Finite dose application of the micelle-based formulation for 6 h resulted in a statistically equivalent deposition of ECZ (p > 0.05) and TBF (p > 0.05) from the 2 systems, and a 2-fold higher accumulation of AMF (p = 0.017). Antifungal concentrations above MIC80 against Trichophyton rubrum were achieved in each skin layer with the “tri-therapy”, which also exhibited a preferential deposition of each antifungal agent in pilosebaceous unit (PSU)-containing biopsies as compared with PSU-free biopsies (p < 0.05). A planned clinical study will test whether these promising results translate to improved therapeutic outcomes in vivo.Lack of accurate diagnosis and the use of formulations designed to address the poor aqueous solubility of antifungal agents, but not optimized for delivery, contribute to unsatisfactory outcomes for topical treatment of cutaneous mycoses. The objective of this study was to develop a micelle-based antifungal formulation containing econazole (ECZ), terbinafine (TBF) and amorolfine (AMF) using D-α-tocopheryl polyethylene glycol succinate (TPGS) for simultaneous cutaneous delivery of three agents with complementary mechanisms of action. The antifungal “tri-therapy” micelle-based formulation containing 0.1% ECZ, 0.1% TBF and 0.025% AMF had a drug loading 10-fold lower than the “reference” marketed formulations (Pevaryl Trichophyton rubrum [Dermatomycosis is a widespread condition characterized by both superficial and subcutaneous infections of keratinous tissues ,2. Appron rubrum ,4,5, andn rubrum .Although standard treatments using azole compounds and terbinafine are effective in many cases of cutaneous dermatophytosis, oral medication for an extensive period of time may lead to significant side effects ,8 and th® using Advanced Chemistry Development (ACD/Labs) Software V11.02 ©1994-2019 ACD/Labs) is the most prominent therapeutic agent employed topically to treat skin infections. However, it has been reported that ~90% of the ECZ present in the marketed formulation ) remained on the skin surface following topical application, potentially leading to irritation, redness, burning or itching [However, the poor aqueous solubility of many antifungal agents makes their formulation difficult and results in limited cutaneous bioavailability. For example, econazole , TBF  and AMF  —it was dTo date, various TPGS-based drug delivery systems, including prodrugs , liposomHere, we present the first report on the simultaneous cutaneous delivery of ECZ, TBF and AMF from a micelle-based formulation. The specific aims of the study were as follows: (i) to develop a triple antifungal micelle-based formulation containing ECZ, TBF and AMF using TPGS; (ii) to quantify the cutaneous delivery of each drug from the micelle-based formulation and to compare it to that from marketed formulations; (iii) to investigate the cutaneous biodistribution  and foll® 80 were purchased from Sigma-Aldrich Chemie . Acetone, methanol and acetonitrile (ACN) of analytical grade were purchased from Fisher Scientific . Formic acid (extra pure 99%) was obtained from Biosolve Chemicals . OCT mounting medium was obtained from VWR Chemicals . All aqueous solutions were prepared using Milli-Q water . Marketed formulations of ECZ-Pevaryl® Cream , TBF-Lamisil® Cream (1% w/w) and AMF-Loceryl® Cream  were purchased from a local pharmacy. The major ingredients of the formulations are: (i) Pevaryl® Cream—macrogols (PEG-6 and PEG-32)/glycol stearate, oleoyl macrogolglycerides, liquid paraffin, butylhydroxyanisole (E320), benzoic acid (E210), flower perfume 4074, purified water; (ii) Lamisil® Cream—sodium hydroxide, benzyl alcohol, sorbitan monostearate, cetyl palmitate, cetyl alcohol, stearyl alcohol, polysorbate 60, isopropyl myristate, demineralised water; (iii) Loceryl® Cream—polyoxyl 40 stearate, stearyl alcohol, paraffin liquid, white soft paraffin, carbomer sodium hydroxide, disodium edetate, 2-phenoxyethanol.TBF hydrochloride and AMF hydrochloride were purchased from Hangzhou Dayang Chem Co., Limited . ECZ nitrate, TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), sodium chloride, isopentane, Dulbecco’s phosphate-buffered saline  and Tween® RP-18, 5 µm, 125 × 40 mm column  that was maintained at 30 °C. The mobile phase consisted of a mixture of acetonitrile and water  containing 12 mM phosphoric acid and 12 mM trimethylamine. The flow rate and injection volume were 1.5 mL/min and 50 µL, respectively. ECZ, TBF and AMF were detected at 2.54 min, 4.18 min and 6.54 min at wavelengths of 214 nm, 224 nm and 219 nm, respectively; the total run time was 10 min. Chromeleon® chromatography management software was used for integration and data analysis. The HPLC-UV method was validated according to ICH guidelines, the lower limits of detection (LOD) and lower limits of quantification (LOQ) were 1.0 and 5.0 µg/mL, respectively, for ECZ, TBF and AMF. Complete details are provided in the A high-performance liquid chromatography (HPLC) system consisting of an LPG-3400A pump equipped with a WPS-3000 autosampler, a thermostatted column compartment TCC-3x00, and a UVD-3000 detector  was used to simultaneously quantify ECZ, TBF and AMF contents in the micelle formulation. Isocratic elution was performed using a Lichrospher® UPLC® system  comprising a binary solvent pump, sample manager and Waters XEVO® TQ-MS detector  was used for sample analysis. Chromatographic separation was performed using a XBridge™ BEH-C18 column , and the column temperature was maintained at 40 °C. The mobile phase consisted of a mixture of acetonitrile and water  containing 0.1% formic acid, under isocratic conditions. Solvents were degassed in an ultrasonic bath before use . The flow rate was set at 0.4 mL/min and the injection volume was 5 µL. Nitrogen and argon were used as the drying gas and collision gas, respectively. The detection was performed by electrospray ionization in positive ion mode using multiple reaction monitoring, the mass transition ion pairs were m/z 381.07→125.01, 292.27→115.04 and 318.35→105.02 for ECZ, TBF and AMF, respectively. The cone voltage and collision energy were 38 V and 26 V, 28 V and 50 V, and 10 V and 38 V for ECZ, TBF, and AMF, respectively. Data processing was performed using MassLynx® software. The method was validated according to ICH guidelines, the LOD and LOQ were 1.0 and 2.0 ng/mL for ECZ, TBF and AMF. Complete details are provided in the An ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) method was developed and validated to evaluate the permeation and cutaneous biodistribution of ECZ, TBF and AMF. A Waters Acquity® S-450D; Carouge, Switzerland) to 8 mL of Milli-Q water. Acetone was evaporated at 40 °C using a rotary evaporator . The micelle solution was equilibrated under vacuum overnight and centrifuged at 10,000 rpm for 20 min to remove the free drug  and the supernatant was collected. TPGS copolymer concentration was fixed at 25 mg/mL, and micelles were prepared with a series of target drug loadings—50, 100, 200 and 400 mg DRUG/g COPOLYMER corresponding to drug:copolymer ratios of 1:20, 1:10, 1:5, 2:5, respectively.ECZ- and TBF-loaded micelles were prepared separately using the solvent evaporation method. Briefly, a given amount of drug and copolymer were dissolved in 4 mL of acetone. The mixture was added dropwise under sonication –(3), respectively, as follows:Size Characterization: Micelle formulations were characterized for their hydrodynamic diameter (Zav), volume-weighted diameter (dv), number-weighted diameter (dn) and polydispersity index (P.I.) using dynamic light scattering (DLS) with a Zetasizer Nano-ZS . Measurements were performed in triplicate at an angle of 90° and at a temperature of 25 °C.Morphology: Micelle morphology was studied by transmission electron microscopy (TEM) using an FEI Tecnai TM G2 Sphera  equipped with a high resolution 2000 × 2000 pixel digital camera. Samples were prepared using the negative staining method. Briefly, 5 µL of micelle solution was placed onto an ionized carbon-coated copper grid , which was then placed in contact with a 100 µL drop of saturated uranyl acetate aqueous solution for 1 s and then another drop of 100 µL for 30 s. The excess staining solution was removed, and the grid was dried under room temperature.Stability of micelle formulations: Micelle solutions were stored at 4 °C for 4 weeks. To investigate the short-term stability, the amount of drug incorporated was quantified by the validated HPLC-UV method at predetermined time-points, on day 1, 7, 14, 21 and 28.ECZ- and TBF-loaded micelles showing at least 4 weeks’ stability at 4 °C were selected and combined with AMF solution by simply diluting the optimal formulations using ultra-pure water. Briefly, AMF was prepared at a concentration of 8.0 mg/mL, then 0.125 mL AMF solution was combined with ECZ-loaded micelles  and TBF-loaded micelles , and ultra-pure water was added to obtain a final volume of 4 mL. Drug content  in the “tri-therapy” was confirmed by HPLC-UV analysis. The triple antifungal micelle-based formulation was characterized in terms of drug content, morphology and stability, as described above.2) with a hydraulic press .Fresh porcine ears were purchased from a local abattoir  shortly after sacrifice. After washing with cold running water, the porcine skin was carefully excised from the external region of the ear and was separated from the underlying cartilage using a scalpel and tweezers. Hair was removed from the skin surface using electric clippers. Then, the full-thickness skin (1.0–1.5 mm) was punched into round discs  and were subsequently horizontally sliced with a Thomas Stadie-Riggs slicer  to a thickness of ∼1000 µm.Human skin samples were collected immediately after surgery from the Department of Plastic, Aesthetic and Reconstructive Surgery, Geneva University Hospital . The study was approved by the Central Committee for Ethics in Research (CER: 08-150 (NAC08-051); Geneva University Hospital). The hypodermis and fatty tissue were removed. The excised skin samples were punched out into 32 mm diameter circular discs  for 30 min before permeation experiment.The processed skin samples were stored at −20 °C for a maximum period of 2 months. Frozen skin samples (porcine or human) were equilibrated in NaCl solution  for 30 min. The micelle-based antifungal “tri-therapy” formulation (50 µL/cm2 for infinite dose conditions and 10 µL/cm2 for finite dose conditions) or marketed formulation (again either 50 mg/cm2 or 10 mg/cm2 for infinite and finite dose conditions) was applied to the exterior surface of porcine or human skin for a given application time . The receptor compartments were filled with 10 mL PBS containing Tween 80  to maintain sink conditions. The receptor compartment was stirred at 250 rpm and maintained at 37 °C throughout the experiment. Aliquots (1 mL) of the receptor phase were withdrawn upon completion of the skin delivery experiment to quantify the amount of drug permeated across the skin using the validated UHPLC-MS/MS method. The diffusion cells were then dismantled, and each skin sample was carefully washed with PBS containing 1% Tween 80 to ensure any residual formulation was completely removed from the surface. Then, the skin samples were dried with a cotton swab and cut into small pieces; drug deposited in the skin was extracted by soaking the pieces in a mixture of 20 mL ACN:H2O  overnight under constant stirring at room temperature. Then, 1.5 mL samples were withdrawn, centrifuged at 10,000 rpm for 20 min and filtered through 0.22 μm nylon membrane filters. The filtrates were then analyzed using the validated UHPLC-MS/MS method. Complete details are provided in the Skin transport studies were performed in vitro using standard Franz vertical diffusion cells  with a formulation contact area of 2 cm2) for 6 h. At the end of the skin delivery experiment, a 0.5 cm2 disc was punched out from the central area of the skin samples. The small skin disc was snap-frozen in isopentane cooled with liquid nitrogen and