Multi‐Drug Resistance (MDR) is one of the most frequent problems observed in the course of cancer chemotherapy. Cells under treatment, tend to develop survival mechanisms to drug‐action thus ...generating drug‐resistance. One of the most important mechanism to get it is the over expression of P‐gp glycoprotein, which acts as an efflux‐pump releasing the drug outside of the cancer cell. A strategy for a succesfull treatment consists in the co‐administration of one compound that acts against P‐gp and another which acts against the cell during chemotherapy. Ningalins are pyrrole‐containing naturally occurring compounds isolated mainly from the marine tunicate Didemnum spp and also they are some of the top reversing agents in MDR treatment acting on P‐gp. Considering the relevance displayed for some of these isolated alkaloids or their core as a drug for co‐administration in cancer therapy, all the total synthesis described to date for the members of ningalins family are reviewed herein.
Abstract
Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that ...deletion of
rfs
, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in
Mucor lusitanicus
, led to a lower virulence in diabetic mice and nematodes. Upregulation of
rfs
correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H
2
O
2
in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing
M. lusitanicus
in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (
pkaR1
), correlated with a decrease in the toxicity of SS, downregulation of
rfs
, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in
M. lusitanicus
. Moreover,
rfs
upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of
rfs
in
M. lusitanicus
infection.
Display omitted
Zygomycetes are ubiquitous saprophytes in natural environments which transform organic matter. Some zygomycetes of gender Mucor have attracted interest in health sector. Due to its ...ability as opportunistic microorganisms infecting immuno-compromised people and to the few available pharmacological treatments, the mucormycosis is receiving worldwide attention. Concerning to the pharmacological treatments, some triazole-based compounds such as fluconazole are extensively used. Nevertheless, we focused in the quinolines since they are broadly used models for the design and development of new synthetic antifungal agents. In this study, the fungistatic activity on M. circinelloides of various 2-aryl-4-aryloxyquinoline-based compounds was discovered, and in some cases, it resulted better than reference compound fluconazole. These quinoline derivatives were synthesized via the Csp2-O bond formation using diaryliodonium(III) salts chemistry. A QSAR study was carried out to quantitatively correlate the chemical structure of the tested compounds with their biological activity. Also, a docking study to identify a plausible action target of our more active quinolines was carried out. The results highlighted an increased activity with the fluorine- and nitro-containing derivatives. In light of the few mucormycosis pharmacological treatments, herein we present some non-described molecules with excellent in vitro activities and potential use in the mucormycosis treatment.
Mucor circinelloides is an opportunistic dimorphic pathogen, with the dimorphic process controlled in parts by fermentative and oxidative metabolisms, which lead to yeast or mycelial growth, ...respectively. Dimorphic transition is important for pathogenesis since the mycelium represents the virulent morphology. We previously reported that the deletion of arl1 or arl2 stimulate anaerobic germination in M. circinelloides, suggesting an augmented fermentative metabolism. In the present study, we demonstrate that the heterokaryon Δarl1(+)(−) and homokaryon Δarl2 strains contain low number of mitochondria, which possibly results in a dysfunctional oxidative metabolism, marked by a low oxygen consumption in glucose and poor growth in glycerol as the unique carbon source. This dysfunction is compensated for by an increase in the glycolysis and fermentation in aerobic conditions, demonstrating growth kinetics similar to that in the wild-type strain. Moreover, as a consequence a high fermentative activity, the Δarl1(+)(−) and Δarl2 strains possibly increased the yeast cell growth during low oxygen concentrations in presence of glucose.
To the best of our knowledge, this is the first study to demonstrate the control of members of Arf family on the mitochondrial population in a Mucor species.
•Arl1 and Arl2 control the mitochondrial content in M. circinelloides.•Δarl1(+)/(−) and Δarl2 decreased the mitochondrial cellular content.•Δarl1(+)/(−) and Δarl2 increased the glycolysis and fermentative metabolism.•Δarl1(+)/(−) and Δarl2 enhance the yeast growth under low oxygen level conditions.•Yeast growth of Δarl1(+)/(−) is more virulent compared to the WT.
The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate ...resistance by exporting chromate ions from the cell's cytoplasm. The Neurospora crassa strain 74-A chr-1 gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the chr-1 gene was up-regulated by chromate exposure of N. crassa cultures. Introduction in N. crassa of sense and antisense fragments of the chr-1 gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in N crassa strains deleted in the genomic chr-1 gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from N. crassa chr-1 gene (Ncchr-1) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast Saccharomyces cerevisiae. Galactose-induced S. cerevisiae transformants expressing Ncchr-1 were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect S. cerevisiae chr-1 transformants from chromate toxicity. These data indicate that the N. crassa CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein.
The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate ...resistance by exporting chromate ions from the cell’s cytoplasm. The
Neurospora crassa
strain 74-A
chr
-
1
gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the
chr
-
1
gene was up-regulated by chromate exposure of
N. crassa
cultures. Introduction in
N. crassa
of sense and antisense fragments of the
chr
-
1
gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in
N crassa
strains deleted in the genomic
chr
-
1
gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from
N. crassa
chr
-
1
gene (Nc
chr
-
1
) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast
Saccharomyces cerevisiae
. Galactose-induced
S. cerevisiae
transformants expressing
Ncchr
-
1
were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect
S. cerevisiae chr
-
1
transformants from chromate toxicity. These data indicate that the
N. crassa
CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein.
