Investigation of skin microbiota reveals Mycobacterium ulcerans-Aspergillus sp. trans-kingdom communication.

Mycobacterium ulcerans secrete a series of non-ribosomal-encoded toxins known as mycolactones that are responsible for causing a disabling ulceration of the skin and subcutaneous tissues named Buruli ulcer. The disease is the sole non-contagion among the three most common mycobacterial diseases in humans. Direct contact with contaminated wetlands is a risk factor for Buruli ulcer, responsible for M. ulcerans skin carriage before transcutaneous inoculation with this opportunistic pathogen. In this study, we analysed the bacterial and fungal skin microbiota in individuals exposed to M. ulcerans in Burkina Faso. We showed that M. ulcerans-specific DNA sequences were detected on the unbreached skin of 6/52 (11.5%) asymptomatic farmers living in Sindou versus 0/52 (0%) of those living in the non-endemic region of Tenkodogo. Then, we cultured the skin microbiota of asymptomatic M. ulcerans carriers and negative control individuals, all living in the region of Sindou. A total of 84 different bacterial and fungal species were isolated, 21 from M. ulcerans-negative skin samples, 31 from M. ulcerans-positive samples and 32 from both. More specifically, Actinobacteria, Aspergillus niger and Aspergillus flavus were significantly associated with M. ulcerans skin carriage. We further observed that in vitro, mycolactones induced spore germination of A. flavus, attracting the fungal network. These unprecedented observations suggest that interactions with fungi may modulate the outcome of M. ulcerans skin carriage, opening new venues to the understanding of Buruli ulcer pathology, prophylaxis and treatment of this still neglected tropical infection.

Maxillary fungus balls due to Fusarium proliferatum.

Fungus ball is the most common form of non-invasive fungal rhinosinusitis. Aspergillusfumigatus (between 44.8% and 75%) and Aspergillusflavus (14%) are the two most common species recovered. However, recent advances in mycological laboratory methods have enhanced the detection and identification of fungi within fungus balls. Fusarium species, sometimes recovered from other forms of fungal rhinosinusitis such as allergic fungal rhinosinusitis or acute invasive fungal rhinosinusitis, are poorly associated with sinonasal fungus ball. Here, we describe two further cases of a fungus ball due to Fusariumproliferatum and provide the first description of this fungal pathogen with a fungus ball of odontogenic origin. These case reports demonstrate that uncommon fungal species such as Fusarium spp. might be underestimated as agents of sinusal cavity fungus ball. Enhanced mycological detection and diagnostic techniques might give rise, in the near future, to the emergence of new or rare fungal species associated with this clinical entity.

Nucleotide Sequence Database Comparison for Routine Dermatophyte Identification by Internal Transcribed Spacer 2 Genetic Region DNA Barcoding.

Conventional dermatophyte identification is based on morphological features. However, recent studies have proposed to use the nucleotide sequences of the rRNA internal transcribed spacer (ITS) region as an identification barcode of all fungi, including dermatophytes. Several nucleotide databases are available to compare sequences and thus identify isolates; however, these databases often contain mislabeled sequences that impair sequence-based identification. We evaluated five of these databases on a clinical isolate panel. We selected 292 clinical dermatophyte strains that were prospectively subjected to an ITS2 nucleotide sequence analysis. Sequences were analyzed against the databases, and the results were compared to clusters obtained via DNA alignment of sequence segments. The DNA tree served as the identification standard throughout the study. According to the ITS2 sequence identification, the majority of strains (255/292) belonged to the genus Trichophyton, mainly T. rubrum complex (n = 184), T. interdigitale (n = 40), T. tonsurans (n = 26), and T. benhamiae (n = 5). Other genera included Microsporum (e.g., M. canis [n = 21], M. audouinii [n = 10], Nannizzia gypsea [n = 3], and Epidermophyton [n = 3]). Species-level identification of T. rubrum complex isolates was an issue. Overall, ITS DNA sequencing is a reliable tool to identify dermatophyte species given that a comprehensive and correctly labeled database is consulted. Since many inaccurate identification results exist in the DNA databases used for this study, reference databases must be verified frequently and amended in line with the current revisions of fungal taxonomy. Before describing a new species or adding a new DNA reference to the available databases, its position in the phylogenetic tree must be verified.

A Case of Fungus Ball-Type Maxillary Sinusitis Due to Penicillium Roqueforti.

The Penicillium genera, encompassing about 225 different species of fungi, are naturally present in the environment. These genera are poorly linked to human disease, except for Penicillium marneffei causing septicemia in immunocompromised hosts. Thus, Penicillium species recovered from respiratory tract samples are often considered as inhaled contaminants in the clinical laboratory. However, we report here a case of fungal maxillary sinusitis due to Penicillium roqueforti diagnosed in a 40-year-old female, a teacher, complaining of moderate pain for months in the maxillary sinus and chronic posterior rhinorrhea. CT scanner and MRI enabled a preliminary diagnosis of left maxillary fungus ball-type sinusitis with calcified material seen on CT and marked very low signal in T2 weighted images seen on MRI. Anatomopathological and mycological examination of sinusal content showed septate hyphae. Direct sequencing of the sinusal content revealed P. roqueforti. P. roqueforti has been traditionally used in France for more than 200 years for cheese ripening. However, to our knowledge, this ascomycetous fungus has very rarely been associated in the literature with human disease. P. roqueforti is associated only with cheese worker's lung, a hypersensitivity pneumonitis affecting employees in blue cheese factories. Other species in the Penicillium genus are reported to cause various disorders such as invasive infection, superficial infection or allergic diseases. P. roqueforti has never previously been reported as a cause of human infection. Thus, we report the first case of fungus ball due to P. roqueforti in an immunocompetent patient.