mounted on a cryotome  to obtain horizontal (XY plane) lamellae with a thickness of 40 µm. Each lamella was collected in a 2 mL Eppendorf tube; all lamellae and the remaining piece of skin were extracted in 1.5 mL of ACN:H2O (90:10) overnight with continuous stirring at room temperature. The samples were centrifuged at 10,000 rpm for 20 min and the concentration of ECZ, TBF and AMF was quantified by the validated UHPLC-MS/MS method. A total of 20 lamellae obtained from each skin sample (from the skin surface to a depth of ~800 µm), provided the biodistribution profile of the 3 antifungal drugs from the stratum corneum, through the viable epidermis and to the upper dermis.Cutaneous biodistribution of each antifungal drug from the micelle-based antifungal “tri-therapy” was investigated. The micelle-based antifungal “tri-therapy” formulation was applied under finite dose conditions (10 µL/cm2) for 6 h, PSU-containing and PSU-free biopsies were punched out using a previously validated punch biopsy method to evaluate the ability of micelles to target the delivery of the antifungal agents to follicular structures [2O  at room temperature on a shaker bath at 150 rpm in an Eppendorf tube. The samples were centrifuged at 10,000 rpm for 15 min; ECZ, TBF and AMF contents were quantified using UHPLC-MS/MS.After application of the micelle-based antifungal “tri-therapy” formulation was completed under finite dose conditions (10 µL/cmructures . Brieflyp < 0.05), which were performed using Sigma Plot 12.5 .Data were expressed as mean ± standard deviation (mean ± SD). Outliers determined using the Dixon test were discarded. Statistical differences were determined by a one-way ANOVA followed by a Student–Newman–Keuls test  on drug incorporation (E1–E4 for ECZ and T1–T4 for TBF). Micelles with incorporation efficiencies (I.E.) ranging from 36.93 ± 0.42 to 109.72 ± 0.64% for ECZ and 92.53 ± 0.58 to 107.57 ± 3.28% for TBF were obtained, corresponding to drug contents (D.C.) from 1.40 ± 0.01 to 5.19 ± 0.02 mg/mL for ECZ and from 1.32 ± 0.02 to 9.50 ± 0.43 mg/mL for TBF. The solubility of ECZ and TBF were significantly increased—from 0.0057 to 5.19 ± 0.02 mg/mL (910-fold) and 0.00093 to 9.50 ± 0.43 mg/mL , respectively. The drug loading (D.L.) for ECZ peaked at 200 mg DRUG/g POLYMER (E3 with a D:P ratio of 1:5) before decreasing sharply; however, for TBF, it continued to increase and a D.L. of 380.00 ± 17.24 mg DRUG/g COPOLYMER was achieved for a theoretical loading of 400 mg DRUG/g COPOLYMER. This suggested that TBF had superior affinity for TPGS compared with ECZ.Size characterization: ECZ- and TBF-loaded micelles were characterized using dynamic light scattering (DLS)  were of a comparatively smaller size than those containing TBF  with hydrodynamic diameters (Zav) from 4.675 to 4.982 nm vs. 7.084 to 9.116 nm, but in all cases < 10 nm. The results were in accordance with number weighted diameters (dn) and volume weighted diameter (dv), which were 3.1–3.3 nm and 3.8–3.9 nm for ECZ, and 5.2–6.7 nm and 6.1–7.9 nm for TBF, respectively. It was reported that micelle size is influenced by structural parameters of the polymer  polymer concentration, drug loading and drug polymer interaction. It was observed that at a given TPGS polymer concentration (25 mg/mL), a greater amount of TBF was solublized in the system, implying there are more drug molecules entrapped in each individual micelle architecture, which is consistent with the larger diameters of the TBF micelles.ng (DLS) . All forMorphology: TEM images of ECZ-loaded  and all formulations were stable after one month. Recent studies have shown that the incorporation efficiency and stability of the micelle formulation were determined by drug–copolymer affinity, which was achieved via more complex molecular mechanisms of interaction than simple hydrophobic interactions [d), log p, number of hydrogen acceptors (Ha) and the aqueous solubility [d group, the higher log p value of TBF may explain the higher drug loading and better stability. However, in order to have the same polymer:drug ratio as that in the optimal ECZ formulation, E2, and thereby avoid any effects due to differences in polymer:drug ratio, T2 was considered as the optimal TBF formulation, corresponding to a 456- and 2548-fold enhancement of aqueous solubility for ECZ (E2 from 0.0057 to 2.60 ± 0.01 mg/mL) and TBF (T2 from 0.00093 to 2.37 ± 0.01 mg/mL), respectively.In the case of TBF b, drug cractions . Accordilubility . Both EC® Cream (1% ECZ), Lamisil® Cream (1% TBF), and Loceryl® Cream (0.25% AMF), it was decided to prepare an antifungal “tri-therapy” formulation, but with 10-fold lower drug concentrations in the antifungal drug-loaded micelles.To carry out comparisons with the marketed formulations, PevarylFormulation B  was obtained by simply diluting Formulation A—the optimal ECZ micelles (E2) and TBF micelles (T2)—using ultra-pure water and combing with AMF solution . The amounts of each antifungal permeated across porcine skin, even after the longest application time from either the triple antifungal micelle-based formulation or the marketed formulations, were below the LOQ (2.0 ng/mL) of the UHPLC-MS/MS method. It should be noted that the micelle-based formulation produced a higher skin deposition than that from the marketed formulations at each time point (av) of ~4 nm vs. Pevaryl® liposomes that are reported to have diameters (Zav) of 160–200 nm [The triple antifungal micelle-based formulation and marketed formulation were applied for three different application times  in order to evaluate antifungal delivery kinetics into and through porcine skin . These fme point , which m0–200 nm ). The mi0–200 nm .Increasing the application time from 1 h to 6 h resulted in a significant increase in antifungal deposition from the micelle-based solution and the marketed creams, but the amounts accumulated from micelle solution after 12 h were statistically equivalent to those obtained at 6 h. It was surprising that AMF—existing as a free drug in the micelle solution—also achieved a higher skin deposition in comparison with the marketed formulation. It was hypothesized that (i) the hydrated environment of the aqueous solution facilitated its penetration, and (ii) TPGS might function as a penetration enhancer promoting diffusion by altering the skin structure .® (3.09 ± 1.06 vs. 1.76 ± 0.59 µg/cm2), Lamisil® (5.23 ± 1.35 vs. 1.09 ± 0.09 µg/cm2), and Loceryl® (1.37 ± 0.34 vs. 0.43 ± 0.14 µg/cm2), respectively (Considering the recommended therapeutic regimen (application 2–3 times per day) and the fact that the formulations might be washed off with showering, it was decided to focus on the experiments using an application time of 6 h—cutaneous delivery of ECZ, TBF and AMF from the “tri-therapy” micelle-based formulation under infinite dose was superior to that from Pevarylectively ; thus de2 or 10 µL/cm2) conditions have been suggested by the OECD guideline for in vitro skin absorption studies to mimic better the actual conditions of use and to decrease the effect of excipients. Therefore, 20 mg of each marketed formulation or 20 µL micelle formulation was applied on the skin (per 2 cm2)—corresponding to 0.2 mg ECZ, 0.2 mg TBF and 0.05 mg AMF in marketed formulations ; 0.02 mg ECZ, 0.02 mg TBF and 0.005 mg AMF in micelle-based formulation . Antifungals incorporated in the micelles—ECZ and TBF—achieved a skin deposition of 1.61 ± 0.62 and 1.71 ± 0.60 µg/cm2, respectively, following finite dose application for 6 h, which was statistically equivalent to the amount delivered from the marketed formulations—1.39 ± 0.58 and 1.70 ± 0.53 µg/cm2 (p > 0.05)—despite the 10-fold lower loading, and AMF resulted in a 2-fold higher accumulation from micelle solution as compared with the marketed cream (0.60 ± 0.23 vs. 0.36 ± 0.09 µg/cm2) (Finite dose (10 mg/cm µg/cm2) . Thus, t µg/cm2) .p > 0.05) in skin deposition upon passing from infinite to finite dose conditions. However, 2–3-fold reductions in antifungal deposition in skin were observed for the micelle-based formulation. It could be explained by water evaporation and subsequent crystallization of drug molecules and/or depletion from the micelle solution under finite dose conditions, which would clearly hinder partitioning and penetration of the antifungal drugs into the stratum corneum. However, the decrease in skin deposition was less than the 5-fold change in the amount of formulation applied. The impact of finite dose on nanocarrier formulation needs to be further elucidated and methods devised to offset the effect of water evaporation.In vitro studies performed under finite dose provide a better simulation of in vivo application. In the case of the marketed formulations, there were no statistically significant differences (2), TBF (0.67 ± 0.22 vs. 0.64 ± 0.16 µg/cm2) and AMF (0.14 ± 0.06 vs. 0.21 ± 0.06 µg/cm2) deposited in epidermis  from marketed and micelle-based formulations were statistically equivalent  against the main dermatophytic pathogen, Trichophyton rubrum  [Although the results confirmed that the triple antifungal micelle-based formulation was able to deliver ECZ, TBF and AMF simultaneously into skin, it is important to know the spatial distribution of the drug molecules and to identify to which skin layer they had been delivered. Therefore, the porcine skin was cryotomed into a series of lamellae, each with a thickness of ~40 µm, following the permeation experiment and samples were analyzed using the validated UHPLC-MS/MS methods. Antifungal deposition in each section of porcine skin was quantified, the amounts of ECZ  will be employed to visualize the spatial distribution of these molecules in the skin and thereby identify penetration pathways—this should also serve to highlight the potential contribution of appendageal routes to drug delivery. We have recently described how this technique was used to visualize the molecular distribution of ECZ and TPGS in 40 µm thick horizontal sections and vertical cross-sections of the skin following topical micelle application; the co-localization of drug and excipient with endogenous skin components provided insight into the respective cutaneous penetration pathways .2, p = 0.0027) than in PSU-free biopsies  after application of micelle formulation under finite dose for 6 h. AMF deposition in PSU-containing biopsies was also higher than that in PSU-free biopsies , which was in accordance with the reports on caffeine (log P = −0.07) transport through hair follicles in vitro [Based on the fact that the spores invade skin tissue by secreting germ tubes, and that the pathogen tends to accumulate in the hair follicle structure during infection, selective delivery of the antifungal agents to the PSU might be expected to produce a better therapeutic effect. Therefore, PSU-containing biopsies or PSU-free biopsies were punched out upon completion of the skin delivery experiment, the integrity of their structure was visually confirmed under light microscopy and drug amounts quantified . In porcin vitro  and in vin vitro ; this po2, respectively) than in PSU-free biopsies   b. Indeed80 against Trichophyton rubrum. A preferential deposition of each antifungal agent in PSU-containing biopsies was observed in porcine, and more importantly, human skin, and a higher therapeutic efficacy might be expected from the synergistic effect of the antifungal combination that target key enzymes involved in ergosterol biosynthesis. In conclusion, the study employed a novel nanocarrier-based formulation to enable polypharmacy through simultaneous delivery of drugs with complementary mechanisms of action and used advanced experimental methods to investigate the cutaneous biodistribution of the three drugs and the ability of the formulation to target appendageal structures. The next steps in the project will involve the following: (i) a preliminary investigation in vitro into the efficacy of this novel formulation against fungal strains; (ii) computational studies to simulate the process of self-assembly and drug loading and distribution in the micelle. These will be extremely useful, together with further experimental data, in elucidating the molecular interactions involved in the formation of drug-loaded polymeric micelles and to predict the compatibility of a specific drug with a given copolymer.An antifungal “tri-therapy” containing 0.1% ECZ, 0.1% TBF and 0.025% AMF was successfully developed using D-α-tocopheryl polyethylene glycol succinate (TPGS) and enabled simultaneous delivery of the three antifungal agents. The novel polymeric micelle carrier system yielded a statistically equivalent deposition of ECZ, TBF and AMF in the treatment compartment, the epidermis, as compared to the marketed formulations under finite dose condition, despite a 10-fold lower drug content. Drug concentrations of each antifungal agent in all the skin layers were significantly higher than their MIC


