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Azoles are currently the mainstay of antifungal treatment both in agricultural and in clinical settings. Although the target site of azole action is well studied, the basis of azole resistance and the ultimate mode of action of the drug in fungi are poorly understood. To gain a deeper insight into these aspects of azole action, restriction-mediated plasmid integration (REMI) was used to create azole sensitive and resistant strains of the clinically important fungus Aspergillus fumigatus. Four azole sensitive insertions and four azole-resistant insertions were characterized. Three phenotypes could be re-created in wild-type AF210 by reintegration of rescued plasmid and a further four could be confirmed by complementation of the mutant phenotype with a copy of the wild-type gene predicted to be disrupted by the original insertional event. Six insertions were in genes not previously associated with azole sensitivity or resistance. Two insertions occur in transporter genes that may affect drug efflux, whereas others may affect transcriptional regulation of sterol biosynthesis genes and NADH metabolism in the mitochondrion. Two insertions are in genes of unknown function., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

This study evaluated the levels of TNFalpha, IL-6, IL-1beta, nitric oxide (NO), CRP, C3 and apoptosis in 36 patients with dengue fever (DF), 34 patients with dengue haemorrhagic fever (DHF) and in virus-infected monocyte cultures. IL-6, TNFalpha, NO (nitrites) and CRP levels were increased and C3 diminished in patients with DF and DHF. IL-6, TNFalpha, CPR and C3 values were associated with disease severity (DHF). Nitrite content was incremented in DF patients. TNFalpha, NO and CRP levels were associated with secondary infection. IL-6 and CRP levels were associated with dengue virus type 4 (DENV-4) and DENV-2, respectively. Low levels of C3 were associated with DENV-2 and DENV-4 infections. Similarly, increased content of TNFalpha, IL-6 and nitrites were observed in supernatants from infected monocyte cultures. IL-6 was associated with DENV-4 infection. The different virus serotypes induced apoptosis in monocyte cultures. Dengue infection did not induce elevated IL-1beta production, either in patients or in infected cultures. These results suggest that TNFalpha, IL-6, NO and CRP are involved in dengue infection and that monocytes could be an important source of cytokine and NO production.

In recent years, the scientific community has provided many tools to assist with pathway analysis. Some of these programs can be used to manage functional annotation of gene products, others are oriented to exploring and analyzing data sets and many allow both possibilities. Potential users of these tools are faced with the necessity to decide which of the existing programs are the most appropriate for their needs. SerbGO is a user-friendly web tool created to facilitate this task. It can be used (i) to search for specific functionalities and determine which applications provide them and (ii) to compare several applications on the basis of different types of functionalities. Iterating and combining both functionalities can easily lead to selecting an appropriate tool. Data required by SerbGO is either the desired capabilities within a defined Standard Functionalities Set or the list of the tools to be compared. The analysis performed carries out a cross-classification that produces an easily readable output with the list of tools that implement the capabilities demanded or a table with the categorization of the GO tools that one wishes to compare. SerbGO is freely available and does not require a login. It can be accessed either directly at our server (http://estbioinfo.stat.ub.es/apli/serbgo) or at the GO Consortium website (http://www.geneontology.org/GO.tools.microarray.shtml#serbgo).

Background: BAL8557 (WSA) is the water-soluble prodrug of the triazole BAL4815 with in vitro anti-Aspergillus activity. We compared the activity of oral BAL8557 with oral itraconazole, oral voriconazole and intravenous caspofungin in a temporarily neutropenic murine model of disseminated Aspergillus flavus., Methods: Mice were immunosuppressed using cyclophosphamide, then infected. Mice were treated either 2 h pre-infection (PRE), or 4 or 24 h post-infection (4POST and 24POST, respectively). Treatment was for 10 days followed by 4 days of observation. Surviving mice were killed and liver, kidneys, lungs and brain cultured. BAL8557 groups included doses corresponding to approximately 30, 15, 6 and 3 mg/kg of the active BAL4815; comparators included itraconazole 25 and 10 mg/kg/dose, voriconazole (plus oral grapefruit) 25 and 10 mg/kg/day or caspofungin 1 mg/kg/day. In a simultaneous tissue burden study mice were treated for 3 days, kidneys removed and homogenized and burden measured by quantitative culture and quantitative PCR using fluorescence resonance energy transfer (FRET)., Results: Control mice had 83-100% mortality. Over 66% of BAL8557-treated mice survived after >6 mg/kg PRE or >15 mg/kg POST. In the PRE models BAL8557 (6 mg/kg) and caspofungin were 100% protective and itraconazole 67% protective, but voriconazole 10 mg/kg had 100% mortality (P = 0.0016). In the 4POST and 24POST models survival was >66% with BAL8557 30 and 15 mg/kg/dose and similar to voriconazole or itraconazole. In the 24POST groups, sterilization of all organs was achieved in 11/16 survivors treated with BAL8557. The quantitative PCR correlated with kidney fungal burden (r2 = 0.59). Earlier treatment reduced burdens., Conclusions: BAL8557 demonstrated impressive antifungal activity against A. flavus in this model, in both survival and tissue burden.