Objective
The goal of this work was to set up a high throughput procedure for the determination of fatty acid methyl esters (FAMEs) in cosmetic castor oils using flow injection–electrospray ...ionization–high‐resolution mass spectrometry, and to demonstrate the need of such analysis for the quality control purposes.
Methods
The sample aliquot was mixed with isooctane:chloroform (1:1) and submitted to transesterification; the obtained FAMEs were appropriately diluted using water:isopropanol:acetonitrile (20:50:30) with addition of sodium formate which served as an internal standard, lock mass calibrant and promoted the formation of sodium adducts during electrospray ionization (ESI). The principle of flow injection analysis (FIA) was applied for sample introduction to an ESI–quadrupole–time of flight mass spectrometer (ESI‐QTOFMS). The carrier solution was composed of water:isopropanol:acetonitrile (20:50:30). From the acquired MS data, flowgrams of the extracted M+Na+ ions were obtained using the following m/z values for individual FAMEs: 293.2451 (C16:0); 315.2295 (C18:3); 317.2451 (C18:2); 319.2608 (C18:1); 321.2764 (C18:0); 335.2557 (C18:1,OH); 349.3077 (C20:0); 377.3390 (C22:0) and m/z 226.9515 for IS. Baseline‐subtracted and filtered signals were integrated, and the list of peaks intensities was exported to Excel, where calibration functions were obtained and quantification carried out. Gas chromatography with a flame ionization detector (GC‐FID) was used as an alternative analytical tool.
Results
The calibration detection limits for FAMEs of unsaturated fatty acids were in the range 3.61–8.62 μg L−1 and for saturated compounds in the range 8.51–82.4 μg L−1. The results obtained for commercial oils were in good agreement with GC‐FID data; among nine cosmetic oils analysed, three contained low concentrations of ricinoleic acid (C18:1, OH), indicating adulteration of castor bean oil with other vegetable oils.
Conclusion
Application of FIA for the sample introduction to ESI‐QTOFMS enabled for reliable determination of FAMEs in cosmetic oils with sampling frequency of thirty per hour as compared to two samples per hour achievable using GC‐FID. The proposed procedure is especially well suited for FAMEs of unsaturated fatty acids that are primary components of castor triacylglycerides and contribute to desirable properties of any cosmetic oil.
The principle of Flow Injection Analysis has been applied for sample introduction to ESI source of the high resolution mass spectrometer enabling for high throughput determination of fatty acid methyl esters in castor bean cosmetic oils (30 injections per hour). The method development included: (i) selection of the composition of carrier solution; (ii) ensuring adequate signal acquisition and integration; (iii) setting three roles for sodium formate. The results obtained were consistent with those obtained by GC‐FID; in the analysis of commercial cosmetic oils, adulteration problem was detected.
Résumé
Objectif
Cette étude avait pour but de mettre en place une procédure de criblage à haut débit pour la détermination des esters méthyliques d'acides gras (EMAG) dans les huiles de ricin cosmétiques, par le biais d'une spectrométrie de masse haute résolution, avec injection en flux et ionisation par électronébulisation, et de démontrer la nécessité d'une telle analyse pour les besoins du contrôle qualité.
Méthodes
Une prise aliquote a été mélangée à une solution d'isooctane:chloroforme (1:1), avant d’être trans‐estérifiée; les EMAG obtenus ont été correctement dilués dans une solution d'eau:isopropanol:acétonitrile (20:50:30), avec l'ajout de formiate de sodium comme étalon interne et de masse de calibration, permettant la formation d'adduits de sodium durant l'ionisation par électronébulisation (ISE). Le principe d'analyse par injection en flux (FIA) a été appliqué pour l'introduction des échantillons dans le spectromètre de masse à temps de vol, quadripolaire, avec ISE (ESI‐QTOFMS). La solution conductrice était composée d'eau:isopropanol:acétonitrile (20:50:30). D'après les données MS acquises, les diagrammes de flux des ions M+Na+ extraits étaient obtenus en utilisant les valeurs m/z suivantes pour chaque EMAG : 293,2451 (C16:0); 315,2295 (C18:3); 317,2451 (C18:2); 319,2608 (C18:1); 321,2764 (C18:0); 335,2557 (C18:1,OH); 349,3077 (C20:0); 377,3390 (C22:0) et m/z 226,9515 pour l’IS. Les signaux filtrés avec soustraction de la ligne de base ont été intégrés, et la liste des intensités des pics a été exportée vers Excel, où les fonctions de calibration ont été obtenues et la quantification effectuée. Une chromatographie en phase gazeuse avec détecteur à ionisation de flamme (GC‐FID) a été utilisée comme outil d'analyse alternatif.
Résultats
Les limites de détection de la calibration étaient comprises dans une plage de 3,61‐8,62 μg/l−1 pour les EMAG non saturés et dans une plage de 8,51‐82,4 μg/l−1 pour les composés saturés. Les résultats obtenus pour les huiles commercialisées étaient conformes aux données de GC‐FID; parmi neuf huiles cosmétiques analysées, trois contenaient de faibles concentrations d'acide ricinoléique (C18:1, OH), indiquant une adultération de l'huile de graine de ricin avec d'autres huiles végétales.
Conclusion
L'application de la FIA pour l'introduction des échantillons dans l’ESI‐QTOFMS a permis une détermination fiable des EMAG présents dans les huiles cosmétiques, avec une fréquence d’échantillonnage de trente par heure, comparativement à deux échantillons par heure avec la GC‐FID. La procédure proposée est particulièrement adaptée aux EMAG non saturés, qui composent principalement les triglycérides contenus dans le ricin et contribuent aux propriétés bénéfiques de toute huile cosmétique.
