECTIVE: To study the distribution of pathogenic Aspergillus strains of otomycosis in central China and the identification of their antifungal sensitivity. METHODS: We collected external ear canal secretions clinically diagnosed as otomycosis from April 2020 to January 2023 from the Department of Otolaryngology-Head and Neck Surgery in central China. The pathogenic Aspergillus strains were identified through morphological examination and sequencing. The antifungal sensitivity was performed using the broth microdilution method described in the Clinical Laboratory Standard Institute document M38-A3. RESULTS: In the 452 clinical strains isolated from the external ear canal, 284 were identified as Aspergillus terreus (62.83%), 92 as Aspergillus flavus (20.35%), 55 as Aspergillus niger (12.17%). In antifungal susceptibility tests the MIC of Aspergillus strains to bifonazole and clotrimazole was high,all the MIC90 is > 16 ug/mL. However, most Aspergillus isolates show moderate greatly against terbinafine, itraconazole and voriconazole. CONCLUSION: A. terreus is the most common pathogenic Aspergillus strain in otomycosis in central China. The selected topical antifungal drugs were bifonazole and clotrimazole; the drug resistance rate was approximately 30%. If the infection is persistent and requires systemic treatment, terbinafine and itraconazole can be used. The resistance of Aspergillus in otomycosis to voriconazole should be screened to avoid the systemic spread of infection in immunocompromised people and poor compliance with treatment. However, the pan-azole-resistant strain of Aspergillus should be monitored, particularly in high-risk patients with otomycosis.

Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

Clotrimazole reverses macrophage M2 polarization by disrupting the PI3K/AKT/mTOR pathway.

Macrophages switch among different activation phenotypes according to distinct environmental stimuli, varying from pro-inflammatory (M1) to alternative (also named resolutive; M2) activation forms. M1-and M2-activated macrophages represent the two extremes of the activation spectrum involving multiple species, which vary in terms of function and the cytokines secreted. The consensus is that molecular characterization of the distinct macrophage population and the signals driving their activation will help in explaining disease etiology and formulating therapies. For instance, myeloid cells residing in the tumor microenvironment are key players in tumor progression and usually display an M2-like phenotype, which help tumor cells to evade local inflammatory processes. Therefore, these specific cells have been proposed as targets for tumor therapies by changing their activation profile. Furthermore, M2 polarized macrophages are phagocytic cells promoting tissue repair and wound healing and are therefore potential targets to treat different diseases. We have already shown that clotrimazole (CTZ) decreases tumor cell viability and thus tumor growth. The mechanism by which CTZ exerts its effects remains to be determined, but this drug is an inhibitor of the PI3K/AKT/mTOR pathway. In this study, we show that CTZ downregulated M2-activation markers in macrophages polarized to the M2 profile. This effect occurred without interfering with the expression of M1-polarized markers or pro-inflammatory cytokines and signaling. Moreover, CTZ suppressed NFkB pathway intermediates and disrupted PI3K/AKT/mTOR signaling. We concluded that CTZ reverses macrophage M2 polarization by disrupting the PI3K/AKT/mTOR pathway, which results in the suppression of NFkB induction of M2 polarization. In addition, we find that CTZ represents a promising therapeutic tool as an antitumor agent.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

Clotrimazole ameliorates chronic mild stress-induced depressive-like behavior in rats; crosstalk between the HPA, NLRP3 inflammasome, and Wnt/ß-catenin pathways.

