Microalga Broths Synthesize Antibacterial and Non-Cytotoxic Silver Nanoparticles Showing Synergy with Antibiotics and Bacterial ROS Induction and Can Be Reused for Successive AgNP Batches.

The era of increasing bacterial antibiotic resistance requires new approaches to fight infections. With this purpose, silver-based nanomaterials are a reality in some fields and promise new developments. We report the green synthesis of silver nanoparticles (AgNPs) using culture broths from a microalga. Broths from two media, with different compositions and pHs and sampled at two growth phases, produced eight AgNP types. Nanoparticles harvested after several synthesis periods showed differences in antibacterial activity and stability. Moreover, an evaluation of the broths for several consecutive syntheses did not find relevant kinetics or activity differences until the third round. Physicochemical characteristics of the AgNPs (core and hydrodynamic sizes, Z-potential, crystallinity, and corona composition) were determined, observing differences depending on the broths used. AgNPs showed good antibacterial activity at concentrations producing no or low cytotoxicity on cultured eukaryotic cells. All the AgNPs had high levels of synergy against Escherichia coli and Staphylococcus aureus with the classic antibiotics streptomycin and kanamycin, but with ampicillin only against S. aureus and tetracycline against E. coli. Differences in the synergy levels were also dependent on the types of AgNPs. We also found that, for some AgNPs, the killing of bacteria started before the massive accumulation of ROS.

Green Extracellular Synthesis of Silver Nanoparticles by Pseudomonas alloputida, Their Growth and Biofilm-Formation Inhibitory Activities and Synergic Behavior with Three Classical Antibiotics.

Bacterial resistance to antibiotics is on the rise and hinders the fight against bacterial infections, which are expected to cause millions of deaths by 2050. New antibiotics are difficult to find, so alternatives are needed. One could be metal-based drugs, such as silver nanoparticles (AgNPs). In general, chemical methods for AgNPs' production are potentially toxic, and the physical ones expensive, while green approaches are not. In this paper, we present the green synthesis of AgNPs using two Pseudomonas alloputida B003 UAM culture broths, sampled from their exponential and stationary growth phases. AgNPs were physicochemically characterized by transmission electron microscopy (TEM), total reflection X-ray fluorescence (TXRF), infrared spectroscopy (FTIR), dynamic light scattering (DLS), and X-ray diffraction (XRD), showing differential characteristics depending on the synthesis method used. Antibacterial activity was tested in three assays, and we compared the growth and biofilm-formation inhibition of six test bacteria: Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We also monitored nanoparticles' synergic behavior through the growth inhibition of E. coli and S. aureus by three classical antibiotics: ampicillin, nalidixic acid, and streptomycin. The results indicate that very good AgNP activity was obtained with particularly low MICs for the three tested strains of P. aeruginosa. A good synergistic effect on streptomycin activity was observed for all the nanoparticles. For ampicillin, a synergic effect was detected only against S. aureus. ROS production was found to be related to the AgNPs' antibacterial activity.

ROS formation is a differential contributory factor to the fungicidal action of Amphotericin B and Micafungin in Candida albicans.

The hypothetical role played by the intracellular formation of reactive oxygen species (ROS) in the fungicidal action carried out by Amphotericin B (AmB) and Micafungin (MF) was examined in Candida albicans, which remains the most prevalent fungal pathogen. The clinical MICs for MF and AmB were 0.016 and 0.12µg/ml, respectively. Whereas AmB (0.5-1.0×MIC) induced a marked production of intracellular ROS accompanied by a high degree of cell killing in the C. albicans SC5314 strain, the fungicidal effect of MF was still operative, but ROS generation was slight. Preincubation with thiourea suppressed the formation of ROS and caused a marked increase in cell viability, regardless of the antifungal used. Simultaneous measurement of several well established antioxidant enzymes (catalase, glutathione reductase and superoxide dismutase) revealed strong AmB-induced activation of the three enzymatic activities, whereas MF only had a weak stimulating effect. Likewise, AmB but not MF promoted a conspicuous rise in the mitochondrial membrane potential together with the intracellular synthesis of trehalose, the non-reducing disaccharide which acts as a specific protector against oxidative stress in C. albicans. Optical and electronic microscopy analysis revealed a significant damage to cell integrity and structural alterations caused by both antifungals. Taken together, our results strongly suggest that the induction of an internal oxidative stress in C. albicans through the accumulation of ROS is a preferential contributory factor to the antifungal action of a widely used polyene (AmB) but not of MF (echinocandin).

