Miguel Ángel Moreno

The regulation of zinc homeostasis by the ZafA transcriptional activator is essential for Aspergillus fumigatus virulence.

We have previously shown that Aspergillus fumigatus is able to grow in zinc‐limiting media and that this ability is regulated at transcriptional level by both the availability of zinc and pH. When A. fumigatus grows as a pathogen, it must necessarily obtain zinc from the zinc‐limiting environment provided by host tissue. Accordingly, the regulation of zinc homeostasis by some zinc‐responsive transcriptional regulator in A. fumigatus must be essential for fungal growth within tissues of an immunocompromised host and, in turn, for pathogenicity. Here we provide evidence of the role of the zafA gene in regulating zinc homeostasis and its relevance in the virulence of A. fumigatus. Thus, we observed that (i) zafA can functionally replace the ZAP1 gene from Saccharomyces cerevisiae that encodes the zinc‐responsive transcriptional activator Zap1 protein; (ii) the expression of zafA itself is induced in zinc‐limiting media and repressed by zinc; (iii) deletion of zafA impairs the germination and growth capacity of A. fumigatus in zinc‐limiting media; and (iv) the deletion of zafA abrogates A. fumigatus virulence in a murine model of invasive aspergillosis. In light of these observations, we concluded that ZafA is a zinc‐responsive transcriptional activator that represents an essential attribute for A. fumigatus pathogenicity. Consequently, ZafA may constitute a new target for the development of chemotherapeutic agents against Aspergillus, because no zafA orthologues have been found in mammals.

Two different pathways are involved in the β-oxidation of n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications

In Pseudomonas putida U, the degradation of n‐alkanoic and n‐phenylalkanoic acids is carried out by two sets of β‐oxidation enzymes (βI and βII). Whereas the first one (called βI) is constitutive and catalyses the degradation of n‐alkanoic and n‐phenylalkanoic acids very efficiently, the other one (βII), which is only expressed when some of the genes encoding βI enzymes are mutated, catabolizes n‐phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the βI genes handicaps the growth of P. putida U in media containing n‐alkanoic or n‐phenylalkanoic acids with an acyl moiety longer than C4. However, all these mutants regained their ability to grow in media containing n‐alkanoates as a result of the induction of βII, but they were still unable to catabolize n‐phenylalkanoates completely, as the βI‐FadBA enzymes are essential for the β‐oxidation of certain n‐phenylalkanoyl‐CoA derivatives when they reach a critical size. Owing to the existence of the βII system, mutants lacking βIfadB/A are able to synthesize new poly 3‐OH‐n‐alkanoates (PHAs) and poly 3‐OH‐n‐phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.

Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development

Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.

The zrfA and zrfB Genes of Aspergillus fumigatus Encode the Zinc Transporter Proteins of a Zinc Uptake System Induced in an Acid, Zinc-Depleted Environment

ABSTRACT Zinc is an essential micronutrient that cells must obtain from the environment in order to develop their normal growth. Previous work performed at our laboratory showed that the synthesis of immunodominant antigens from Aspergillus spp., including A. fumigatus, was up-regulated by a low environmental concentration of zinc. These results suggested that a tightly regulated system for the fungus to grow under zinc-limiting conditions must underlie the ability of A. fumigatus to acquire zinc in such environments. In this work, we show that zrfA and zrfB are two of the genes that encode membrane zinc transporters from A. fumigatus in this system. Expression of these genes is differentially down-regulated by increasing concentrations of zinc in the medium. Thus, the transcription of zrfB is turned off at a concentration 50-fold higher than that for zrfA transcription. In addition, phenotypic analyses of single zrfAΔ and zrfBΔ mutants and a double zrfAzrfBΔ mutant revealed that the deletion of zrfB causes a greater defect in growth than the single deletion of zrfA. Deletion of both genes has a dramatic effect on growth under acid, zinc-limiting conditions. Interestingly, in neutral or slightly alkaline zinc-depleted medium, the transcriptional expression of both genes is down-regulated to such an extent that even in the absence of a supplement of zinc, the expression of zrfA and zrfB is strongly reduced. This fact correlates with the growth observed in alkaline medium, in which even a zrfAzrfBΔ double mutant was able to grow in a similar way to the wild-type under extremely zinc-limiting conditions. In sum, the zinc transport proteins encoded by zrfA and zrfB are members of a zinc uptake system of A. fumigatus that operates mainly under acid, zinc-limiting conditions.