Depression is a major emotional disorder that has a detrimental effect on quality of life. The chronic mild stress (CMS)-depression model was adopted in rats to evaluate the neurotherapeutic effect of Clotrimazole (CLO) and investigate the possible mechanisms of its antidepressant action via its impact on the hypothalamic pituitary adrenal (HPA) axis and the stress hormone, cortisol. It was found that azole antifungals affect steroidogenesis and the HPA axis. Behavioral, histopathological, inflammatory, and apoptotic pathways were assessed. Serum cortisol, inflammasome biomarkers, hippocampal NLRP3, caspase-1, and IL-18, and the canonical Wnt/ß-catenin neurogenesis biomarkers, Wnt3a, and non-phosphorylated ß-catenin levels were also determined. Different stressors were applied for 28 days to produce depressive-like symptoms, and CLO was administered at a daily dose of 30 mg/kg body weight. Subsequently, behavioral and biochemical tests were carried out to assess the depressive-like phenotype in rats. Stressed rats showed increased immobility time in the forced swimming test (FST), decreased grooming time in the splash test (ST), increased serum cortisol levels, increased inflammasome biomarkers, and decreased neurogenesis. However, administration of CLO produced significant antidepressant-like effects in rats, which were accompanied by a significant decrease in immobility time in FST, an increase in grooming time in ST, a decrease in serum cortisol level, a decrease in inflammasome biomarkers, and an increase in neurogenesis biomarkers. The antidepressant mechanism of CLO involves the HPA axis and the anti-inflammatory effect, followed by neurogenesis pathway activation. Therefore, CLO may have the potential to be a novel antidepressant candidate.

Antimicrobial Activity of Manganese(I) Tricarbonyl Complexes Bearing 1,2,3-Triazole Ligands.

BACKGROUND: Antimicrobial resistance is one of the most pressing health issues of our time. The increase in the number of antibiotic-resistant bacteria allied to the lack of new antibiotics has contributed to the current crisis. It has been predicted that if this situation is not dealt with, we will be facing 10 million deaths due to multidrug resistant infections per year by 2050, surpassing cancer-related deaths. This alarming scenario has refocused attention into researching alternative drugs to treat multidrug-resistant infections. AIMS: In this study, the antimicrobial activities of four manganese complexes containing 1,2,3,-triazole and clotrimazole ligands have been evaluated. It is known that azole antibiotics coordinated to manganese tricarbonyl complexes display interesting antimicrobial activities against several microbes. In this work, the effect of the introduction of 1,2,3,-triazole-derived ligands in the [Mn(CO)3(clotrimazole)] fragment has been investigated against one Gram-positive bacterium and five Gram-negative bacteria. METHODS: The initial antimicrobial activity of the above-mentioned complexes was assessed by determining the minimum inhibitory and bactericidal concentrations using the broth microdilution method. Growth curves in the presence and absence of the complexes were performed to determine the effects of these complexes on the growth of the selected bacteria. A possible impact on cellular viability was determined by conducting the MTS assay on human monocytes. RESULTS: Three of the Mn complexes investigated (4-6) had good antimicrobial activities against all the bacteria tested, with values ranging from 1.79 to 61.95 µM with minimal toxicity. CONCLUSIONS: Due to the increased problem of antibiotic resistance and a lack of new antibacterial drugs with no toxicity, these results are exciting and show that these types of complexes can be an avenue to pursue in the future.

IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration.

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

Adjunct antiseizure effect of clotrimazole in a rotenone corneal kindling mouse model of mitochondrial drug-resistant epilepsy.

This study aimed to investigate the therapeutic potential of clotrimazole, an inhibitor of the transient receptor potential cation channel, for treating mitochondrial drug-resistant epilepsy and to understand its underlying neurochemical mechanisms. Adult albino mice underwent rotenone-corneal kindling, receiving daily electric shocks (15 mA, 20 V, 6-Hz for 3 s) through a corneal electrode, to induce mitochondrial drug-resistant epilepsy. The onset of drug resistance was confirmed by the significant (p < 0.05) lack of seizure control with standard antiseizure medications including levetiracetam (40 mg/kg), valproate (250 mg/kg), phenytoin (35 mg/kg), lamotrigine (15 mg/kg), and carbamazepine (40 mg/kg). Drug-resistant mice were then classified into one vehicle-treated group and three groups treated with varying doses of clotrimazole (40, 80, and 160 mg/kg orally). Neurochemical analysis of the seizurogenic hippocampus and cerebral cortex was conducted using high-performance liquid chromatography with an electrochemical detector. Administration of clotrimazole alongside standard antiseizure medications led to a significant decrease (p < 0.05) in seizure scores suggesting the restoration of antiseizure effects. Neurochemicals, including tryptophan, serotonin, kynurenine, serine, taurine, gamma-aminobutyric acid, and glutamate, were significantly restored post-clotrimazole treatment. Overall, the present study underscores the adjunct antiseizure effect of clotrimazole in a rotenone corneal kindling mouse model of mitochondrial drug-resistant epilepsy, emphasising its role in neurochemical restoration.