Gyrovirus of the Anelloviridae family, has been described in multiple hosts. Epidemiologically, there are suggestions that GyV3 is associated with diarrhea/proventriculitis, however, no direct causal evidence exists between GyV3 infection and specific clinical diseases. Herein, we infected special pathogen-free (SPF) chickens with GyV3, and then assessed the pathogenicity and tissue tropism. The results revealed that GyV3 induced persistent infection characterized by diarrhea, aplastic anemia, immunosuppression, and persistent systemic lymphocytic inflammation. Clinically, the infected chickens presented ruffled feathers, diarrhea, anemia, and weight loss. Aplastic anemia was characterized by progressive depletion of hematopoietic cells in the bone marrow, immunosuppression was associated with atrophy of the thymus, spleen, and bursa of Fabricious, progressive lymphocytic inflammations were characterized by proventriculitis, adrenalitis, pancreatitis, hepatitis, nephritis, and bronchitis. Viral loads of GyV3 in tissues exhibited “M”, “N”, “W” or “V” type dynamic changes. The highest level of viral loads was reported in bone marrow at 7dpi, followed by the adrenal gland at 2 dpi, the sciatic nerve at 7 dpi, and bile at 35 dpi. The bone marrow and kidney demonstrate the strongest immunostaining of GyV3-VP1 antigen and were suggested as the target tissues of GyV3. Collectively, GyV3 is an immunosuppressive pathogenic virus that targets the bone marrow and kidney in chickens. Exploring the pathogenicity and tissue tropism of GyV3 will guide the basic understanding of the biology of GyV3 and its pathogenesis in chickens.Gyrovirus 3 (GyV3), the third novel emerging species of the genus  Gyrovirus (GyV) of the Anelloviridae family, comprises a group of small, non-enveloped, icosahedral viruses with a circular single-stranded approximately 2.3-kb long DNA (ssDNA) genome (negative-sense). The chicken anemia virus (CAV) prototype and thirteen other unofficially recognized species  belongs to the GyV genus that have been described in humans, ferrets, cats, wild birds, sea birds, chickens, pigeons, and ducks .GyV3-SDAU-1 strain  was stored in our Molecular Pathology Laboratory. Based on previous reports, we isolated GyV3 from the kidney. Briefly, kidneys from GyV3 positive chicks were homogenated in DMEM (1 g kidney + 5 mL DMEM) with 30 μL penicillin (3000 IU/mL) and 30 μL streptomycin (3 mg/mL). The homogenate was placed in an ice bath. Then, using an ultrasonic breaker, the tissue was crushed for 3 s at intervals of 3 s, 99 × 3 times, differential centrifugation at 4 °C . Rabbits were immunized with the protein three times to obtain antiserum. Then, we used the purified antiserum as the primary antibody for immunohistochemistry (IHC). Serum collected from VP1-free rabbits served as the negative control.An anti-GyV3 polyclonal antibody was prepared from rabbits. Briefly, recombinant fusion proteins of pET32a-GyV3-VP1 were expressed in 7 viral copies of the GyV3-SDAU-1 strain quantified via quantitative real-time PCR (qPCR) from the supernatant of kidney homogenate of the GyV3 infected SPF chicks. The weights and deaths of the birds were recorded daily. Sera were used to detect the viral infection rate through PCR 7 days post-infection (dpi). Three chickens were euthanized and necropsied at 2, 3, 5, 7, 14, 21, 28, and 35 dpi, respectively. In the control group, three animals were euthanized and necropsied at the same duration intervals, respectively. We collected 29 tissues from feather pulp, skin, pancreas, ovary or testis, footpad, trachea, lung, bone marrow, spleen, thymus, bursa of Fabricius, heart, pectoralis, crureus, kidney, adrenal gland, cerebrum, cerebellum, sciatic nerve, liver, bile, proventriculus, ventriculus, duodenum, jejunum, ileum, cecum, colon and rectum for histopathological examination, qPCR and IHC assay.Sixty 1-day-old SPF chicks were categorized into the control and GyV3 infected groups. On day 1, each chick of the infection group was intraperitoneally infected with 200 µL of 3.37 × 10Upon necropsy, tissues were fixed in 10% neutral formalin for 48 h at room temperature, embedded in paraffin, and cut into 4-μm-thick sections. After deparaffinization, the sections were subjected to hematoxylin and eosin (H&E) staining and examined using a light microscope .–9/[(molecular weight of vector + molecular weight of inserted fragment) × 660] (g/mol)). To test the sensitivity of the established real-time PCR, the constructed plasmids were diluted from 109 to 103 in TE buffer , and then used as templates to generate a standard curve for GyV3. The threshold cycle (Ct) of these standard dilutions was plotted against the log value of the copy number of the corresponding standard plasmid. Real-time PCR was performed using a Roche LightCycler96 machine . The real-time PCR mixture comprised 10 μL TB Green®Premix Ex Taq™ , 0.4 μL forward primer (10 μmol/L), 0.4 μL reverse primer (10 μmol/L), 1 μL template DNA, and sterile water to attain a final volume of 20 μL. The amplification parameters were set as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s, 95 °C 1 s, 60 °C 15 s, 95 °C 1 s. Roche fluorescence quantitative PCR system automatically generated a standard curve.Viral DNA copy number from tissues of GyV3 infected chickens was established through extrapolation from a GyV3 plasmid-based standard curve. We applied the DNASTAR software  to verify the highly conserved regions in the GyV3-SDAU-1 genomes. Primers were designed using the Primer 5.0 software . The following primer pair was used: Forward, GyV3-F: 5′-ACCGGGACTTGGACACC-3′, reverse, GyV3-R: 5′-AGCCAGGAAGCGATACG-3′. The GyV3 amplicons were 155 base pairs in length. The PCR products were cloned into the pMD18-T vector. The constructed plasmids pMD18-T were transformed into DH5α competent cells  and then extracted using QIAGEN Plasmid Midi Kits. The plasmids served as templates for real-time PCR optimization. An OSE260 spectrophotometer  was used to quantify the plasmids. The copy numbers of the plasmids were calculated as follows: log10 copies/μL = 6.02 × 1023 (copies/mol) × plasmid concentration (ng/μL) × 10Serial sections (4 µm) of selected tissues from chickens were immunolabelled for GyV3 VP1 antigen using polyclonal antibodies specific for VP1 protein following a routine process. The labeling intensity and the number of immuno-labeled GyV3-positive cells in the tissues were recorded as previously described by Castaño et al. .t-test was applied to analyze the differences in mean values between groups. A P value < 0.05 denoted statistical significance.  All values are presented as mean ± SEM.All statistical data were analyzed in Graphpad Prism software, version 7.00. A Student Chickens infected with GyV3 developed an acute course of disease with 90% (27/30) morbidity. They presented ruffled feathers, diarrhea and anemia Figure A, and siGyV3 infected chickens presented with progressive immunosuppression, aplastic anemia, and systematic lymphocytic inflammation from 7 dpi. The gross lesions reported in GyV3 infected birds were characterized by yellowish-white bone marrow Figure A, atroph10 copies/µL), followed by an adrenal gland at 2 dpi (6.80 ± 0.08 log10 copies/µL) and bile at 35 dpi (6.57 ± 0.28 log10 copies/µL). The lowest viral loads in nearly all tissues were reported on 21 dpi, which implicated 21 dpi as the critical point of GyV3 infection.Using qPCR, copies of the GyV3 genome were quantified in 29 tissues of infected chickens on 2, 3, 5, 7, 14, 21, 28, and 35 dpi. Figure To understand the tissue tropism and target organs of GyV3, the distribution and localization of GyV3-VP1 were evaluated via IHC in various tissues at 27 dpi. GyV3 displayed restricted tissue tropism. The strongest immunostaining was detected in myeloid cells of bone marrow Figure A, epitheGyrovirus genus of the Anelloviridae family have been reported in multiple natural hosts. Compelling evidence shows that several GyV are associated with gastroenteritis or neurological signs but their potential pathogenicity remains elusive. Previously, we identified GyV3 from commercial broiler chickens with TVP and confirmed its epidemic in chicken flocks [Large-scale and highly diverse novel Anelloviruses have presently been identified through viral metagenomics of different hosts or environments . Howevern flocks . In the As for the source of GyV3, Niu et al.  had specAnelloviridae family are in most cases, asymptomatic [Gyrovirus, chicken anemia virus (CAV), which potentially can cause illness in young chickens [Infections by viruses of the ptomatic , except chickens . Like CASeveral specific lesions induced by GyV3 should be of high concern. We reported a majority of bone marrow failures characterized by aplastic anemia during GyV3 infection. The high GyV3 loads and strong VP1 immunolabelling further confirmed that the bone marrow is a potential direct target for GyV3, inducing aplastic anemia. Viral metagenomics revealed 46 different species of Anelloviruses in bone marrow specimens from hematologic patients , suggestGyroviruses, GyV8 was described in a case of ataxia in a northern fulmar [Chauna torquata, associated with a neurological disease with clinical and pathological characteristics [Besides, the high neuroendocrine-tropism of GyV3 is an apathogenic feature. We found that high-level GyV3 loads or VP1 expression in the cerebellum, sciatic nerve, cerebellum, adrenal gland, and pancreas were associated with viral encephalitis, adrenalitis, and pancreatitis. Previously, in genus n fulmar , howevereristics . Other seristics , 31. TheDetection of viral loads and GyV3-VP1 antigen expression revealed that GyV3 exhibits a persistent active replication and broad tropism in body tissues. These events contribute to the clinical manifestations and epidemiology observed in the current epidemic. As such, most researchers believed that fecal–oral is the transmission route of Gyroviruses. In our study, early and late stages of infection, and high GyV3 loads were reported in the ovary, trachea, and feather pulp, respectively. We concluded that GyV3 can efficiently spread via vertical and horizontal transmission routes. In addition, we found no significant correlation between the severe gastroenteritis and the low viral load of the digestive canal, an implication that local GyV3 replication in the digestive canal occurred in a few target cells. Thus, further exploration is warranted to identify the concrete target cells. The timing of peak viral loads (1 dpi) did not coincide with the timing of peak disease severity (21 dpi). Conversely, at the lowest point, 21 dpi, GyV3 infected chickens were characterized by severe lesions. Therefore, the impact of GyV3 load on histopathological changes should be further explored.The present study implicates GyV3 as an immunosuppressive pathogenic virus with bone marrow as the principal target organ that mediates the immunosuppression. The basic understanding of the pathogenicity and tissue tropism of GyV3 will be vital in the understanding of the biology of GyV3, and reveal the pathogenesis in chickens.


