In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

A complex biogeographic history of diversification in Neotropical lancehead pitvipers (Serpentes,Viperidae)

Based on the literature, we had predicted that the diversification within the Neotropical snake genus Bothrops occurred along a latitudinal gradient from north to south, with diversification into unoccupied niches through ecological opportunity, not correlated with geoclimatic events. Using a dated phylogeny and estimating likelihoods of ancestral states at cladogenesis events, we reconstructed ancestral areas and assessed major events of the diversification of Bothrops clades, and we also discuss systematic implications for this group. Based on the phylogeny we produced, B. lojanus was not considered as part of the genus Bothrops since the results recovered this species nested within the Bothrocophias clade. We infer that the diversification of the Miocene Bothrops pictus and Bothrops alternatus clades may be related to the uplift of the western slopes of the Andes and the Argentinian Patagonian Andes, respectively. The Pliocene Bothrops taeniatus and Bothrops osbornei clades may be related to the uplift of the eastern and northern Andes, respectively. The Plio-Pleistocene Bothrops neuwiedi clade may be related to the habitat transitions from a warmer and forested environment to a cooler and open landscape; the Bothrops jararaca (i.e. island endemic species) and Bothrops lanceolatus clades to over-water dispersal with island speciation; and Bothrops atrox clade to the appearance of the Panamanian land bridge. We found that a multitemporal and multidirectional history of diversification may be correlated with geoclimatic and dispersalist events. We argue that the vacant niche hypothesis by itself does not explain Bothrops diversification.     

Use of capillary electrophoresis in the study of ligand-DNA interactions.

Free solution capillary electrophoresis (FSCE) has been used to separate two non-self-complementary 12mer oligonucleotide duplexes: d(AAATTATATTAT).d(ATAA-TATAATTT) and d(GGGCCGCGCCGC).d(GCGGCGCGGCCC). Titration of mixtures of the two oligonucleotides with model intercalators (ethidium bromide andactinomycin D) and minor groove binders (netropsin, Hoechst 33258 and distamycin) has shown the suitability of FSCE as a method to study the sequence selectivity of DNA binding agents. Binding data have shown cooperativity of binding for netropsin and Hoechst 33258 and have provided ligand:DNA binding ratios for all five compounds. Cooperativity of netropsin binding to a 12mer with two potential sites has been demonstrated for the first time. Ligands binding in the minor groove caused changes in migration time and peak shape which were significantly different from those caused by intercalators.     

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.     

Characterization of a novel serotonin receptor from Caenorhabditis elegans: cloning and expression of two splice variants

Serotonin [5-hydroxytryptamine (5-HT)] modulates feeding activity, egg-laying, and mating behavior in the free-living nematode, Caenorhabditis elegans. We have cloned a novel receptor cDNA from C. elegans (5-HT2Ce) that has high sequence homology with 5-HT2 receptors from other species. When transiently expressed in COS-7 cells, 5-HT2Ce exhibited 5-HT binding activity and activated Ca2+-mediated signaling in a manner analogous to other 5-HT2 receptors. However, 5-HT2Ce displayed unusual pharmacological properties, which resembled both 5-HT2 and 5-HT1-like receptors but did not correlate well with any of the known 5-HT2 subtypes. Two splice variants of 5-HT2Ce that differ by 48 N-terminal amino acids were identified. The two isoforms were found to have virtually identical binding and signaling properties but differed in their levels of mRNA expression, with the longer variant being four times more abundant than the shorter species in all developmental stages tested. Taken together, the results describe two variants of a novel C. elegans 5-HT receptor, which has some of the properties of the 5-HT2 family but whose pharmacological profile does not conform to any known class of receptor.     