Homozygous deletion of ATC1 and NTC1 genes in Candida parapsilosis abolishes trehalase activity and affects cell growth, sugar metabolism, stress resistance, infectivity and biofilm formation.

A double homozygous atc1Δ/atc1Δ/ntc1Δ/ntc1Δ mutant (atc1Δ/ntc1Δ KO) was constructed in the pathogen opportunistic yeast Candida parapsilosis by disruption of the two chromosomal alleles coding for NTC1 gene (encoding a neutral trehalase) in a Cpatc1Δ/atc1Δ background (atc1Δ KO strain, deficient in acid trehalase). The Cpatc1Δ/ntc1Δ KO mutant failed to counteract the inability of Cpatc1Δ cells to metabolize exogenous trehalose and showed a similar growth pattern on several monosaccharides and disaccharides. However, upon prolonged incubation in either rich medium (YPD) or nutrient-starved medium the viability of Cpatc1Δ cells exhibited a sensitive phenotype, which was augmented by further CpNTC1/NTC1 disruption. Furthermore, Cpatc1Δ/ntc1Δ KO cells had difficulty in resuming active growth in fresh YPD. This homozygous mutant also lacked any in vitro measurable trehalase activity, whether acid or neutral, suggesting that a single gene codes for each enzyme. By contrast, in Cpatc1Δ/ntc1Δ KO strain the resistance to oxidative and heat stress displayed by atc1Δ mutant was suppressed. Cpatc1Δ/ntc1Δ KO cells showed a significant decrease in virulence as well as in the capacity to form biofilms. These results point to a major role for acid trehalase (Atc1p) in the pathobiology of C. parapsilosis, whereas the activity of neutral trehalase can only partially counteract Atc1p deficiency. They also support the use of ATC1 and NTC1 genes as interesting antifungal targets.

Specific stress-induced storage of trehalose, glycerol and D-arabitol in response to oxidative and osmotic stress in Candida albicans.

Candida albicans exponential yeast cells are able to face environmental challenges by mounting a rapid and efficient "general stress response". Here we show that one of the main components of this response consists of the intracellular protective accumulation of the non-reducing disaccharide trehalose and two polyols, glycerol and D-arabitol, an accumulation that occurs in a stress-specific dependent manner. Thus, oxidative exposures promoted a marked increase in both trehalose and D-arabitol in the wild type strain, RM-100, whereas the glycerol content remained virtually unaffected with respect to basal levels. In contrast, osmotic challenges induced the significant storage of glycerol accompanied by minor changes, or even a slight drop, in the intracellular content of trehalose and D-arabitol. We examined the hypothetical role in this process of the MAP kinase Hog1, which regulates the protective responses in C. albicans against both oxidative and osmotic stress. Interestingly, unlike glycerol synthesis, the stress-induced trehalose accumulation was always Hog1-independent, whereas the ability to synthesize D-arabitol was only partially dependent on a functional Hog1 pathway.

Evolution of genes and genomes on the Drosophila phylogeny.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Insight into the Antifungal Effects of Propolis and Carnosic Acid-Extension to the Pathogenic Yeast Candida glabrata: New Propolis Fractionation and Potential Synergistic Applications.

Fungi have traditionally been considered opportunistic pathogens in primary infections caused by virulent bacteria, protozoan, or viruses. Consequently, antimycotic chemotherapy is clearly less developed in comparison to its bacterial counterpart. Currently, the three main families of antifungals (polyenes, echinocandins, and azoles) are not sufficient to control the enormous increase in life-threatening fungal infections recorded in recent decades. Natural substances harvested from plants have traditionally been utilized as a successful alternative. After a wide screening of natural agents, we have recently obtained promising results with distinct formulations of carnosic acid and propolis on the prevalent fungal pathogens Candida albicans and Cryptococcus neoformans. Here, we extended their use to the treatment against the emerging pathogenic yeast Candida glabrata, which displayed lower susceptibility in comparison to the fungi mentioned above. Taking into account the moderate antifungal activity of both natural agents, the antifungal value of these combinations has been improved through the obtention of the hydroethanolic fractions of propolis. In addition, we have demonstrated the potential clinical application of new therapeutical designs based on sequential pre-treatments with carnosic/propolis mixtures, followed by exposure to amphotericin B. This approach increased the toxic effect induced by this polyene.