Topical delivery systems containing clotrimazole for the management of candidiasis: Effect of different excipients and enhanced antifungal activity of nanovesicles.

WHO classified Candida albicans as one of the four critical priority fungi for public health worldwide in 2022. Conventional topical formulations commercially available for the treatment of cutaneous candidiasis are associated with low drug bioavailability at the infection site and the lack of a sustained therapeutic effect. The main objectives of this work were to develop new topical administration systems of clotrimazole (CLT) and study the influence of surfactants on the antifungal inhibitory efficacy. Therefore, the minimum concentration of CLT required to inhibit 50 % of growth (MIC50) was determined, obtaining a value of approximately 15 ng/mL. A non-ionic emulsion type 1, Beeler base cream, hydrogel and liposomes containing CLT were designed, prepared, characterized and their antifungal activity against C. albicans was tested. CLT loaded liposomes were small in size (102 nm), homogeneous (polydispersity index = 0.3) and uncharged (+0.07 mV), showing higher antifungal activity against C. albicans than that of the commercially available cream Canesten®. Furthermore, the antifungal activity of CLT was reduced in combination with surfactants such as Tween-80/Span-80 or Brij-S10. Sodium lauryl sulphate showed a fungicidal effect that disappeared when formulated as part of the Beeler base cream.

Efficacy of manuka honey with conventional antifungals on Malassezia pachydermatis.

Yeast infections such as otitis externa and seborrheic dermatitis in dogs and cats are frequently associated with Malassezia pachydermatis secondary infection. It is part of the normal cutaneous microflora of most warm-blooded vertebrates, however, under certain conditions, it can become a causative agent of infection that needs to be treated pharmacologically. Azole derivatives are the drugs of the first choice. An interesting trend in developing resistance is the use of natural substances, which include manuka honey with confirmed antimicrobial properties. The main intention of this research was to evaluate the mutual effect of manuka honey in combination with four conventional azole antifungals - clotrimazole, fluconazole, itraconazole, and miconazole - on 14 Malassezia pachydermatis isolates obtained from dogs and 1 reference strain. A slightly modified M27-A3 method (CLSI 2008) and the checkerboard test (Nikolic et al. 2017) were used for this purpose. Our results show an additive effect of all 4 antifungals with manuka honey concurrent use. Based on the determined values of fractional inhibitory concentration index (FICI - 0.74±0.03 when manuka honey combined with clotrimazole, 0.96±0.08 with fluconazole, 1.0±0 with miconazole and 1.16±0.26 with itraconazole), it was found in all cases that the effect of substances used is more pronounced in mutual combination than when used separately.

Development of invaethosomes and invaflexosomes for dermal delivery of clotrimazole: optimization, characterization and antifungal activity.