A remote Raman prototype with a function of excitation energy adjusting for the purpose of obtaining a Raman signal with good signal-to-noise ratio (SNR), saving power consumption, and possibly avoiding destroying a target by high energy pulses, which may have applications for Chinese planetary explorations, has been setup and demonstrated for detecting different minerals. The system consists of a spectrograph equipped with a thermoelectrically cooled charge-coupled device (CCD) detector, a telescope with 150 mm diameter and 1500 mm focus length, and a compact 1064 nm Nd:YAG Q-switched laser with an electrical adjusted pulse energy from 0 to 200 mJ/pulse. A KTP crystal was used for second harmonic generation in a 1064 nm laser to generate a 532 nm laser, which is the source of Raman scatting. Different laser pulse energies and integration time were used to obtain distinguishable remote Raman spectra of various samples. Results show that observed remote Raman spectra at a distance of 4 m enable us to identify silicates, carbonates, sulfates, perchlorates, water/water ice, and organics that have been found or may exist on extraterrestrial planets. Detailed Raman spectral assignments of the measured planetary materials and the feasible applications of remote Raman system for planetary explorations are discussed. As a powerful spectroscopic analysis technique, Raman spectroscopy that has been applied into many geoscientific areas including mineralogy, gemology, planetary analyses and space exploration, astrobiology and biomineralization, cultural heritage and archaeometry, etc., can provide accurate and detailed molecular and structural information of Earth and planetary materials. Owing to its advantages in no sample preparations, quick and non-destructive analyses, unambiguous phase identifications, as well as low-mass and robust behaviors on the mineralogy and mineral chemistries of rock and soil samples, many Raman spectroscopic studies on returned samples , meteori2 and CO2 from an oil smoke plume using a remote Raman system at a distance of 20 m [3, NaNO2, and acetaminophen at a distance of ten meters [However, almost all of these studies used traditional micro-Raman with a working distance of several centimeters or less. During planetary exploration, tools with long detective distance are desirable. In the early 1960s, remote Raman was developed and employed in the detection of gases. Kobayasi and Inaba successfully observed Raman features of SO of 20 m . In 1992n meters . To daten meters ,8,9, espn meters ,17,18,19Wiens et al. detailed and introduced the SuperCam Instrument Suite including remote Raman spectrometer from the aspect of optical description, mechanical and thermal description, electronics, software, operation, and model development and environmental testing . They prLaser power is an adjustable parameter in a traditional Micro-Raman spectrometer. Using proper laser power would obtain Raman signals with good SNR, but could also protect samples from being destroyed by high laser energy. Furthermore, many dark materials need higher laser energy than bright materials to excite Raman scattering. Hence, a remote Raman system with a function of excitation energy adjusting will not only obtain Raman signals of both brightness and dark materials without burning them but also reduce power consumption, which is significant while designing missions for planetary exploration. However, a remote Raman system with a function of excitation energy adjusting has been seldomly discussed.Based on these laboratory studies and practical planetary exploration applications related to Raman spectrometers, herein, we aim to build a remote Raman system with a function of excitation energy adjusting. During planetary explorations, using different pulse energies for different types of minerals will help obtain Raman signal with good SNR, save power consumption, and possibly avoid destroying the target by high energy pulses. Raman peaks may be shifted by the environment of exoplanets; the calibration targets  carried with the rover from Earth are necessary for remote Raman to calibrate on extraterrestrial planets. The system we built showed an ability of obtaining hydrous/anhydrous minerals, water/water ice, and organics Raman spectra of high quality, which are comparable to the remote Raman spectra obtained by other groups. China has developed several successful missions to the Moon (Chang’e 1–5) and has an ongoing mission to Mars   with an exchange efficiency of 45–50%, was selected as the light source. The 532 nm green laser used in oblique geometry to excite Raman scattering was reflected onto samples by two 532 nm mirrors (45°). The oblique geometry delivers all the laser pulse power to the target and creates less near-field scattering at a short distance [In this work, we built a remote Raman system in Shandong University using a compact Nd:YAG Q-switched laser source , a spectrograph equipped with a CCD  detector, and a telescope  shown in −1 in the wavelength region of 531–614 nm and 4.8 cm−1 in the wavelength region of 605–699 nm, respectively. Raman scattering was first focused to pass through a 50 µm slit by lens combination and then transmitted into a collimated beam by lens, finally irradiated on the volume holographic grating, which will disperse Raman scattering onto CCD. While conducting Raman scattering measurement, CCD worked in CW mode, which means the detector is “on” during the integration time. All of the Raman measurements were conducted in our lab at 4 m and 20 Hz with different pulse energies as well as different integration time in a dark environment for obtaining a better SNR.When the laser hits the samples, Raman signal will be generated and collected by the telescope and then focused onto a fiber probe (FP) by the convex lens (L). The reflected 532 nm laser is removed by a 532 nm Notch filter (NF) fixed between the telescope and L. An optical fiber with 600 µm diameter core transferred the Raman signal into the spectrograph, in which two volume holographic gratings are employed. The spectral resolutions of the two volume holographic gratings with a 50 µm slit were 5.48 cm4·7H2O), KClO4, melanterite (FeSO4·7H2O), gypsum (CaSO4·2H2O), alunogen (Al2(SO4)3·18H2O), potassium nitrate (KNO3), potassium carbonate (K2CO3), NaClO4·H2O, Mg(ClO4)2·6H2O, L-Alanine, L-Phenylalanine, L-Glutamine and ethanol (C2H6O)) of analytical grade were purchased from the Sinopharm Chemical Reagent Beijing Co., Ltd. Rhomboclase (FeHSO4·4H2O) was synthesized using the method mentioned by Ling et al. [With the purpose of testing the performance of this remote Raman system, we conducted a series of remote Raman measurements on minerals, analytical grade chemicals, deionized water, and water ice. Mineral samples  were collected by petrographic laboratory in Shandong University. No cutting or polishing was performed with these mineral samples. Analytical grade chemicals  incident to sample. According to previous works [3 in remote Raman spectrum can be identified as follows: the strongest Raman peaks 1052 cm−1 are assigned to ν1(Ag) mode of NO3−1; another Ag mode observed at 1362 cm−1 is assigned to ν3 mode of NO3−1; peak at 1346 cm−1 belongs to ν3 (B1g) mode of NO3−1; peak at 716 cm−1 is attributed to ν4 (Ag + B1g) mode of NO3−1. Raman peaks in wavenumber region of 190–150 cm−1 and 310–285 cm−1 are attributed to relative translations between the cations and anionic groups [−1 are asymmetric bending mode of ν4 of K2CO3 and CaCO3, respectively. Symmetric stretching mode of ν1 are observed at 1063 cm−1 for K2CO3 and at 1085 cm−1 for CaCO3. Asymmetric stretching mode of ν1 are identified by peaks near 1407 and 1755 cm−1 in remote Raman spectra of K2CO3 and CaCO3 respectively. An amount of 1786 cm−1 in K2CO3 and 1755 cm−1 in CaCO3 are ν1 + ν4 modes. Detailed assignments of Raman spectra of KNO3, CaCO3 and K2CO3 are shown in 1, ν3) of Raman peak seems to be lower for NO3 (1052 cm−1 and 1362 cm−1) than that of CO3 (1063 cm−1 and 1407 cm−1).us works ,27, Rama4·H2O, Mg(ClO4)2·6H2O, and KClO4 are shown in 1 (A1)) in the wavenumber region of 950–930 cm−1, Raman active deformation (ν4 (T2)) of Cl-O between 635 and 625 cm−1, and a theoretical Raman inactive deformation (ν2 (E)) belonging to Cl-O within range of 470–445 cm−1. A theoretical inactive vibrational mode, which displays a negligible intensity as the fourth Raman band of perchlorates caused by the anti-symmetric stretching vibration (ν3 (T2)), was observed between 1150–1040 cm−1. Raman peak positions of KClO4, NaClO4·H2O, and Mg(ClO4)22O agree well with previous works [4−1 also has been found in Apollo lunar samples and carbonaceous chondrite meteorites [The remote Raman spectra of three perchlorates NaClOus works  and are us works , Gale Crus works , Viking us works ,35, and us works  and ClO4teorites . Remote 42O, MgSO42O, Al2(SO4)32O, CaSO42O, and FeHSO42O from 100 to 4000 cm−1. The strongest peaks of five hydrous sulfates at 977, 985, 992, 1008, and 1101 cm−1 are caused by symmetrical stretching mode (ν1(SO42−)) of SO42− ions in FeSO42O, MgSO42O, Al2(SO4)32O, CaSO42O, and FeHSO42O, respectively. Another ν1(SO42−) mode of FeHSO42O was observed near 1032 cm−1. These frequency shifts of the strongest Raman peaks of covalent groups SO4 usually indicate different strong metallic ions , which share the coordinating O with S, within their structure. As a result, the vibrational frequencies of S-O would be affected by different metallic ions because of their difference in atomic weights and attractive forces between the Fe, Mg, Ca, Al and O. The remote Raman spectra of these five hydrous sulfates caused by ν3(SO42−) modes are different from each other. MgSO42O produces three peaks at 1061, 1096, and 1145 cm−1. FeSO42O has two peaks near 1101 and 1139 cm−1. CaSO42O and FeHSO42O display only one peak around 1136 and 1183 cm−1 respectively. Al2(SO4)32O has two peaks at 1087 and 1127 cm−1. The other two fundamental vibrational modes ν2 and ν4 of SO42− were found at: ν2-446 and 461 cm−1, ν4-613 cm−1 in MgSO4·7H2O; ν2-445 and 465 cm−1, ν4-618 cm−1 in FeSO42O; ν2-415 and 493 cm−1, ν4-621 and 671 cm−1 in CaSO42O; ν2-451 and 472 cm−1, ν4-597 cm−1 in FeHSO42O; ν2-412, 469 and 529 cm−1, ν4-612 cm−1 in Al2(SO4)32O. In shorter wavenumber region, the vibration of the  in MgSO42O is near 366 cm−1, in FeSO42O are at 140, 238, and 375 cm−1, in FeHSO42O are at 239 and 378 cm−1, and in Al2(SO4)32O is around 310 cm−1. Structural water in sulfates could produce different Raman bands. The O-H stretching modes have been observed at 3297 and 3421 cm−1 in MgSO42O, at 3240, 3360, and 3426 cm−1 in FeSO42O, at 3407 and 3495 cm−1in CaSO42O, at 3350 cm−1 in FeHSO42O, at 3252 cm−1 in Al2(SO4)32O. The detailed assignments have been concluded in As a significant secondary mineral, sulfates may have important implications for past and present environmental evolutions of Mars. Different types of hydrous/anhydrous sulfates  have been found on Mars by orbital remote sensing and roving missions . We cond−1 which are attributed to the symmetric (ν1) and antisymmetric stretching (ν3) vibrational modes of the water molecule, respectively [−1 [Water displays strong Raman signals in longer wavenumber region due to intramolecular stretching such as the symmetric and