A <i>Falciformispora senegalensis</i> grain model in <i>Galleria mellonella</i> larvae

Eumycetoma is a subcutaneous implantation mycosis often found in the foot. One of the hallmarks of eumycetoma is the formation of grains. These grains are either black or white, and the consistency and morphology differs per causative agent. The two most common causative agents of black-grain eumycetoma are Madurella mycetomatis and Falciformispora senegalensis. Since grains cannot be formed in vitro, in vivo models are needed to study grain formation. Here, we used the invertebrate Galleria mellonella to establish an in vivo grain model for F. senegalensis. Three different F. senegalensis strains were selected, and four different inocula were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 10 mg/larvae. Larval survival was monitored for 10 days. Grain formation was studied macroscopically and histologically. The efficacy of antifungal therapy was determined for itraconazole, amphotericin B, and terbinafine. A concentration of 10 mg F. senegalensis per larva was lethal for the majority of the larvae within 10 days. At this inoculum, grains were formed within 24 h after infection. The grains produced in the larvae resembled those formed in human patients. Amphotericin B given at 1 mg/kg 4 h, 28 h, and 52 h after infection prolonged larval survival. No enhanced survival was noted for itraconazole or terbinafine. In conclusion, we developed a F. senegalensis grain model in G. mellonella larvae in which grains were formed that were similar to those formed in patients. This model can be used to monitor grain formation over time and study antifungal efficacy.</p

The performance and costs of XTT, resazurin, MTS and luciferin as viability dyes in <i>in vitro</i> susceptibility testing of <i>Madurella mycetomatis</i>

Background: in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis. Methods: To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared. Results: Each of the four dyes gave highly reproducible results with &gt;85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 &lt;euro&gt;/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 &lt;euro&gt;/MIC. Conclusion: All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns. Lay summary: To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them

The performance and costs of XTT, resazurin, MTS and luciferin as viability dyes in <i>in vitro</i> susceptibility testing of <i>Madurella mycetomatis</i>

Background: in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis. Methods: To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared. Results: Each of the four dyes gave highly reproducible results with &gt;85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 &lt;euro&gt;/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 &lt;euro&gt;/MIC. Conclusion: All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns. Lay summary: To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them

Complete Genome Sequence of the Itraconazole Decreased Susceptible <i>Madurella fahalii</i> Type-Strain CBS 129176

Madurella fahalii is a causative agent of the implantation mycosis mycetoma with decreased susceptibility to itraconazole, the preferred therapeutic drug to combat mycetoma. Here, we report the M. fahalii type-strain CBS 129176 genome assembly and annotation to identify a glutamic acid insert near the azole-binding pocket in the Cyp51A protein

Complete Genome Sequence of the Itraconazole Decreased Susceptible Madurella fahalii Type-Strain CBS 129176

Madurella fahalii is a causative agent of the implantation mycosis mycetoma with decreased susceptibility to itraconazole, the preferred therapeutic drug to combat mycetoma. Here, we report the M. fahalii type-strain CBS 129176 genome assembly and annotation to identify a glutamic acid insert near the azole-binding pocket in the Cyp51A protein

Glueball calculations in large-N_c gauge theory

We use the light-front Hamiltonian of transverse lattice gauge theory to compute from first principles the glueball spectrum and light-front wavefunctions in the leading order of the 1/N_c colour expansion. We find 0^{++}, 2^{++}, and 1^{+-} glueballs having masses consistent with N_c=3 data available from Euclidean lattice path integral methods. The wavefunctions exhibit a light-front constituent gluon structure.Comment: 4 pages, 2 figures, uses macro boxedeps.tex, minor corrections in revised versio

Determining the effects of clumping and porosity on the chemistry in a non-uniform AGB outflow

(abridged) In the inner regions of AGB outflows, several molecules have been detected with abundances much higher than those predicted from thermodynamic equilibrium (TE) chemical models. The presence of the majority of these species can be explained by shock-induced non-TE chemical models, where shocks caused by the pulsating star take the chemistry out of TE in the inner region. Moreover, a non-uniform density structure has been detected in several AGB outflows. A detailed parameter study on the quantitative effects of a non-homogeneous outflow has so far not been performed. We implement a porosity formalism for treating the increased leakage of light associated with radiation transport through a clumpy, porous medium. The effects from the altered UV radiation field penetration on the chemistry, accounting also for the increased reaction rates of two-body processes in the overdense clumps, are examined. We present a parameter study of the effect of clumping and porosity on the chemistry throughout the outflow. Both the higher density within the clumps and the increased UV radiation field penetration have an important impact on the chemistry, as they both alter the chemical pathways. The increased amount of UV radiation in the inner region leads to photodissociation of parent species, releasing the otherwise deficient elements. We find an increased abundance in the inner region of all species not expected to be present assuming TE chemistry, such as HCN in O-rich outflows, H$_2$O in C-rich outflows, and NH$_3$ in both. Outflows whose clumps have a large overdensity and that are very porous to the interstellar UV radiation field yield abundances comparable to those observed in O- and C-rich outflows for most of the unexpected species investigated. The inner wind abundances of H$_2$O in C-rich outflows and of NH$_3$ in O- and C-rich outflows are however underpredicted.Comment: 33 pages, 20 figures, 15 tables, accepted for publication in Astronomy & Astrophysic

Regression of fibrous dysplasia in response to denosumab therapy: a report of two cases

We present two patients with fibrous dysplasia who showed a decrease in lesional size and activity after denosumab therapy. Both patients also experienced a reduction in pain and bone turnover markers, which had not been accomplished during previous bisphosphonate therapy. These cases highlight the potential of denosumab to decrease lesional size in fibrous dysplasia. This finding has been reported in mice, but not in humans. Denosumab may be considered when bisphosphonates are not tolerated or not effective (enough), or in severe cases as neoadjuvant therapy to improve surgical possibilities and outcome. In addition, these results show that Na[F-18]F PET-CT is suitable for detecting change in each fibrous dysplasia lesion distinctively.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Proteomic analysis of the processes leading to Madurella mycetomatis grain formation in Galleria mellonella larvae

Mycetoma is a neglected chronic and granulomatous infection primarily associated with the fungal pathogen Madurella mycetomatis. Characteristic of this infection is the formation of grains. However, the processes leading to grain formation are not known. In this study, we employed a proteomic approach to characterise M. mycetomatis grain formation in Galleria mellonella larvae and map the processes leading to grain formation over time. For this, at 1 day, 3 days and 7 days post-inoculation, proteins from grains and hemolymph were extracted and analysed by label-free mass spectrometry. A total of 87, 51 and 48 M. mycetomatis proteins and 713, 997, 18 G. mellonella proteins were found in grains on day 1, 3 and 7 post-inoculation respectively. M. mycetomatis proteins were mainly involved in cellular metabolic processes and numerous enzymes were encountered. G. mellonella proteins were primarily involved in the nodulation process. The proteins identified were linked to nodulation and grain formation and four steps of grain formation were identified. The results of this proteomic approach could in the future be used to design novel strategies to interfere with mycetoma grain formation and to combat this difficult to treat infection