The objective of this study was to develop novel invaethosomes (I-ETS) and invaflexosomes (I-FXS) to enhance the dermal delivery of clotrimazole (CZ). Twenty model CZ-loaded I-ETS and I-FXS formulations were created according to a face-centered central composite experimental design. CZ-loaded vesicle formulations containing a constant concentration of 0.025% w/v CZ and various amounts of ethanol, d-limonene, and polysorbate 20 as penetration enhancers were prepared using the thin film hydration method. The physicochemical characteristics, skin permeability, and antifungal activity were characterized. The skin permeability of the experimental CZ-loaded I-ETS/I-FXS was significantly higher than that of conventional ethosomes, flexosomes, and the commercial product (1% w/w CZ cream). The mechanism of action was confirmed to be skin penetration of low ethanol base vesicles through the disruption of the skin microstructure. The optimal I-ETS in vitro antifungal activity against C. albicans differed significantly from that of ETS and the commercial cream (control). The response surface methodology predicted by Design Expert® was helpful in understanding the complicated relationship between the causal factors and the response variables of the 0.025% w/v CZ-loaded I-ETS/I-FXS formulation. Based on the available information, double vesicles seem to be promising versatile carriers for dermal drug delivery of CZ.

Cross-linkable star-hyperbranched unimolecular micelles for the enhancement of the anticancer activity of clotrimazole.

Clotrimazole, a hydrophobic drug routinely used in the treatment of vaginal candidiasis, also shows antitumor activity. However, its use in chemotherapy has been unsuccessful to date due to its low solubility in aqueous media. In this work, new unimolecular micelles based on polyether star-hyperbranched carriers of clotrimazole are presented that can enhance solubility, and consequently the bioavailability, of clotrimazole in water. The amphiphilic constructs consisting of a hydrophobic poly(n-alkyl epoxide) core and hydrophilic corona of hyperbranched polyglycidol were synthesized in a three-step anionic ring-opening polymerization of epoxy monomers. The synthesis of such copolymers, however, was only possible by incorporating a linker to facilitate the elongation of the hydrophobic core with glycidol. Unimolecular micelles-clotrimazole formulations displayed significantly increased activity against human cervical cancer HeLa cells compared to the free drug, along with a weak effect on the viability of the normal dermal microvascular endothelium cells HMEC1. This selective activity of clotrimazole on cancer cells with little effect on normal cells was a result of the fact that clotrimazole targets the Warburg effect in cancer cells. Flow cytometric analysis revealed that the encapsulated clotrimazole significantly blocks the progression of the HeLa cycle in the G0/G1 phase and induces apoptosis. In addition, the ability of the synthesized amphiphilic constructs to form a dynamic hydrogel was demonstrated. Such a gel facilitates the delivery of drug-loaded single-molecule micelles to the affected area, where they can form a continuous, self-healing layer.

Clotrimazole is effective, safe and tolerable for the treatment of endometriosis and functions by downregulating inducible nitric oxide synthase and modulating oxidative stress biomarkers.

This study investigated the mechanism of action of clotrimazole (CTZ) and its adverse effects in a model of endometriosis. After autologous endometrial implantation, 18 rats were randomized into two treatment groups: 200 mg/kg CTZ or vehicle for 15 consecutive days. The lesion growth, implant size, glandular atrophy, nitric oxide (NO) serum levels, number of macrophage cells and inducible nitric oxide synthase (iNOS) immunoreactivity were significantly reduced in the CTZ group compared with the control. CTZ (p < 0.05) reduced the lipid peroxidation and protein carbonylation levels in the liver but did not alter the superoxide dismutase (SOD), glutathione (GSH) or glutathione S-transferase (GST) levels in the brain; however, the drug significantly reduced SOD activity and enhanced GST activity in the liver. These results suggest that CTZ interferes with reactive nitrogen species production by downregulating iNOS expression and thus enhances the antioxidant system to promote atrophy and regression of endometriotic lesions, without adverse effects on the brain and/or liver.

Clotrimazole-Based Modulators of the TRPM3 Ion Channel Reveal Narrow Structure-Activity Relationship.