antisymmetric stretching vibrational modes. In deionized water Raman spectra obtained at room temperature, two strong broad Raman bands were observed around 3223 and 3448 cmectively . The incvely [−1 , which ively [−1 ,43,44. A2H6O), solid L-alanine (C3H7NO2), L-phenylalanine (C9H11NO2), and L-glutamine (C5H10N2O3), shown in −1 in −1 is attributed to stretching vibrational mode of CCO and peaks at 1053 and 1097 cm−1 are assigned to deformation mode of CCO. Peak around 1456 cm−1 is produced by bending vibrational modes of CH3 and CH2. The shoulder at 1486 cm−1 is bending vibrational mode of CH3.The symmetric stretching mode of CH3 is observed at 2873 cm−1. The symmetric and asymmetric CH3 stretching modes produce peaks at 2924 and 2972 cm−1, respectively [Organics, which might be indicative for life, are always attractive targets in planetary explorations. Definite detection of organics on extraterrestrial planets and moons might suggest the presence of life. We conducted remote Raman measurements on liquid ethanol (C3H7NO2), L-phenylalanine (C9H11NO2), and L-glutamine (C5H10N2O3) serving as basic elements for the various proteins would display many Raman peaks due to many different kinds of vibrational modes. The Raman spectra of them are shown in −1 is attributed to the (CCH3) symmetric stretching vibration, the 532 cm−1 peak is due to the rocking motion of the CO2− group. The rocking motion of the CH3 group appeared at 1010 and 1024 cm−1. A Raman peak of 1112 cm−1 is attributed to CN stretching vibration and 1304 cm−1 is caused by CH deformation. The CH3 symmetric deformation band (1357 cm−1) and asymmetric deformation bands (1457 and 1478 cm−1) were also observed. In Raman spectra of L-Phenylalanine, the intense Raman peaks at 1004 cm−1 of L-Phenylalanine is caused by C-C symmetric stretching in ring. Peak at 621 cm−1 exhibits the presence of O-C=O in-plane bending modes. Wagging vibration of CH2 is observed near 832 cm−1. 854 cm−1 is assigned to NH2 deformation. 1035 cm−1 is distributed to C-H plane deformation in ring. Raman band corresponding to C-N stretching is observed at 1163 cm−1. Raman peaks at 1216, 1309, 1414, and 1450 cm−1 are, respectively, identified to ring breathing, CH2 wagging vibration, symmetric stretching vibration of COO−, and CH2 scissor vibration. C-C stretching in ring displays two Raman peaks around 1589 and 1605 cm−1. From Raman spectra of L-Glutamine, we can find that the ν(CN) vibrational band can be found at 843 cm−1 and the ν(CC) vibrational bands are at 995 and 1047 cm−1. The δ(CH2) rocking vibrations (891 cm−1) and δ(CH2) twist vibrations were also obtained. Raman peaks at 1093 and 1328 cm−1 are attribute to the symmetric ν(CN) stretching vibrational band and the CH deformation vibration. Two δ(NH3+) rocking vibrations were observed at 1129 and 1162 cm−1. The detailed assignments of these three organics are listed in [L-alanine  and E  modes. Feldspars, which include different members, are the most abundant among all minerals and are thought to be a primary tool for classifying igneous rock. Raman spectra of feldspars should be similar with each other due to closely related members. Alkali feldspar series are between Na(Si3Al)O8 and K(Si3Al)O8, and the plagioclase feldspar series are between Na(Si3Al)O8 and Ca(Si2Al2)O8. It is a significant index whether remote Raman system is sufficiently sensitive to distinguish the small changes in structure and chemistry occurring between members of feldspars. K-feldspar and albite were selected as two feldspar members to conduct remote Raman measurement. In the region of 600 to 450 cm−1, the double Raman bands caused by a mixed Si-O-Si (or Si-O-Al) bending/stretching mode are observed at 481 and 508 cm−1 in albite, but triple characters near 457, 476 and 512 cm−1 are found in K-feldspar [ studies ,48. In ofeldspar . Compari−1 has been used to estimate impact pressure [2 structural framework. Applications for estimating impact pressure using this Raman peak shift have been demonstrated in planetary materials, e.g., lunar soils [Raman features of olivine, albite, and K-feldspar are useable for determining mineralogy, providing mineral chemistries as well as aiding lithologic distinction ,50. The pressure , which war soils , lunar mar soils . The ins−1 in KNO3 obtained with 50 mJ/pulse laser (532 nm) and 10–15 s integration time, 942 cm−1 in KClO4 obtained with 55–65 mJ/pulse laser (532 nm) and 10 s integration time. Thus, long integration time and/or high pulse energy would cause higher power consumption and unnecessary saturated Raman signal. Distinguishable Raman spectra can be obtained using 25 mJ/pulse laser (532 nm) and 5 s for KNO3, 40 mJ/pulse laser (532 nm) and 5 s for KClO4, 28 mJ/pulse laser (532 nm) and 15 s for CaSO42O, and 30 mJ/pulse laser (532 nm) and 30 s for albite in our experiment. It is not our goal to find out the minimum pulse energy/integration time for excitation of Raman signal for each sample, but to propose a remote Raman system with a function of excitation energy adjusting for reducing power consumption, obtaining Raman spectra with good SNR, and possibly avoiding damage of targets. The root mean square (RMS) SNR values shown in The Raman spectra of four samples in  were selpeakI, and backgroundI, which were the strongest background intensity in a region where no Raman signal was detected. rms, backgroundN is the RMS value of the region mentioned before. The RMS SNR values of some Raman peaks were not calculated due to the saturation that led to flat-top peaks with no accurate intensity value.The strongest peaks were selected as 2 and H2S) on the Moon, Mars, Venus, asteroids, and icy satellites, etc.A remote Raman prototype working in oblique geometry was developed in Shandong University, aiming at providing suggestions on planetary exploration analysis techniques for Chinese planetary exploration. The Raman spectra acquired by our remote Raman system at a distance of 4 m demonstrates the ability of remote Raman for detecting silicates, carbonates, anhydrous/hydrous minerals, water/water ice, and organics. With its character of being sensitive to the molecular structure of a sample, Raman spectra enable us to identify the mineralogy and deduce the mineral chemical composition. For planetary mineralogy detection and survey, a remote Raman system has advantage on no sample preparations, quick and remote analyses, and unambiguous phase identifications that can help us identify minerals, organics, water/water ice, and other volatiles (e.g., CO


Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in ex vivo mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na+ channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. Our data establish osmotic pressure as a mechanism that initiates egg activation in Drosophila and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants.Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In  Stocks were raised on standard cornmeal-agar medium at 21°C or 25°C. For dissection of mature oocytes, mated females were fattened on yeast for 48 h at 25°C.The following fly stocks were used: 31B::GFP ; Nudel R. 4.2BAPTA (Sigma-Aldrich) was used at a final concentration of 10 µM; BAPTA-AM + PF-127 (Sigma-Aldrich) was used at a final concentration of 30 µM; carvacrol (Sigma-Aldrich) used at 300–700 µm; copper sulfate (Sigma-Aldrich) was used at a final concentration of 2 mm. Standard preparation protocols were used as according to Sigma-Aldrich.2PO4, 16.6 mm KH2PO4, 10 mm NaCl, 50 mm KCl, 5% polyethylene glycol 8000, 2 mm CaCl2, brought to pH 6.4 with a 1 : 5 ratio of NaOH : KOH [Drosophila Medium, approximately 360 mOsm, a standard Drosophila culture medium that does not initiate egg activation (Thermo Fisher); Series95 halocarbon oil ; EZ-Squeeze tube 125 µm . For osmolarity experiments, sucrose (Sigma-Aldrich) was directly dissolved into distilled water and the osmolarity was measured using an osmometer (Löser).AB, 260 mOsm, containing 3.3 mm NaHOH : KOH ; Gibco S. 4.3ex vivo [Mature oocytes were dissected from the ovaries from fattened flies using a probe and fine forceps . Dissectex vivo .. 4.4Time series were acquired with an inverted Leica SP5, under 20 × 0.7NA immersion objective. The Z-stacks were acquired at 2 µm steps from the first visible plane to 40 µm deep. The Z-stacks were presented as maximum projections of the 40 µm unless stated otherwise.. 4.5Preparation for microinjection was carried out with a Femtotips II microinjection needle (Eppendorf) and a gas pressure injection system were used to inject oil into the Stage 14 egg chambers . Imaging. 4.6p-values (p < 0.05 considered significantly different) [t-test with p < 0.05 values showing significant difference. The number of asterisks represents the p-value: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.The calcium wave data were analysed statistically using Fisher's exact test with fferent) . The spi


Shewanella are among the most frequently identified psychrotrophic bacteria. Here, we have studied the cellular properties, growth dynamics, and stress conditions of cold-active Shewanella strain #4, which was previously isolated from Baltic Sea ice. The cells are rod-shaped of ~2μm in length and 0.5μm in diameter, and they grow between 0 and 25°C, with an optimum at 15°C. The bacterium grows at a wide range of conditions, including 0.5–5.5% w/v NaCl (optimum 0.5–2% w/v NaCl), pH 5.5–10 (optimum pH 7.0), and up to 1mM hydrogen peroxide. In keeping with its adaptation to cold habitats, some polyunsaturated fatty acids, such as stearidonic acid (18:4n-3), eicosatetraenoic acid (20:4n-3), and eicosapentaenoic acid (20:5n-3), are produced at a higher level at low temperature. The genome is 4,456kb in size and has a GC content of 41.12%. Uniquely, strain #4 possesses genes for sialic acid metabolism and utilizes N-acetyl neuraminic acid as a carbon source. Interestingly, it also encodes for cytochrome c3 genes, which are known to facilitate environmental adaptation, including elevated temperatures and exposure to UV radiation. Phylogenetic analysis based on a consensus sequence of the seven 16S rRNA genes indicated that strain #4 belongs to genus Shewanella, closely associated with Shewanella aestuarii with a ~97% similarity, but with a low DNA–DNA hybridization (DDH) level of ~21%. However, average nucleotide identity (ANI) analysis defines strain #4 as a separate Shewanella species (ANI score=76). Further phylogenetic analysis based on the 92 most conserved genes places Shewanella strain #4 into a distinct phylogenetic clade with other cold-active marine Shewanella species. Considering the phylogenetic, phenotypic, and molecular characterization, we conclude that Shewanella strain #4 is a novel species and name it Shewanella glacialimarina sp. nov. TZS-4T, where glacialimarina means sea ice. Consequently, S. glacialimarina TZS-4T constitutes a promising model for studying transcriptional and translational regulation of cold-active metabolism.Species of genus  Shewanella belongs to the order Alteromonadales of class Gammaproteobacteria. In 1985, Shewanellaceae  and mesophilic  bacteria , Mn(IV), Fe(III), U(VI) (The genus ellaceae . Membersellaceae . Shewanebacteria . Shewane), U(VI) , as well), U(VI) , which aShewanella spp. are saprophytic and form an