Resveratrol lacks antifungal activity against Candida albicans.

The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10-40 µg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 µg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 µg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.

Cell death induction in Aspergillus fumigatus: accentuating drug toxicity through inhibition of the unfolded protein response (UPR).

One of the most potent opportunistic fungal pathogens of humans is Aspergillus fumigatus, an environmental mold that causes a life-threatening pneumonia with a high rate of morbidity and mortality. Despite advances in therapy, issues of drug toxicity and antifungal resistance remain an obstacle to effective therapy. This underscores the need for more information on fungal pathways that could be pharmacologically manipulated to either reduce the viability of the fungus during infection, or to unleash the fungicidal potential of current antifungal drugs. In this review, we summarize the emerging evidence that the ability of A. fumigatus to sustain viability during stress relies heavily on an adaptive signaling pathway known as the unfolded protein response (UPR), thereby exposing a vulnerability in this fungus that has strong potential for future therapeutic intervention.

Pleiotropic Effects of the P5-Type ATPase SpfA on Stress Response Networks Contribute to Virulence in the Pathogenic Mold Aspergillus fumigatus.

Aspergillus fumigatus is a human-pathogenic mold that extracts nutrients from the environment or from host tissues by secreting hydrolytic enzymes. The ability of A. fumigatus to adjust secretion levels in proportion to demand relies on the assistance of the unfolded protein response (UPR), an adaptive stress response pathway that regulates the unique protein-folding environment of the endoplasmic reticulum (ER). The P5-type ATPase Spf1 has recently been implicated in a novel mechanism of ER homeostasis that involves correcting errors in ER-membrane protein targeting. However, the contribution of this protein to the biology of A. fumigatus is unknown. Here, we employed a gene knockout and RNA sequencing strategy to determine the functional role of the A. fumigatus gene coding for the orthologous P5 ATPase SpfA. The data reveal that the spfA gene is induced by ER stress in a UPR-dependent manner. In the absence of spfA, the A. fumigatus transcriptome shifts toward a profile of altered redox and lipid balance, in addition to a signature of ER stress that includes srcA, encoding a second P-type ATPase in the ER. A ΔspfA deletion mutant showed increased sensitivity to ER stress, oxidative stress, and antifungal drugs that target the cell wall or plasma membrane. The combined loss of spfA and srcA exacerbated these phenotypes and attenuated virulence in two animal infection models. These findings demonstrate that the ER-resident ATPases SpfA and SrcA act jointly to support diverse adaptive functions of the ER that are necessary for fitness in the host environment. IMPORTANCE The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus.

A Human IRE1 Inhibitor Blocks the Unfolded Protein Response in the Pathogenic Fungus Aspergillus fumigatus and Suggests Noncanonical Functions within the Pathway.

The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacAu into its active form, hacAi, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4µ8C prevented A. fumigatus from increasing the levels of hacAi mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4µ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4µ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4µ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAi mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacAu mRNA in ER stress response.IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.

The Fungicidal Action of Micafungin is Independent on Both Oxidative Stress Generation and HOG Pathway Signaling in Candida albicans.