TRPM3 is an ion channel that is highly expressed in nociceptive neurons and plays a key role in pain perception. In the presence of the endogenous TRPM3 ligand, pregnenolone sulfate (PS), the antifungal compound clotrimazole (Clt) augments Ca2+ signaling and opens a non-canonical pore, permeable to Na+, which aggravates TRPM3-induced pain. To date, little is known about structural features that govern the Clt modulatory effect of TRPM3. Here, we synthesized and evaluated several Clt analogues in order to gain insights into their structure-activity relationship. Our results reveal a tight SAR with the three phenyl rings on the trityl moiety being essential for the activity, as well as the presence of fluorine or chlorine substituents on the trityl group. Imidazole as a heterocycle is also necessary for activity. Interestingly, we identified a pentafluoro-trityl analogue (29a) that is able to act as a TRPM3 agonist in the absence of PS. The compounds we report in this work will be useful tools for the further study of TRPM3 modulation and its effect on pain perception.

Dynamic nitric oxide/drug codelivery system based on polyrotaxane architecture for effective treatment of Candida albicans infection.

The low permeability of antifungal agents to fungal biofilms, which allows the continued survival of the fungus inside, is a key issue that makes fungal infections difficult to cure. Inspired by the unique dynamic molecule motion properties of the polyrotaxane (PR) nanomedicine, herein, a dynamic delivery system Clo@mPRP/NONOate was fabricated by co-loading nitric oxide (NO) and the antifungal drug clotrimazole (Clo) onto the α-cyclodextrin (α-CD) PR modified mesoporous polydopamine (mPDA) nanoparticles, in which pentaethylenehexamine (PEHA) was grafted to α-CDs. The cationic α-CDs endowed this dynamic NO/Clo codelivery system with the ability to effectively attach to fungal biofilms through electrostatic interaction, while the introduction of PRs with flexible molecule motion (slide and rotation of CDs) enhanced the permeability of nanoparticles to biofilms. Meanwhile, NO could effectively inhibit the formation of fungal hyphae, showing an dissipating effect on mature biofilms, and could be further combined with Clo to completely eradicate fungi inside the biofilms. In addition, the dynamic system Clo@mPRP/NONOate could efficiently and synergistically eliminate planktonic Candida albicans (C. albicans) in a safe and no toxic side effect manner, and effectively cured C. albicans-induced vaginal infection in mice. Therefore, this dynamic NO/Clo codelivery system provided an effective solution to the clinical treatment of C. albicans-induced vaginal infection, and the application prospect could even be extended to other microbial infectious diseases. STATEMENT OF SIGNIFICANCE: A dynamic codelivery system based on cationized cyclodextrin polyrotaxane combining nitric oxide and antifungal drugs clotrimazole was prepared to deal with the issue of clinical fungal biofilm infection. This dynamic codelivery system could be attached to the Candida albicans biofilms and penetrate into biofilm via flexible molecular mobility to effectively eradicate the fungi. This dynamic codelivery system could synergistically and efficiently eliminate planktonic-state Candida albicans, but did not show significant cytotoxicity to normal somatic cells.

A Comprehensive in vitro and in silico Assessment on Inhibition of CYP51B and Ergosterol Biosynthesis by Eugenol in Rhizopus oryzae.

Mucormycosis, also known as Zygomycosis, is a disease caused by invasive fungi, predominantly Rhizopus species belonging to the Order of Mucorales. Seeing from the chemistry perspective, heterocyclic compounds with an "azole" moiety are widely employed as antifungal agent for minimising the effect of mucormycosis as a prescribed treatment. These azoles serve as non-competitive inhibitors of fungal CYP51B by predominantly binding to its heme moiety, rendering its inhibition. However, long-term usage and abuse of azoles as antifungal medicines has resulted in drug resistance among certain fungal pathogens. Hence, there is an unmet need to find alternative therapeutic compounds. In present study, we used various in vitro tests to investigate the antifungal activity of eugenol against R. oryzae/R. arrhizus, including ergosterol quantification to test inhibition of ergosterol production mediated antifungal action. The minimum inhibitory concentration (MIC) value obtained for eugenol was 512 µg/ml with reduced ergosterol concentration of 77.11 ± 3.25% at MIC/2 concentration. Further, the molecular interactions of eugenol with fungal CYP51B were meticulously studied making use of proteomics in silico study including molecular docking and molecular dynamics simulations that showed eugenol to be strongly interacting with heme in an identical fashion to that shown by azole drugs (in this case, clotrimazole was evaluated). This is the first of a kind study showing the simulation study of eugenol with CYP51B of fungi. This inhibition results in ergosterol synthesis and is also studied and compared with keeping clotrimazole as a reference.