integral part of the marine microflora. Certain Shewanella species, such as S. putrefaciens and S. baltica, have been associated with spoilage of fishery food products due to their psychrotrophic nature, which permits growth at low temperatures (N-oxide (TMAO) to trimethylamine (TMA), which generates a pungent odor that alongside hydrogen sulfide (H2S) gas, produced by degradation of amino acids, further exacerbates the spoilage process  was previously isolated from a Baltic Sea ice sample , 1.5g yeast extract (Fisher Bioreagents), and 9.35g marine broth (BD-Difco) in 1L of ddH2O ] agar (15g/L) and stored in 30% v/v glycerol stock at −80°C.The bacterial strain #4   for Gram staining. These slides were flooded with Gram’s iodine [1g iodine (Riedel-de Haen) and 2g potassium iodide (Merck) in 300ml distilled water] and counterstained with safranin (Merck)  of 0.2, and the cells were grown until the desired OD600 was reached. The turbidity was measured using an Eppendorf Biophotometer.The cells were cultured on rMB agar. After 48h of growth, a single colony of 2S production (Blood agar plates (3% v/v sheep blood with agar base no.2) (Labema) were used to check hemolytic activity. Iron agar test was performed to determine bacterial motility and Hoduction . Both bl2O2)  (p-phenylenediamine dihydrochloride) (Acros Organics) was used for the oxidase test  , and oxiase test . Both ca600 values.The cells were prepared in rMB media as described in section Growth Conditions, except for the starter culture to assess growth at 0°C, which was grown at 4°C for 48h (necessary for growth at 0°C). Various growth conditions, namely, temperature , pH (4.5–10.5), salinity (5–55g/L NaCl), and hydrogen peroxide (0.5–4mM)-induced oxidative stress, were evaluated as a time-course series by determining the change in ODS. glacialimarina TZS-4T on M-9 minimal media  in the presence of a sole carbon source, namely, 200mM glucose , fructose , maltose (Alfa Aesar), or galactose (Fluka), respectively. The cells were grown, as described in section Growth Conditions, in 10ml of M-9 minimal media using starter cultures grown in rMB. Bacterial growth was monitored by measuring OD600. M-9 media without any carbon source was used as negative control.Carbohydrate assimilation was determined by growing g, 4°C, after which 10ml of supernatant was collected. Subsequently, 1ml of Nessler’s reagent was added to the supernatant and the formation of a dark blue color indicating the presence of ammonia.Ammonia production was analyzed by colorimetric assays using Nessler’s reagent . The cells were grown in 50ml rMB for 24 or 48h, as described in section Growth Conditions. Cells were removed by centrifugation for 5min at 3200Biolog GENIII microplates were used for initial phenotypic characterization, providing a standardized platform to test 94 different phenotypic characteristics, including 71 carbon utilization assays and 23 chemical sensitivity tests. Samples were prepared and tested according to the manufacturer’s instructions. All assays were performed in triplicates.2O), kanamycin (50μg/ml in ddH2O), tetracycline (10μg/ml in ddH2O), and chloramphenicol (25μg/ml in 99.5% EtOH). The growth on antibiotic media was observed after 24h, and results were interpreted as resistant (+) or susceptible (−).The bacteria were grown in both rMB media and rMB agar in the presence of different antibiotics, such as ampicillin . Phylogenetic trees based on maximum likelihood, neighbor-joining, and maximum parsimony methods using the consensus sequence of the seven gene copies of 16S rRNA were constructed on MEGA software with 100 replicates as bootstrap value. To infer genome-based phylogeny, we constructed a concatenated bacterial core-gene set phylogenetic tree by the up-to-date bacterial core-gene set (UBCG) method  method . A model) method . OrthoAN) method  as an adion 3.25  applyingS. glacialimarina TZS-4T were determined by transmission electron microscopy (TEM). The cells were grown in rMB media at 15°C until they reached an OD600 of 0.6, which corresponds to approximately 108 cells/ml. Later, 2μl of the culture was incubated on carbon-coated Cu mesh grids for 1min and then negative stained with filtered, neutral 2% uranyl acetate for 15s. TEM examination was conducted using a JEOL JEM-1400 microscope  operating at 80kV and equipped with Gatan Orius SC 1000B bottom-mounted CCD camera .The size and shape of 600 of 0.6, as described in section Growth Conditions. Cells were harvested from 10ml of culture by centrifugation  and resuspended in the original volume with phosphate buffer saline (PBS). Cells were fixed with 2.5% v/v glutaraldehyde at 4°C for 20h. After fixation, the cells were washed twice with PBS using centrifugation and resuspended in half of the original volume of PBS. Cell suspension (100μl) was spread on concanavalin A-coated glasses, fixed with osmium tetroxide, and dehydrated through ethanol series . Dehydrated cells were incubated in hexamethyldisilazane overnight and coated with platinum. Finally, SEM examination was conducted using FEI Quanta 250 Field Emission Gun (FEG) Scanning Electron Microscope.For scanning electron microscopy (SEM), cells were grown to ODS. glacialimarina TZS-4T was used to inoculate a 50ml culture as described in section Growth Conditions and grown until it reached OD600 of 0.8. Samples were prepared at different temperature conditions, i.e., 5, 15, and 25°C, and the pellet from 5ml culture was washed five times with PBS. Finally, the washed pellet was used for the analysis of fatty acid (FA) composition by gas chromatography. The bacterial pellets were subjected to transmethylation by heating with 1% v/v H2SO4 in methanol under nitrogen, and the formed FA methyl esters were extracted with hexane  and compared to the spectra of several authentic standard mixtures  and published reference mass spectra  and 800μl of BCP (Acros organic). Glass beads were added to break the bacterial cells and vortexed for 10min at room temperature (RT). The lysate was centrifuged at 10000g for 10min at RT, and aqueous phase was collected. The aqueous phase was re-extracted twice with phenol:BCP (2ml of acidic phenol and 400μl of BCP). Total RNA from the aqueous phase was precipitated by adding 2.5 vol of 99.6% v/v EtOH, at −20°C overnight and pelleted by centrifugation at 10000g for 20min at 4°C. The RNA pellets were air-dried and resuspended in RNAase-free ddH2O water. RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific) and run on 2% w/v agarose, Tris-borate EDTA (TBE) gel with of Midori green (4μl per 100ml of 2% agarose) for quality assessment. The images were captured on Gel-doc XR (Biorad).RNA isolation was performed using acidic phenol:bromochloropropane BCP; . The cel2O water. Control samples were included to check amplification arising from contaminating genomic DNA and from the primer-dimer formation. All the samples were run in technical triplicates on Quantstudio 3 Real-time PCR system (Thermo Fisher Scientific). The transcript was amplified using the following conditions: 95°C for 3min followed by 50cycles of 95°C for 30s, 62°C for 30s, and 72°C for 30s. A melting curve profile was generated to determine the formation of a single amplification product. Primer efficiency for all the genes was determined using the formula Efficiency (%)=100×(10-1/slope-1) with 5-fold dilution series of template cDNA. Comparison of fold change between different target genes was using ∆∆CT method, and gyrase A (gyrA) gene was selected as reference for normalization  were tested on cDNA using end-point PCR. The PCR reaction conditions were 95°C for 2min followed by 35cycles of 95°C for 30s, 62°C for 30s and 72°C for 30s. The amplification temperature of the primer pairs was confirmed using gradient PCR from 60 to 64°C (data not shown). The products of PCR reactions were analyzed on a 2% agarose TBE gel. The images were captured using a Gel-doc XR (Biorad).The RT-qPCR primers  analysis  and S. denitrificans OS217 (96.53%) branching next to it  of 55 Shewanella strains available in NCBI. The result showed that S. aestuarii yielded the highest ANI score with 611 matching sequences S. glacialimarina TZS-4T, followed by S. Arc9-Lz and S. frigidimarina  along with S. aestuarii  values for dimarina . FurtherS. glacialimarina TZS-4T, we grew the bacteria on rMB-agar where they form smooth, circular, and convex colonies with a diameter of 1–2mm and a milky white appearance. Gram staining confirmed that S. glacialimarina TZS-4T is a Gram-negative, rod-shaped bacterium. Interestingly, after 3days of growth the colonies turn reddish-brown. A similar phenomenon was observed when the bacteria were grown for up to 29h in rMB-broth at 15 and 25°C . This showed a rod-shaped bacterium 0.5μm in diameter and ~2μm in length  and the mesophilic reference bacterium S. baltica  than at 15°C (~8 ×109 cfu/ml), and the cells lose their ability to grow altogether at 28°C (data not shown). Indeed, a significant loss of viability is also observed during the 24–48h post-inoculation window , putative collagenase genes, and clp proteases, were also identified from the genomic sequence.Finally, we investigated the antibiotic resistance of picillin . Indeed,S. glacialimarina TZS-4T, we analyzed the FA modulation as it is a well-known strategy of cold-active bacteria to maintain membrane fluidity and viability. Our analysis revealed that palmitoleic acid (16:1n-7) was the most prevalent FA component of S. glacialimarina TZS-4T, constituting 32–37mol% of the total FAs, with the highest values detected at 5°C. In general, iso-13:0 and iso-15:0 accounted for more than 10mol% and were followed by 12:0, 14:0, and 16:0 that constituted close to 5%  and store solutes, such as glycerol, sucrose, or mannitol, which provide both cryoprotection and yield an additional carbon source  and Academy Professor program ; the Novo Nordisk Foundation Emerging Investigator grant (#NNF18OC0054454 to LPS); and the Sigrid Jusélius Foundation Young Group Leader grant (to LPS). Open access fees are covered by the Helsinki University Library.