In fungi, the Mitogen-Activated Protein kinase (MAPK) pathways sense a wide variety of environmental stimuli, leading to cell adaptation and survival. The HOG pathway plays an essential role in the pathobiology of Candida albicans, including the colonization of the gastrointestinal tract in a mouse model, virulence, and response to stress. Here, we examined the role of Hog1 in the C. albicans response to the clinically relevant antifungal Micafungin (MF), whose minimum inhibitory concentration (MIC) was identical in the parental strain (RM100) and in the isogenic homozygous mutant hog1 (0.016 mg/L). The cell viability was impaired without significant differences between the parental strain, the isogenic hog1 mutant, and the Hog1+ reintegrant. This phenotype was quite similar in a collection of hog1 mutants constructed in a different C. albicans background. MF-treated cells failed to induce a relevant increase of both reactive oxygen species (ROS) formation and activation of the mitochondrial membrane potential in parental and hog1 cells. MF was also unable to trigger any significant activation of the genes coding for the antioxidant activities catalase (CAT1) and superoxide dismutase (SOD2), as well as on the corresponding enzymatic activities, whereas a clear induction was observed in the presence of Amphotericin B (AMB), introduced as a positive control of Hog1 signaling. Furthermore, Hog1 was not phosphorylated by the addition of MF, but, notably, this echinocandin caused Mkc1 phosphorylation. Our results strongly suggest that the toxic effect of MF on C. albicans cells is not mediated by the Hog1 MAPK and is independent of the generation of an internal oxidative stress in C. albicans.

Novel Bi-Factorial Strategy against Candida albicans Viability Using Carnosic Acid and Propolis: Synergistic Antifungal Action.

The potential fungicidal action of the natural extracts, carnosic acid (obtained from rosemary) and propolis (from honeybees' panels) against the highly prevalent yeast Candida albicans, used herein as an archetype of pathogenic fungi, was tested. The separate addition of carnosic acid and propolis on exponential cultures of the standard SC5314 C. albicans strain caused a moderate degree of cell death at relatively high concentrations. However, the combination of both extracts, especially in a 1:4 ratio, induced a potent synergistic pattern, leading to a drastic reduction in cell survival even at much lower concentrations. The result of a mathematical analysis by isobologram was consistent with synergistic action of the combined extracts rather than a merely additive effect. In turn, the capacity of SC5314 cells to form in vitro biofilms was also impaired by the simultaneous presence of both agents, supporting the potential application of carnosic acid and propolis mixtures in the prevention and treatment of clinical infections as an alternative to antibiotics and other antifungal agents endowed with reduced toxic side effects.

Bacteria From the Multi-Contaminated Tinto River Estuary (SW, Spain) Show High Multi-Resistance to Antibiotics and Point to Paenibacillus spp. as Antibiotic-Resistance-Dissemination Players.

Bacterial resistance to antibiotics is an ever-increasing phenomenon that, besides clinical settings, is generally assumed to be prevalent in environmental soils and waters. The analysis of bacteria resistant to each one of 11 antibiotics in waters and sediments of the Huelva's estuary, a multi-contaminated environment, showed high levels of bacteria resistant mainly to Tm, among others. To further gain knowledge on the fate of multi-drug resistance (MDR) in environmental bacteria, 579 ampicillin-resistant bacteria were isolated tested for resistance to 10 antibiotics. 92.7% of the isolates were resistant to four or more antibiotic classes, indicating a high level of multi-resistance. 143 resistance profiles were found. The isolates with different MDR profiles and/or colony morphologies were phylogenetically ascribed based on 16S rDNA to phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, including 48 genera. Putative intrinsic resistance was detected in different phylogenetic groups including genera Altererythrobacter, Bacillus, Brevundimonas, Erythrobacter, Mesonia, Ochrobactrum, and Ponticaulis. Correlation of the presence of pairs of the non-intrinsic-resistances in phylogenetic groups based on the kappa index (κ) highlighted the co-habitation of some of the tested pairs at different phylogenetic levels. Maximum correlation (κ = 1.000) was found for pairs CzR/TcR in Betaproteobacteria, and CcR/TcR and EmR/SmR in Sphingobacteriia at the class level, while at the genus level, was found for CcR/TcR and NxR/TmR in Mesonia, CzR/TmR and EmR/KmR in Paenibacillus, and CcR/EmR and RpR/TcR in Pseudomonas. These results could suggest the existence of intra-class and intra-genus-transmissible genetic elements containing determinants for both members of each pair. Network analysis based on κ values higher than 0.4 indicated the sharing of paired resistances among several genera, many of them centered on the Paenibacillus node and raising the hypothesis of inter-genera transmission of resistances interconnected through members of this genus. This is the first time that a possible hotspot of resistance interchange in a particular environment may have been detected, opening up the possibility that one, or a few, bacterial members of the community could be important promoters of antibiotic resistance (AR) dissemination in this environment's bacterial population. Further studies using the available isolates will likely give insights of the possible mechanisms and genetic elements involved.