Treatment of otomycosis with clotrimazole: results accordingly with the fungus isolated.

BACKGROUND: Otomycosis is usually caused by Candida spp or Aspergillus spp. While Candida is usually multissensitive to available antifungals, Aspergillus is not. Topical antifungals for otomycosis that are available in Portugal are scarce, and systemic treatments have too many interactions and contraindications. OBJECTIVES: Determine otomycosis epidemiology, microbiology and treatment results. METHODS: Observational study that included patients followed in Professor Doutor Fernando Fonseca Hospital, between 2011 and 2020. Otomycosis diagnosis was obtained through ear drainage culture, and every case was treated with 1% clotrimazole ear drops plus ear cleaning once per week. RESULTS: Aspergillus was found in ear drainage culture in 43.9% of patients and Candida in the remaining. There was a significant statistical difference between patients with otomycosis caused by Aspergillus versus Candida in treatment duration from 25.0 days (16.5-43.0) versus 14.0 days (7.0-18.5) (p < .001), respectively. CONCLUSIONS: Otomycosis was more frequently caused by Candida, and this type of otomycosis is treated faster with clotrimazole 10 mg/dL plus ear cleaning, when compared with otomycosis by Aspergillus. SIGNIFICANCE: If otomycosis causative agent is identified or suspected, a prediction of the time needed till the resolution of otomycosis can be made, when clotrimazole ear drops are used.

Self-Healable, Injectable Hydrogel with Enhanced Clotrimazole Solubilization as a Potential Therapeutic Platform for Gynecology.

Injectable, self-healing hydrogels with enhanced solubilization of hydrophobic drugs are urgently needed for antimicrobial intravaginal therapies. Here, we report the first hydrogel systems constructed of dynamic boronic esters cross-linking unimolecular micelles, which are a reservoir of antifungal hydrophobic drug molecules. The selective hydrophobization of hyperbranched polyglycidol with phenyl units in the core via ester or urethane bonds enabled the solubilization of clotrimazole, a water-insoluble drug of broad antifungal properties. The encapsulation efficiency of clotrimazole increases with the degree of the HbPGL core modification; however, the encapsulation is more favorable in the case of urethane derivatives. In addition, the rate of clotrimazole release was lower from HbPGL hydrophobized via urethane bonds than with ester linkages. In this work, we also revealed that the hydrophobization degree of HbPGL significantly influences the rheological properties of its hydrogels with poly(acrylamide-ran-2-acrylamidephenylboronic acid). The elastic strength of networks (GN) and the thermal stability of hydrogels increased along with the degree of HbPGL core hydrophobization. The degradation of the hydrogel constructed of the neat HbPGL was observed at approx. 40 °C, whereas the hydrogels constructed on HbPGL, where the monohydroxyl units were modified above 30 mol %, were stable above 50 °C. Moreover, the flow and self-healing ability of hydrogels were gradually decreased due to the reduced dynamics of macromolecules in the network as an effect of increased hydrophobicity. The changes in the rheological properties of hydrogels resulted from the engagement of phenyl units into the intermolecular hydrophobic interactions, which besides boronic esters constituted additional cross-links. This study demonstrates that the HbPGL core hydrophobized with phenyl units at 30 mol % degrees via urethane linkages is optimal in respect of the drug encapsulation efficiency and rheological properties including both self-healable and injectable behavior. This work is important because of a proper selection of a building component for the construction of a therapeutic hydrogel platform dedicated to the intravaginal delivery of hydrophobic drugs.

yEvo: experimental evolution in high school classrooms selects for novel mutations that impact clotrimazole resistance in Saccharomyces cerevisiae.