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Candida auris is a recently discovered human fungal pathogen and has shown an alarming potential for developing multi- and pan-resistance toward all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome-wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes, and echinocandins. We show the stepwise accumulation of copy number variations and novel mutations in genes both known and unknown in antifungal drug resistance. Echinocandin resistance was accompanied by a codon deletion in FKS1 hot spot 1 and a substitution in FKS1 “novel” hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1, a segmental duplication of chromosome 1 containing ERG11, and a whole chromosome 5 duplication, which contains TAC1b. The latter was associated with increased expression of ERG11, TAC1b, and CDR2 but not CDR1. The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross-resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential for and diversity of MDR development in C. auris, even in a clade until now not associated with MDR (clade II), stressing its clinical importance and the urge for future research. Candida auris has emerged in at least 39 countries in every inhabited continent  nosocomial pathogen  , echinocandins , and polyenes .Over the course of a decade since its discovery , Candidaontinent , occasio threats . C. auriC. auris isolates are estimated to have acquired resistance to at least one drug, while 30 to 41% are resistant to two drugs, and roughly 4% are pan-resistant   . These n species , 13. Thed so far .ERG11, the gene encoding the fluconazole target lanosterol 14-α-demethylase. The ATP binding cassette (ABC) transporter Cdr1 was proven to act as an efflux pump of azoles in C. auris   –9, 14 an–C. auris –18, and f action . A recenf action . Reducedids S639 , 20 and ids S639  and a deids S639  in Fks1,e stress , rather  general , 23. So d ERG16)  and SNPs6) drug resistance.Overall, few studies have actually been able to validate the proposed drug resistance mechanisms in C. auris , 26, 27,r clades  and has r clades , 29. Pre of SNPs  and ofte of SNPs , 29. ThiC. auris strain B11220, the original type strain described by Satoh et al.  to fluconazole , caspofungin , and amphotericin B . Based on these MIC50 values, the wild type strain was exposed (in triplicate) to three concentrations of each drug as follows: 2× MIC50, 1× MIC50, and 0.5× MIC50, or no drug, representing three selective pressures and a control, respectively. Serial transfer with conditional drug treatment (A single colony of reatment  was mainfluconazole (F-lineage), caspofungin (C-lineage), and amphotericin B (A-lineage). Next, these single resistant strains were exposed to a second drug to acquire multidrug resistance, yielding the FC-lineage for the F (fluconazole-resistant) strain that was given caspofungin, and the CF-lineages for the C (caspofungin-resistant) strain that was given fluconazole, respectively. The name of each strain represents the experimental lineage (letter which refers to the treatment) and day of isolation (number), respectively. 50 values for each drug of each endpoint evolved strain . The length of the evolution experiment ranged from 16 (CF-lineage) to 30 days (F-lineage), although later it was shown that resistant clones emerged quite early  was exposed to FKS1 (see below) were novel to C. auris based on literature review and comparison with a set of 304 globally distributed C. auris isolate sequences representing clades I, II, III, and IV  Summary of MIC50 values for each strain and indication of (relatively low [↑] or high [↑↑]) increase or decrease (↓) of MIC in respect to the parental strain (wt) MIC. Download FIG S1, TIF file, 0.2 MB.Resistance profiles of endpoint evolved strains. Growth profiles in a broth dilution assay (BDA) of the endpoint evolved strains  and control strain (wt) for fluconazole (A), caspofungin (B), and amphotericin B (C). The percentage of growth was calculated from growth without drug and based on ODCopyright © 2021 Carolus et al.2021Carolus et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03333-20.2FIG S2MEC3 allele-specific primers on gDNA of the wild-type strain (wt) and strain A29, containing the gCg/gTg|A272V mutation in MEC3  and mutated allele (CauMEC3_B11220_PCR/Seq1_F and CauMEC3_SNP_A272V_R), respectively, and are given in FIG S2, TIF file, 0.1 MB.Example of the temperature specificity range of allele-specific primers. Here, a temperature gradient PCR was performed using the MEC3 see . Primer Copyright © 2021 Carolus et al.2021Carolus et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03333-20.3FIG S3TAC1b  by qPCR on gDNA. CNVs were determined for 1 biological repeat represented by 3 technical repeats. Download FIG S3, TIF file, 0.03 MB.Copy number variation quantification of Copyright © 2021 Carolus et al.2021Carolus et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03333-20.6TABLE S1Table S1, DOCX file, 0.02 MB.All primers used in this study. Primers are arranged per marker. “Purpose” indicates whether the primer was used for PCR and sequencing (PCR/seq), CNV or expression analysis (qPCR), or allele-specific PCR (AS). Primer pairs for AS-PCR consist of a universal PCR-sequencing primer (indicated by “PCR/seq/AS”) or universal AS-primer (indicated by “AS”) and one allele-specific primer . Download Copyright © 2021 Carolus et al.2021Carolus et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50, >64 μg/ml)  in ERG3, encoding sterol Δ5,6-desaturase  and ERG3 (present in C20) is associated with the further increase of the caspofungin MIC50, exceeding 64 μg/ml  . Whole-g-lineage . Two add64 μg/ml . The eme64 μg/ml . AcquireI strain . The samed prior . The FKSic fungi  and seemic fungi .FKS1 gene do not seem to have a significant effect on the fitness of the strains as shown in growth , oxidative stress (B), membrane stress (C), osmotic stress (D), and pH stress (E), as described in Materials and Methods. Only 1 of 3 biological repeats and 9 technical repeats is shown, as all repeats showed similar results. Stress conditions without any growth or with no differential growth between the strains are not shown. Download Copyright © 2021 Carolus et al.2021Carolus et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03333-20.5FIG S5P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Download FIG S5, TIF file, 0.03 MB.Cytotoxicity of evolved strains. The relative cytotoxicity of the endpoint evolved strains toward HeLa cells after 24 h (A) and 72 h (B) was evaluated using an LDH cytotoxicity assay as described in Materials and Methods. Bars represent means with standard deviation accounting for data obtained from 3 biological repeats, each represented by the mean of 3 technical repeats. Asterisks indicate a significant difference in relative cytotoxicity; *, Copyright © 2021 Carolus et al.2021Carolus et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 600) values measured after 36 h in RPMI-MOPS medium supplemented with 2% glucose compared to the wt strain  to an MIC50 of 4 μg/ml  region of ERG11 in azole-resistant C. albicans  that have been linked to drug (azole) resistance in C. auris so far , depicted in Additionally, a nonsense mutation (Cag/Tag|Q384*) was found in the transcription factor encoding the TAC1b gene was identified  and strain F13 (TAC1b mutation obtained) confirms that the acquisition of this mutation corresponds to a significantly increased expression of CDR1 (B9J08_000164), as shown in CDR1 is maintained in strain F30 and in the multidrug-resistant strain FC17, as shown in C. auris, it is located in a region of TAC1b that is known to harbor gain-of-function mutations in fluconazole-resistant clinical isolates of C. auris, as shown by Rybak et al.  in the conazole . Tac1b iC. auris , 26. GenB9J08_0004, as shoTwo aneuploidies independently emerged during fluconazole resistance evolution. Read coverage of whole-genome sequencing was used to analyze copy number variation (CNV) by calculating the normalized depth read coverage per 5 kb window . A visual representation of this normalized coverage for each chromosome in all endpoint sequenced strains is displayed in CP043531 to CP043537), including ERG11. During further evolution to caspofungin resistance in the FC-lineage (see ERG11). The segmental Chr1 duplication is the only genetic difference between strain F13 and strain F30 that can be attributed to the increase in MIC50 of 32 μg/ml (in strain F13) to a MIC50 of >64 μg/ml (in strain F30), as shown in ERG11, not present in strain F13 , but not CDR1 in strain CF16 (The whole chromosome 5 (Chr5) duplication in the CF16 strain contained a region of 933 kb encompassing 405 protein-encoding genes, including  strains . Expressain CF16 .The mutation in TAC1b and segmental duplication of Chr1 did not seem to have any major effect on the growth , stress C. auris clade II strains, 62.3% of a total of 61 isolates proved to be fluconazole resistant, although only 3 isolates harbored a known azole-conferring mutation in ERG11 (K143R) ] has been reported to confer azole resistance in vitro and in vivo  and TAC1b but not CDR1 (Chr5)  . This inclade II . Here, w species –38, inclC. auris , through Candida . Rybak eof TAC1b . One sucC. auris , lies inC. auris , 26. Ano in vivo . Based oof ERG11 , 15, 41,ericin B . Recentl1 (Chr5) . In thisof TAC1b  does notg et al. . TAC1b mCandida spp., including alterations in ERG2  between generation A20 and A21 had either both or no nonsense mutations can be explained by simultaneous emergence or an epistatic effect  of ERG3 and ERG11 LOF mutations in strain A21  sequenced isolates harbored a nonsense mutation in MSH2, a DNA mismatch repair gene  of CIS2 encodes a γ-glutamyltranspeptidase, involved in glutathione degradation . This might be linked to the mutation in CIS2 and an increased oxidative stress tolerance. Nevertheless, in the study by Maras and colleagues  are still scarce and the database of 304 sequenced clinical isolates of C. auris  background and/or editing mutant alleles to the wild type form in evolved strains. For this, a CRISPR-based gene editing system would be ideal. So far, no scarless allele-specific CRISPR gene editing system in C. auris has been optimized or reported, although alleles have been deleted, replaced, and complemented with an inducible promoter  from the Westerdijk Fungal Biodiversity Center (wi.knaw.nl/). Strains were grown on yeast extract-peptone-dextrose (YPD) agar (2% glucose) at 37°C and enriched in RPMI 1640 (Thermo Fisher Scientific)-MOPS (morpholinepropanesulfonic acid) liquid medium containing 2% glucose at 37°C in a shaking incubator overnight. All strains, including daily aliquots of serially transferred populations in the microevolution assay, were stored at −80°C in RPMI-MOPS medium containing 25% glycerol.All experiments were performed with 600 in a SPECTRAmax Plus 384 microplate reader (Molecular Devices). Resistance was determined through tentative breakpoints provided by the CDC (The MIC was determined using a broth dilution assay (BDA) based on Clinical and Laboratory Standards Institute (CLSI) guidelines . In shor6 cells are diluted in a 5 ml volume of RPMI-MOPS medium (2% glucose) containing no drug (control) or a drug at a concentration of 0.5× MIC50, 1× MIC50, or 2× MIC50. All conditions were performed in triplicate (3 evolving populations per condition). After 24 h of incubation at 37°C in a shaking incubator, growth of each population was compared to the average growth of 3 controls (no drug) by spectrophotometric quantification (OD600). Next, 500 μl of each population was transferred to 4,500 μl of fresh medium with a concentration of drug equal to the previous culture when OD600 (evolving population) ≤ OD600 (average control) or double compared to the previous culture when OD600 (evolving population) > OD600 (average control). The experiment was terminated after 30 days or if the MIC50 exceeded the resistance breakpoint value, as evaluated by intermediate MIC testing (using BDA). At the end of each evolution experiment, a single colony was picked as the progenitor for a consecutive evolution experiment.An overview of the design of the experimental evolution assay is given in 600) over time in a Multiskan GO (Thermo Scientific) automated plate reader using flat-bottom 96-well plates and intermittent  pulsed shaking . Cultures were diluted in RPMI-MOPS medium containing 0.2% or 2% glucose, to a final volume of 106 cells per well. Growth was measured