Habitat preference behavior may play an important role in nonallopatric speciation. However, most examples of habitat preference contributing to differentiation within natural populations correspond to parasites or herbivores living in the discrete environments constituted by their animal or plant hosts. In the present study we investigated migration guided by habitat preference in the intertidal snail Littorina saxatilis in a hybrid zone associated with an ecotone across the shore, which is therefore a continuously varying environment. First, we found evidence for this behavior in one of the two locations studied. Second, we made reciprocal transplants to suppress the phenotypic gradient observed across the hybrid zone and measured the relative contributions of selection and migration to its regeneration. Selection played an important role at the two locations studied, but migration was only important at one, where it accounted for between a third and a half of the regenerated gradient. This overall minor effect of migration was relevant for theoretical models dealing with nonallopatric speciation, because it suggested that variation for habitat preference did not have an important role in the initiation of the differentiation process. The preference behavior observed in the hybrid zone would have evolved secondarily, as a consequence of habitat-dependent fitness differences between phenotypes.

Background: The anti-apoptotic properties of melatonin have been demonstrated previously in several in vivo and in vitro studies. Previous reports have shown increased apoptosis during puromycin aminonucleoside nephrosis (PAN). The aim of this study was to determine if melatonin (MEL) can prevent apoptosis and modify oxidative stress, an apoptosis inducer, in this experimental model., Methods: Rats were injected intraperitoneally with puromycin aminonucleoside. In addition, by the intragastric route they received 1 mg/kg/day of MEL or vehicle 3 days before puromycin injection and throughout the experiment. Animals were sacrificed at weeks 1 and 2 of nephrosis and frozen renal sections were studied for apoptosis by TUNEL, for apoptosis-associated proteins by monoclonal and polyclonal antibodies, and for superoxide anion (O(2)(-)) by a histochemical method. Nitric oxide (NO), malondialdehyde (MDA) and reduced glutathione (GSH), and the activities of superoxide dismutase (SOD) and catalase were measured in homogenized kidney tissue by appropriate biochemical and enzymatic methods., Results: Increases in apoptosis, p53, Fas and Fas-ligand were observed in nephrotic animals. MEL treatment decreased apoptosis at weeks 1 and 2 in the glomerular, interstitial and tubular compartments. This was accompanied by decreased expression of p53 (glomerulus, week 1; tubules, weeks 1 and 2), Fas (glomerulus and interstitium, week 2; tubules, weeks 1 and 2) and Fas-ligand (interstitum and tubules, week 2). Increased expression of Bcl-2-positive cells was observed at week 2 in all renal compartments in MEL-treated animals. High levels of O(2)(-) and NO generation and lipid peroxidation (MDA) were found in nephrotic animals. SOD and GSH remained unchanged, and only decreased catalase activity (week 1) was observed in PAN animals. Tendencies toward decreased values of O(2)(-) and MDA content along with recovery of catalase activity (week 1) were observed in MEL-treated nephrotic animals, but were insignificant in magnitude. MEL, however, did significantly downregulate pro-apoptotic genes and upregulated anti-apoptotic genes., Conclusions: The data demonstrate that, in PAN, melatonin has anti-apoptotic effects, which might in part be independent of the modulation of the oxidative status.