Nonuniversality of front fluctuations for compact colonies of nonmotile bacteria.

The front of a compact bacterial colony growing on a Petri dish is a paradigmatic instance of non-equilibrium fluctuations in the celebrated Eden, or Kardar-Parisi-Zhang (KPZ), universality class. While in many experiments the scaling exponents crucially differ from the expected KPZ values, the source of this disagreement has remained poorly understood. We have performed growth experiments with B. subtilis 168 and E. coli ATCC 25922 under conditions leading to compact colonies in the classically alleged Eden regime, where individual motility is suppressed. Non-KPZ scaling is indeed observed for all accessible times, KPZ asymptotics being ruled out for our experiments due to the monotonic increase of front branching with time. Simulations of an effective model suggest the occurrence of transient nonuniversal scaling due to diffusive morphological instabilities, agreeing with expectations from detailed models of the relevant biological reaction-diffusion processes.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Strong correlation between the antifungal effect of amphotericin B and its inhibitory action on germ-tube formation in a Candida albicans URA⁺ strain.

The hypothetical capacity of amphotericin B to suppress the formation of germ-tubes, which is the first step of yeast-to-hypha conversion in Candida albicans, has been investigated in the wild-type strain CEY.1 (CAI.4-URA⁺). Exponential cells exposed to concentrations of amphotericin B below or around the MIC90, exhibited a weak reduction in the percentage of human serum-induced germ-tube formation at 37ºC compared with a non-exposed control. However, the dimorphic transition was drastically suppressed after addition of potentially lethal doses of amphotericin B, which also caused severe cell killing. In contrast, an identical experimental approach carried out with the fungistatic compound 5-fluorocytosine had no significant effect on the level of the germ-tube formation. Together, these results strongly point to a close correlation between the fungicidal action of amphotericin B and its ability to impair morphogenetic conversion in C. albicans.

Analysis of validamycin as a potential antifungal compound against Candida albicans.

Validamycin A has been successfully applied in the fight against phytopathogenic fungi. Here, the putative antifungal effect of this pseudooligosaccharide against the prevalent human pathogen Candida albicans was examined. Validamycin A acts as a potent competitive inhibitor of the cell-wall-linked acid trehalase (Atc1p). The estimated MIC50 for the C. albicans parental strain CEY.1 was 500 mg/l. The addition of doses below MIC50 to exponentially growing CEY.1 cells caused a slight reduction in cell growth. A concentration of 1 mg/ml was required to achieve a significant degree of cell killing. The compound was stable as evidenced by the increased reduction of cell growth with increasing incubation time. A homozygous atc1delta/atc1delta mutant lacking functional Atc1p activity showed greater resistance to the drug. The antifungal power of validamycin A was limited compared with the drastic lethal action caused by exposure to amphotericin B. The endogenous content of trehalose rose significantly upon validamycin and amphotericin B addition. Neither serum-induced hypha formation nor the level of myceliation recorded in macroscopic colonies were affected by exposure to validamycin A. Our results suggest that, although validamycin A cannot be considered a clinically useful antifungal against C. albicans, its mechanism of action and antifungal properties provide the basis for designing new, clinically interesting, antifungal-related compounds.

Telomere maintenance in Drosophila: rapid transposon evolution at chromosome ends.

The maintenance of terminal sequences is an important role of the telomere, since it prevents the loss of internal regions that encode essential genes. In most eukaryotes, this is accomplished by the telomerase. However, telomere length can also be maintained by other mechanisms, such as homologous recombination and transposition of telomeric retrotransposons to the chromosome ends. A remarkable situation is the case of Drosophila, where telomerase was lost, and thus telomeres managed to be maintained by occasional retrotransposition of telomeric elements to the receding ends. In the recent analysis of 12 Drosophila genomes, the multiplicity of autonomous and non-autonomous telomere-specific retrotransposons has revealed extensive and rapid evolution of telomeric DNA. The phylogenetic relationship among these telomeric retrotransposons is congruent with the species phylogeny, suggesting that they have been vertically transmitted from a common ancestor. In this review, we also suggest that the formation of a non-canonical DNA structure at Drosophila telomeres could be the way to protect the ends.