for 72 h at 37°C. Growth curves were plotted as an average value of 3 biological repeats with 3 technical repeats per biological repeat.Growth was assessed by spectrophotometric observation  at concentrations of 107, 106, 105, and 104 cells/ml and 1 μl of each dilution was spotted on YPD agar containing 2% glucose. Plates were incubated for 48 h at 37°C or at 30°C, 37°C, and 42°C for the evaluation of temperature stress. Oxidative stress was evaluated on agar containing 6, 8, or 10 mM H2O2 (Sigma-Aldrich). Membrane stress was evaluated on agar containing 0.005, 0.05, or 0.2% sodium dodecyl sulphate . Osmotic stress was evaluated on agar containing 1, 1.75, or 2.5 M NaCl (Sigma-Aldrich). pH stress was evaluated on agar adjusted to pH 4, 5, 6, or 7 by supplementing the agar with HCl or KOH. Agar adjusted to pH 4 and 5 was buffered with 50 mM citrate buffer, while agar adjusted to pH 6 was buffered with 50 mM morpholineethanesulfonic acid (MES) buffer, and agar adjusted to pH 7 was buffered with 100 mM MOPS buffer. All spot assays were performed on 3 biological repeats with 3 technical repeats per biological repeat, of which one example is shown.The survival of 2, using 25-cm2 polystyrene culture flasks. Upon reaching 80% confluence, cells were washed twice with PBS, trypsinized using 0.5% trypsin EDTA (Gibco), and replenished to allow cell adhesion in polystyrene 96-well plates as a 100-μl suspension of 105 cells/ml fresh DMEM culture medium for 24 h, at 37°C and 5% CO2. A 10 μl volume of 105C. auris cells or PBS (negative control) was added to the HeLa cells, followed by incubation at 37°C and 5% CO2 for 24 h or 72 h. Cytotoxicity of C. auris toward HeLa cells was assessed using a lactate dehydrogenase (LDH) cytotoxicity assay as follows: 10 μl of cell lysis buffer (Invitrogen) was added to cultures of HeLa cells with C. auris or pure HeLa cells (positive control), followed by incubation for 45 min at 37°C and 5% CO2. Then, 50 μl of supernatant (lysate) was mixed with 50 μl of substrate mix (Invitrogen) in a separate 96-well plate and incubated for 30 min at room temperature in darkness. Next, 50 μl of stop solution (Invitrogen) was added, followed by spectrophotometric quantification of the OD490 and OD680 in a Synergy H1 hybrid plate reader (BioTek). LDH activity was calculated by subtracting the OD680 from the OD490, and relative toxicity was calculated as HeLa cells (Thermo Fisher Scientific) were cultured in complete Dulbecco’s modified Eagle medium (DMEM) at 37°C and 5% COTM Classic lysis system  . After cell lysis, DNA was isolated and purified using ethanol precipitation. The resulting DNA was diluted to a concentration of 200 ng/μl in milliQ H2O, based on the DNA concentration measured through absorbance at 260 nm with a NanoDrop spectrophotometer (Isogen).Genomic DNA for whole-genome sequencing was extracted using the MasterPure yeast DNA purification kit  following the manufacturer’s protocol. For (AS-)PCR and Sanger sequencing, DNA was isolated from the cells through phenol chloroform isoamyl alcohol (PCI) extraction. Cells were dissolved in 300 μl Tris EDTA (TE) buffer with 300 μl PCI solution  and lysed by microbead shearing in a FastPrep-24GCA_002759435.2 ) was plotted from log2(Y) transformed data and compared statistically (using a one-way ANOVA with multiple comparisons in respect to wt) with GraphPad Prism. Expression analysis in each strain was performed using 3 biological repeats each represented by the average of 2 technical repeats.For gene expression analysis (RNA extraction and RT-qPCR), cells were resuspended in 1 ml TRIzol (Thermo Fisher Scientific) and lysed by microbead shearing in FastPrep-24TAC1b and ERG11) by qPCR using the same protocol and analysis as described above.For copy number variation analysis, gDNA was extracted as described in “DNA Extraction,” above, and standardized concentrations of 0.5 ng/μl of gDNA were used to quantify target markers (


The NCDs showed excellent anti-interference ability and selectivity for different emissions. In addition, a good linear relationship was observed between the fluorescence intensity (FI) of the NCD solutions in different emissions with Cu2+ (0–90 μM) and ClO− (0–75 μM). The limits of both Cu2+ detection and ClO− were very low, at 17.7 and 11.6 nM, respectively. The NCDs developed herein also showed a good recovery rate in water for Cu2+ and ClO− detection. Hence, they are expected to have a more extensive application prospect in real samples.The use of carbon dots (CDs) with dual emission based on ratiometric fluorescence has been attracting attention in recent times for more accurate ion detection since they help avoid interference from background noise, probe concentration, and complexity. Herein, novel dual-emission nitrogen-doped CDs (NCDs) were prepared by a simple method for Cu Carbon dots (CDs) are widely used for ion detection ,2,3, bio2+, while that at 644 nm maintained its original intensity throughout. Thus, these CDs could be used as a ratiometric fluorescent sensor for Cu2+ ions. Some ratiometric probes can be used to detect different ions simultaneously [3+ was added to the CD solutions, the emission center of the CDs at 655 nm displayed a clear quenching. Similarly, when Zn2+ was added to the CD solutions, the emission center of the CDs at 470 nm displayed an obvious quenching effect. Thus, the CDs can be used as a good dual-ratiometric probe for simultaneous detection of Zn2+ and Fe3+.Recently, dual-emission CDs based on ratiometric fluorescence have been used to develop novel methods for more accurate ion detection ,25,26,27aneously ,30,31,32aneously , which d2+ and ClO−.Although dual-ratiometric probes have significant advantages, very few such probes can accurately detect the common ions. Therefore, it is necessary to develop suitable dual-ratiometric probes for the accurate detection of common ions. This work explores novel dual-ratiometric nitrogen-doped carbon dots (NCDs) and uses them to effectively and simultaneously detect Cu4, MnCl2·4H2O, NaSO3, KH2PO4, Pb(NO3)2, (CH3COO)2Zn, NaF, KCl, NaBr, NaHCO3, BaCl2·2H2O, AgNO3, Na2H2PO2, Ni(NO3)2·6H2O, CaCl2, MgCl2·6H2O, CrCl3·6H2O), and other chemicals such as K2CO3, NaOH, and HCl were supplied by Macklin . All raw chemicals were of analytical grade and were used without further purification. Ultrapure water was prepared using a Milli-Q system .For this study, 2,4-dihydroxybenzoic acid and glycine were supplied by Macklin . Metal salts  was performed using FEI Tecnai G2 F20 to observe the morphologies of the NCDs. The chemical composition of the NCDs was analyzed by XPS (Thermo ESCALAB 250Xi). Fourier transform infrared spectroscopy  was used to identify the chemical structures of the NCDs. Ultraviolet–visible (UV–vis) spectroscopy  and fluorescence spectroscopy  were used to determine the optical properties of the NCDs.First, 2,4-dihydroxybenzoic acid (2 g), glycine (1 g), and ultrapure water (40 mL) were weighed and placed in a Teflon-lined autoclave. Next, the mixture was heated to 200 °C for 24 h, and the primary product was obtained after allowing the mixture to cool naturally to room temperature. Finally, the NCD solutions were obtained by performing dialysis for 3 days and lyophilized to obtain the final NCD powders.−, Cu2+, Mn2+, SO32-, K+, H2PO4−, NO3−, Zn2+, F−, Cl−, Br−, HCO3−, Ba2+, Pb2+, Ag+, H2PO2−, Ni2+, Ca2+, K+, Mg2+, SO42−, AC−, Cr3+, and CO32−. Their effect on the FI of NCDs was observed at 311 and 497 nm under 458 nm and 515 nm excitation sources, respectively. All experiments were performed more than thrice.To elucidate the specificity of the NCDs as probes for ion detection, different ion solutions (2 mM) and NCD solutions (1.0 mg/mL) were added to a 5 mL colorimetric tube. The following ions were used to make the ion solutions: ClO- and NCD solutions were added in different concentrations  to a 5 mL colorimetric tube, and their FI spectra were recorded under 311 and 497 nm excitations. All experiments were performed more than thrice.ClO2+ and NCD solutions were added in different concentrations  to a 5 mL colorimetric tube, and their FI spectra were also recorded under 311 and 497 nm excitations. All experiments were performed more than thrice.Similarly, Cu− and Cu2+ to obtain different concentrations of ClO− and Cu2+ solution . Then, NCDs were added into each of these solutions, and the content of ClO− and Cu2+ in the solutions was quantified by the FI, respectively. All experiments were performed in triplicate.Real samples (tap water) were first filtered through the 0.22 μm PES water phase membrane. The filtered water samples were then spiked with ClO−1 in the FT–IR spectrum of the CDs correspond to –OH stretching vibrations and –NH2 stretching vibrations, respectively [−1, respectively. This observation indicated the presence of hydrophilic groups  on the surface of the NCDs, which was further confirmed by the XPS spectrum [The morphology of the as-prepared NCDs was observed using TEM. As shown in ectively ,35. The spectrum ,38,39,40n−π*), and 478 nm (excitonic absorption band) were clearly observed.The optical properties of the NCDs were investigated using fluorescence spectra and UV–visible absorption spectra. The excitation–emission 3D color map of the NCDs is shown in 2+ was added to the NCDs, the FI rapidly quenched (<1 min)  c. The FIthe NCDs d. These  of 4–14 e. Hence,1 and 14 f.-  were estimated to be 0.9945 within 0–75 μM by linear fitting. Based on the “three times the standard deviation” rule [− was studied. When ClO− was added to the NCD solution, the strong oxidation destroyed the two emission centers (372 and 454 nm) of the NCDs and enhanced the nonradiative carrier depopulation, leading to a further weakening of the fluorescence intensity. Compared with other probes reported previously, the NCDs probe demonstrated lower LOD and wider range for ClO− detection [NCDs have good selectivity and sensitivity for the detection of Cu of ClO− a. In conobserved d. The coon” rule , the limetection ,14,42.2+ concentrations were studied. The FI of NCDs in the 515 nm emission center gradually weakened with increasing Cu2+, whereas that of NCDs in the 454 nm emission center showed a little change with the same increase in Cu2+  were estimated to be 0.9974 by linear fitting within 0–90 μM. The LOD of this probe was calculated to be 17.7 nM  and home (tap water 2) were used. Cu2+ and ClO− in different concentrations were added to the NCD solutions in tap water. The concentrations of Cu2+ and ClO− in different tap water samples were calculated using the additive recovery method. The above results exhibited a good recovery rate and good accuracy for both Cu2+ and ClO− detection (NCDs have high selectivity, sensitivity, wide detection range, and low detection limit for Cuetection . Thus, N2+ and ClO− detection in different emissions. In addition, NCDs have a considerably wide detection range and low detection limit for Cu2+ and ClO− detection based on ratiometric fluorescence, respectively. These results imply that NCDs can be used for the detection of Cu2+ and ClO−, with potential applications in real-life water samples.NCDs were synthesized from 2,4-dihydroxybenzoic acid and glycine via a simple hydrothermal method. The NCDs were found to have high selectivity and sensitivity for Cu