A study of the interaction of CO2 with water ice

Aims. We studied the interaction between CO2 (guest) and H2O (host) molecular ices. Methods. Ices of CO2 and H2O are prepared by four di erent deposition techniques: sequential deposition (amorphous water ice followed by addition of CO2), co-deposition (both gases added simultaneously), inverse sequential deposition (carbon dioxide ice fol- lowed by addition of water) and crystalline sequential deposition (crystalline water ice is prepared first and CO2 is added afterwards). Samples are deposited at 80 K and are studied by temperature programmed desorption and transmission infrared spectroscopy. Results. Two slightly di erent varieties of association of CO2 and H2O are revealed from the di erent spectroscopic properties of the asymmetric stretching band of 12 CO2 and 13 CO2. The two varieties are found to co-exist in some of the samples at 80 K, whereas only the so-called internal CO2 remains after heating at 105 K. At 80 K carbon dioxide is able to adhere to a crystalline water ice surface. Activation energies for the desorption of CO2 from amorphous (Ed = 20:7 2 kJ mol 1 ) and crystalline (Ed = 19:9 2 kJ mol 1 ) water ice are derived from measurements of the sticking of CO2 as a function of ice temperature. Conclusions. These findings may have implications for the study of icy bodies of the Solar System.

The structure and vibrational frequencies of crystalline nitric acid

Ab initio calculations and reflection–absorption infrared (RAIR) spectroscopy have been used for the study of the structure and vibrational frequencies of crystalline nitric acid. The crystallographic data on the unit cell structure have been refined and the vibrational frequencies of the crystal have been determined in the calculation. A good general coincidence is found between the location of the calculated vibrational frequencies and the absorption bands in the IR spectra. Some discrepancies with previous empirical assignments are discussed. The results provide a useful base to support studies of more complex hydrous phases of nitric acid of great relevance for atmospheric chemistry. 2003 Elsevier B.V. All rights reserved.

A cryostat for low-temperature spectroscopy of condensable species

A simple experimental setup for the production of cold samples for spectroscopy is described. The samples are deposited under vacuum on a cold metallic surface whose temperature is controlled between 80 and 323 K by varying the heat flow balance between a liquid nitrogen reservoir and a power transistor. Tests of temperature stability and thermal inertia, as well as a set of reflection–absorption infrared and thermal desorption spectra, are reported as a demonstration of the performance of the system.

Ices of CO2/H2O Mixtures. Reflection−Absorption IR Spectroscopy and Theoretical Calculations

Ice mixtures of CO2 and H2O are studied using Fourier transform reflection-absorption infrared (RAIR) spectroscopy. Mixtures are prepared by sequential deposition or co-deposition of the two components from the gas phase onto an Al plate kept at 87 K inside a low-pressure chamber. Two CO2 structures are found in most experiments: a crystalline form similar to pure CO2, which evaporates when warming at 105 K, and a noncrystalline species which remains embedded in amorphous water ice after warming. Significant spectral variations are found depending on the deposition method and the thickness of the solid. Features characteristic of the RAIR technique appear in the spectral regions of the normal modes of the bending and asymmetric stretching CO2 vibrations. Simulations using Fresnel theory and Mie scattering are carried out with acceptable agreement with the experimental spectra of solids of variable thickness, from approximately 1 microm to the limit of nanoparticles. Theoretical calculations of a pure CO2 crystal are performed. The relaxed geometry of the solid and its vibrational spectrum are determined and compared to the experimental results.

Stability of carbonaceous dust analogues and glycine under UV irradiation and electron bombardment

The effect of UV photon (120-200 nm) and electron (2 keV) irradiation of analogues of interstellar carbonaceous dust and of glycine were investigated by means of IR spectroscopy. Films of hydrogenated amorphous carbon (HAC), taken as dust analogues, were found to be stable under UV photon and electron bombardment. High fluences of photons and electrons, of the order of 10(19) cm(-2), were needed for a film depletion of a few percent. UV photons were energetically more effective than electrons for depletion and led to a certain dehydrogenation of the HAC samples, whereas electrons led seemingly to a gradual erosion with no appreciable changes in the hydrocarbon structure. The rates of change observed may be relevant over the lifetime of a diffuse cloud, but cannot account for the rapid changes in hydrocarbon IR bands during the evolution of some proto-planetary nebulae. Glycine samples under the same photon and electron fluxes decay at a much faster rate, but tend usually to an equilibrium value different from zero, especially at low temperatures. Reversible reactions re-forming glycine, or the build-up of less transparent products, could explain this behavior. CO2 and methylamine were identified as UV photoproducts. Electron irradiation led to a gradual disappearance of the glycine layers, also with formation of CO2. No other reaction products were clearly identified. The thicker glycine layers (a few hundred nm) were not wholly depleted, but a film of the order of the electron penetration depth (80 nm), was totally destroyed with an electron fluence of -1 x 10(18) cm(-2). A 60 nm ice layer on top of glycine provided only partial shielding from the 2 keV electrons. From an energetic point of view, 2 keV electrons are less efficient than UV photons and, according to literature data, much less efficient than MeV protons for the destruction of glycine. The use of keV electrons to simulate effects of cosmic rays on analogues of interstellar grains should be taken with care, due to the low penetration depths of electrons in many samples of interest.

A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

The 5′ untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II–IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg2+-induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg2+ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg2+. This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4–6 mM Mg2+ in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I–II)+(V–VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.