We investigated the in vitro interaction of terbinafine with itraconazole, fluconazole, amphotericin B and 5-flucytosine, against Aspergillus spp. We tested three isolates of Aspergillus fumigatus (one resistant to itraconazole), and two each of Aspergillus flavus, Aspergillus niger and Aspergillus terreus. We employed a broth microdilution-based method derived from an in vivo validated method capable of detecting itraconazole resistance in A. fumigatus. We studied the effect on the MICs by calculation of the fractional inhibitory concentration (FIC) and fractional fungicidal concentration (FFC) (99.99% kill). Itraconazole and terbinafine were synergic or additive in all strains (FIC = 0.15-1.0). Fluconazole and terbinafine were synergic with A. fumigatus, A. terreus and A. flavus (FIC = 0.3-0.5) and indifferent with A. niger (FIC = 2) isolates. Amphotericin B and terbinafine were mostly indifferent or antagonistic (FIC = 1.0-4.02). Flucytosine and terbinafine were usually indifferent or antagonistic (FIC = 0.63-8.5). FFCs were generally in accord with FICs. The use of terbinafine in combination therapy for Aspergillus infections with azoles seems promising, whereas terbinafine and amphotericin B or flucytosine in combination were less effective.

The activities of amphotericin B and itraconazole were studied in a temporarily neutropenic murine model of disseminated Absidia corymbifera infection, caused by two different strains. Amphotericin B MICs were 0.25 mg/L for both strains and itraconazole MICs were 1 and 2 mg/L. Amphotericin B was effective in vivo with both isolates. Itraconazole was less effective.

Background: Leukocyte infiltration is a common feature in renal biopsies from patients with acute poststreptococcal glomerulonephritis (APSGN). Cationic streptococcal erythrogenic toxin type B (ETB) and its precursor (ETBP) have been implicated in the pathogenesis of the disease, and the presence of ETB has been evidenced in renal biopsies from patients with APSGN. The present studies were performed to determine the effect of the ETBP and ETB on renal leukocyte infiltration and the mechanism(s) implicated in the phenomenon., Methods: Male Sprague-Dawley rats were injected intrarenally with 100 microg of ETB or ETBP. Animals were sacrificed at 1, 6 and 24 h after injection and renal samples were studied by indirect immunofluorescence for the presence of leukocyte common antigen (LCA+) cells, C3, monocyte chemotactic protein-1 (MCP-1) and intercellular adhesion molecule-(ICAM-1), and by direct immunofluorescence for the presence of immunoglobulins. ETB and ETBP were tested for chemotactic effect and migration inhibition factor (MIF) activity by chemotaxis under agarose and agarose microdroplet methods, respectively. Streptococcal proteins were also tested for the capacity to induce MIF activity in rat glomerular cultures. To test for the influence of cationic charge on renal LCA+ cell infiltration, rats were injected with cationized ferritin or polyethyleneimine (PEI) and sacrificed 1 h later., Results: An increased number of LCA+ cells was found in glomeruli and interstitial areas in ETB- or ETBP-injected animals. ETB and ETBP showed chemotactic and MIF activity on neutrophils and macrophages, and ETBP induced MIF activity in supernatants of glomerular cultures. Data obtained from C3, MCP-1, ICAM-1 or immunoglobulin renal staining in experimental animals were not significantly different when compared to control values. Cationized compounds failed to induce LCA+ cell infiltration; however, an increased number of glomerular LCA+ cells was observed after PEI perfusion., Conclusions: ETB and ETBP induce renal LCA+ cell infiltration during a short period after intrarenal injection, and this finding could be mediated by chemotactic and MIF activities. These observations could be relevant in the early events of pathogenesis of APSGN.

Background: Apoptosis is a mechanism of cellular death involved in the deletion of cells in hyperplasic processes which has been observed in proliferative glomerulonephritis. Patients with systemic lupus erythematosus (SLE) are known to have defective apoptosis but there is scarce information about apoptosis in the renal lesions of this disease. The present studies were done to evaluate apoptosis in relation to the intensity of proliferation in several glomerulonephritis and the possible association between this relationship and the chronic histologic changes in lupus nephritis., Methods: Studies were done in renal biopsies from 19 patients with lupus nephritis (types, IV, Va, Vb) classified with respect to chronicity and activity indexes, five patients with acute poststreptococcal glomerulonephritis, five with idiopathic mesangioproliferative glomerulonephritis, four with membranous glomerulonephritis, four with minimal-change nephrotic syndrome, and three patients with focal segmental sclerosis. Seven normal kidneys which could not be used for transplantation were used as a control group. Apoptosis was determined by in situ DNA nick-end labelling techniques and proliferating cells were identified with a monoclonal antibody antiproliferating cell nuclear antigen (PCNA)., Results: Decreased number of apoptotic cells and a high ratio of PCNA: apoptosis were observed in glomerulus and tubulointerstitium of patients with lupus nephritis when compared to other proliferative glomerulonephritis and controls. In lupus nephritis, glomerular apoptosis had a negative correlation with the chronicity index (r = -0.606 P = 0.005). The number of apoptotic cells in the glomeruli was not correlated with the number of PCNA positive cells in lupus nephritis, in contrast with a striking linear correlation observed in acute poststreptococcal nephritis (r = 0.925, P = 0.024), a disease with excellent prognosis., Conclusions: Apoptosis is decreased in proliferative lupus nephritis. Intense proliferation without increment in apoptosis (a high PCNA: apoptosis ratio) is a characteristic of lupus nephritis associated with chronic renal histological changes.