Proteomic analysis of an orthotopic neuroblastoma xenograft animal model

Neuroblastoma is the most common extracranial solid tumour of childhood and comprises up to 50% of malignancies among infants. There is a great need of designing novel therapeutic strategies and proteome analysis is one approach for defining markers useful for tumour diagnosis, as well as molecular targets for novel experimental therapies. We started by comparing healthy adrenal glands (which are the election organs developing primary neuroblastoma, NB, tumours) and adrenal glands carrying primary NB tumours, taken from nude mice. Standard maps of healthy and tumour samples were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups, were analysed by MALDI-TOF mass spectrometry and 14 of these spots were identified so far. Among these proteins, of particular interest are the down-regulated proteins adrenodoxin (21-folds), carbonic anhydrase III (eight-folds) and aldose reductase related protein I (eight-folds), as well as the up-regulated protein peptidyl-propyl cis-trans isomerase A (five-folds). Moreover new proteins, which were absent in control samples, were expressed in tumour samples, such as nucleophosmin (NPM) and stathmin (oncoprotein 18).     

Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.     

Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor

In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII. Cysteine residues were substituted, one at a time, into 10 consecutive positions of TM I (Ala71-Val80) of a modified version of the M(3) muscarinic receptor that lacked most endogenous cysteine residues and contained a factor Xa cleavage site within the third intracellular loop. Following their expression in COS-7 cells, the 10 resulting cysteine mutant receptors were oxidized in their native membrane environment, either in the absence or in the presence of muscarinic ligands. Disulfide cross-link formation was monitored by examining changes in the electrophoretic mobility of oxidized and factor Xa-digested receptors on SDS gels. When molecular iodine was used as the oxidizing agent, the L77C receptor (position 1.42) was the only mutant receptor that displayed significant disulfide cross-linking, either in the absence or in the presence of muscarinic agonists or antagonists. On the other hand, when the Cu(II)-(1,10-phenanthroline)(3) complex served as the redox catalyst, muscarinic ligands inhibited disulfide cross-linking of the L77C receptor, probably because of impaired access of this relatively bulky oxidizing agent to the ligand binding crevice. The iodine cross-linking data suggest that M(3) muscarinic receptor activation is not associated with significant changes in the relative orientations of the outer and/or central segments of TM I and VII. In bovine rhodopsin, the residues present at the positions corresponding to Cys532 and Leu77 in the rat M(3) muscarinic receptor are not located directly adjacent to each other, raising the possibility that the relative orientations of TM I and VII are not identical among different class I GPCRs. Alternatively, dynamic protein backbone fluctuation may occur, enabling Cys532 to move within cross-linking distance of Leu77 (Cys77).     

Mutations in DOCK7 in Individuals with Epileptic Encephalopathy and Cortical Blindness

Epileptic encephalopathies are increasingly thought to be of genetic origin, although the exact etiology remains uncertain in many cases. We describe here three girls from two nonconsanguineous families affected by a clinical entity characterized by dysmorphic features, early-onset intractable epilepsy, intellectual disability, and cortical blindness. In individuals from each family, brain imaging also showed specific changes, including an abnormally marked pontobulbar sulcus and abnormal signals (T2 hyperintensities) and atrophy in the occipital lobe. Exome sequencing performed in the first family did not reveal any gene with rare homozygous variants shared by both affected siblings. It did, however, show one gene, DOCK7, with two rare heterozygous variants (c.2510delA [p.Asp837Alafs^∗48] and c.3709C>T [p.Arg1237^∗]) found in both affected sisters. Exome sequencing performed in the proband of the second family also showed the presence of two rare heterozygous variants (c.983C>G [p.Ser328^∗] and c.6232G>T [p.Glu2078^∗]) in DOCK7. Sanger sequencing confirmed that all three individuals are compound heterozygotes for these truncating mutations in DOCK7. These mutations have not been observed in public SNP databases and are predicted to abolish domains critical for DOCK7 function. DOCK7 codes for a Rac guanine nucleotide exchange factor that has been implicated in the genesis and polarization of newborn pyramidal neurons and in the morphological differentiation of GABAergic interneurons in the developing cortex. All together, these observations suggest that loss of DOCK7 function causes a syndromic form of epileptic encephalopathy by affecting multiple neuronal processes.