Antifungal resistance in pathogenic fungi is a growing global health concern. Nonpathogenic laboratory strains of Saccharomyces cerevisiae are an important model for studying mechanisms of antifungal resistance that are relevant to understanding the same processes in pathogenic fungi. We have developed a series of laboratory modules in which high school students used experimental evolution to study antifungal resistance by isolating azole-resistant S. cerevisiae mutants and examining the genetic basis of resistance. We have sequenced 99 clones from these experiments and found that all possessed mutations previously shown to impact azole resistance, validating our approach. We additionally found recurrent mutations in an mRNA degradation pathway and an uncharacterized mitochondrial protein (Csf1) that have possible mechanistic connections to azole resistance. The scale of replication in this initiative allowed us to identify candidate epistatic interactions, as evidenced by pairs of mutations that occur in the same clone more frequently than expected by chance (positive epistasis) or less frequently (negative epistasis). We validated one of these pairs, a negative epistatic interaction between gain-of-function mutations in the multidrug resistance transcription factors Pdr1 and Pdr3. This high school-university collaboration can serve as a model for involving members of the broader public in the scientific process to make meaningful discoveries in biomedical research.

[Analysis of resistance and sensitivity of 1 200 strains of vulvovaginal candidiasis in China to five common antifungal drugs].

Objective: To test the antibiotic susceptibility of vulvovaginal candidiasis pathogenic strains to 5 antifungal drugs commonly used in clinic. Methods: A total of 1 200 vulvovaginal candida patients from 23 gynecological and family planning outpatient departments in China were enrolled. Their vaginal secretions were collected for candida strain isolation and species identification. According to Clinical and Laboratory Standards Institute (CLSI) M27-S3, the sensitivity of 1 200 strains to clotrimazole, fluconazole, miconazole, itraconazole and nystatin was tested. Results: (1) The sensitivity and resistance of 1 200 vulvovaginal candidiasis pathogens to 5 antifungal drugs were statistically different (χ2=3 513.201, P<0.01). (2) All strains had higher sensitivity to nystatin [99.92% (1 199/1 200)], followed by miconazole [92.25% (1 107/1 200)] and clotrimazole [87.17% (1 046/1 200)]. All strains had higher resistance to fluconazole [69.17% (830/1 200)], while itraconazole was 50.83% (610/1 200). (3) There was no significant difference between candida albicans and non-candida albicans in drug sensitivity to nystatin (P=0.315) and miconazole (P=0.425). (4) Candida albicans and non-candida albicans showed different sensitivity to clotrimazole, fluconazole and itraconazole, respectively. Compared with non-candida albicans, candida albicans showed higher sensitivity to clotrimazole [susceptibility rate: 73.01% (165/226) vs 90.45% (881/974); P<0.001] and higher resistance to fluconazole [resistance rate: 50.88% (115/226) vs 73.41% (715/974); P<0.001]. Although the drug sensitivity of itraconazole was not high, the susceptibility rate of candida albicans to itraconazole was slightly higher than that of non-candida albicans [37.68% (367/974) vs 23.89% (54/226)], and the drug resistance rate was lower [49.28% (480/974) vs 57.52% (130/226)]. Conclusions: The sensitivity of 1 200 strains of candida to 5 antifungal drugs is significantly different, the sensitivity rate of nystatin, miconazole and clotrimazole are higher, but the resistance rate of fluconazole and itraconazole are higher. The sensitivity of candida albicans and non-candida albicans to the same drug is also significantly different. It is suggested that in clinical diagnosis and treatment, we should pay attention to the identification of candida and drug sensitivity test, so as to select antifungal drugs rationally.