Background: Reactive oxygen radicals are probably involved in the pathogenesis of human and experimental models of renal disease, yet current methods are inadequate to quantify and identify the cells producing reactive oxygen radicals., Methods and Results: We used the nitroblue tetrazolium reaction to determine superoxide anion production in glomerular cells in phorbol myristate-stimulated glomerular suspensions and in isolated glomeruli from rats with nephrotoxic nephritis, ureteral obstruction, and puromycin aminonucleoside nephrosis. We were also able to identify these nitroblue tetrazolium + cells using specific appropriate antibodies. When the technique was tested in conditions known to increase reactive oxygen radicals, as phorbol myristate-stimulated glomeruli and glomeruli from animals with nephrotoxic nephritis and ureteral obstruction, increased number of nitroblue tetrazolium + cells were found. These cells were identified as glomerular intrinsic cells (Thy-1 +) or infiltrating leukocytes (leukocyte common antigen + or antineutrophil +)., Conclusion: This method may be useful to determine cells participating in glomerular damage induced by reactive oxygen radicals.

The relationship between nutritional status and cutaneous leishmaniasis (CL) was evaluated in 230 children living in a rural subtropical rainforest in Northwest Ecuador. One-third of the subjects had evidence of either current (13 per cent) or past CL infection (21 per cent). Subjects with current (4.71 +/- 0.44 mg) or previous disease (4.29 +/- 0.35 mg) had lower mean daily dietary iron intakes than non-infected children (5.45 +/- 0.2 mg; chi 2 = 0.048), but not energy, protein, or other micronutrients. The low dietary iron intake data was corroborated by the reduced mean haemoglobin values observed in children with current (11.7 +/- 0.3 mg/dL) or past infection (11.3 +/- 0.2 mg/dL) compared to non-infected subjects (12.7 +/- 0.15 mg/dL; F-ratio = 17.0, P < 0.0001). Mean hematocrit values were also lower in the two infected groups (37.4 +/- 0.9 per cent and 37.4 +/- 0.6 per cent v. 39.5 +/- 0.5 per cent; F-ratio = 4.23, P = 0.0175). Furthermore, they were more likely to suffer from iron-deficiency anaemia than their non-infected counterparts (chi 2 = 4.64, P = 0.03). However, the children with active disease accounted for most of the excess risk for anemia (Fisher's exact test P = 0.009; OR = 10.0, exact 95 per cent CI = 1.37-111.8). Finally, growth stunting (< -2SD height-for-age) was more common in subjects with current (54 per cent) or past infection (51 per cent) compared to those without CL history (31 per cent; chi 2 = 8.03, P = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)

Four patients with a previous inflammatory rheumatic disease developed a peripheral tuberculous (TB) arthritis in a joint apparently affected by a rheumatic disease. The single most important factor in the diagnosis of TB was the presence of past or present pulmonary TB or a family history on a background of steroid use. Clinical presentation, disease evolution, and routine laboratory tests were unhelpful. The most effective method of diagnosis was synovial biopsy.

Autoantigenic events in acute poststreptococcal glomerulonephritis (APSGN) may result from neuraminidase-induced alterations in immunoglobulin levels. Whether streptococci from patients with APSGN produced neuraminidase and, if so, what substrates could be used for screening purposes and what were of potential clinical relevance were determined. Group A streptococci cultured from 20 patients with APSGN and four patients with acute rheumatic fever and group B, C, D, and G streptococci cultured from other individuals were reacted with the substrates to determine neuraminidase activity by the thiobarbituric acid assay. Neuraminidase production was detected in 16 of 20 streptococci from patients with APSGN. Partial purification by Sephadex G-150 chromatography gave two peaks, and polyacrylamide gel electrophoresis gave two bands with neuraminidase activity. Bovine mucin was the most useful substrate to detect neuraminidase production by nephritogenic streptococci, and with respect to human substrates, IgM was the most sensitive and renal basement membrane the least sensitive to enzyme action.