Validation of a New Web Application for Identification of Fungi by Use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has emerged as a reliable technique to identify molds involved in human diseases, including dermatophytes, provided that exhaustive reference databases are available. This study assessed an online identification application based on original algorithms and an extensive in-house reference database comprising 11,851 spectra (938 fungal species and 246 fungal genera). Validation criteria were established using an initial panel of 422 molds, including dermatophytes, previously identified via DNA sequencing (126 species). The application was further assessed using a separate panel of 501 cultured clinical isolates (88 mold taxa including dermatophytes) derived from five hospital laboratories. A total of 438 (87.35%) isolates were correctly identified at the species level, while 26 (5.22%) were assigned to the correct genus but the wrong species and 37 (7.43%) were not identified, since the defined threshold of 20 was not reached. The use of the Bruker Daltonics database included in the MALDI Biotyper software resulted in a much higher rate of unidentified isolates (39.76 and 74.30% using the score thresholds 1.7 and 2.0, respectively). Moreover, the identification delay of the online application remained compatible with real-time online queries (0.15 s per spectrum), and the application was faster than identifications using the MALDI Biotyper software. This is the first study to assess an online identification system based on MALDI-TOF spectrum analysis. We have successfully applied this approach to identify molds, including dermatophytes, for which diversity is insufficiently represented in commercial databases. This free-access application is available to medical mycologists to improve fungal identification.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: revolutionizing clinical laboratory diagnosis of mould infections.

The clinical diagnosis of mould infections currently involves complex species identification based on morphological criteria, which is often prone to error. Employing an extensive mould species reference spectral library (up to 2832 reference spectra, corresponding to 708 strains from 347 species), we assessed the extent to which matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) enhanced the accuracy of species identification. MALDI-TOF MS data were validated against morphology-based and DNA sequence-based results with 262 clinical isolates collected over a 4-month period in 2013. The implementation of MALDI-TOF MS resulted in a dramatic improvement in mould identification at the species level (from 78.2% to 98.1%) and a marked reduction in the misidentification rate (from 9.8% to 1.2%). We then compared the mould identification results obtained before (i.e. 2011) and after (i.e. 2013) the implementation of MALDI-TOF MS in routine identification procedures, which showed an improvement from 64.57% to 100%. Reassessment of a set of isolates from 2011 with this procedure, including MALDI-TOF MS, yielded an increase in species diversity from 16 to 42 species. Finally, application of this procedure during a 16-month period (2012-2013) enabled the identification of 1094 of 1107 (98.8%) clinical mould isolates corresponding to 107 distinct species. MALDI-TOF MS-based mould species identification may soon challenge traditional techniques in the clinical laboratory, as patient prognosis is largely contingent on rapid and accurate diagnosis.

Identification of Leishmania at the species level with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.

Diode-pumped 99 fs Yb:CaF2 oscillator.

We demonstrate the generation of 99 fs pulses by a mode-locked laser oscillator built around a Yb:CaF(2) crystal. An average power of 380 mW for a 13 nm bandwidth spectrum centered at 1053 nm is obtained. The short-pulse operation is achieved thanks to a saturable absorber mirror and is stabilized by the Kerr lens effect. We investigated the limits of the stabilization process and observed a regime slowly oscillating between mode locking and Q switching.

Phosphatidylserine increase in rat liver endomembranes during the acute phase response.

The acute phase response (APR) is responsible for great changes in protein and lipid metabolism. For example, marked changes are observed in the metabolism of fatty acids, triglycerides, cholesterol and sphingolipids. Those lipids are partly recovered in the lipoproteins and subsequently in the plasma. Beside these lipid families, nothing is known about phospholipids and their synthesis in endomembranes during the APR. Our studies show that phosphatidylserine synthesis is stimulated during the APR and that this lipid is increased in the endoplasmic reticulum (ER) and the ER-derived vesicles.

The homeotic protein dlk is expressed during peripheral nerve development.

To investigate the molecular events controlling myelination of the peripheral nervous system, we compared gene expression of normal mouse sciatic nerves to that of the trembler mouse, whose Schwann cells are blocked in a pre-myelinating phenotype. Using cDNA array, we assessed expression levels of 1176 genes, and we found that delta-like protein (dlk), an epidermal growth factor-like homeotic protein, was expressed in the normal developing nerves, but at a low level in the dysmyelinating mutant trembler. Moreover, dlk expression was down-regulated when myelin protein expression was up-regulated, and no expression was observed in the developing brain. These results suggest that dlk expression is required for Schwann cell acquisition of the myelinating phenotype.

Autoantibodies to lipids in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome.

OBJECTIVE: To investigate the presence of autoantibodies to lipids in the bronchoalveolar lavage (BAL) fluid from adult patients with acute respiratory distress syndrome (ARDS). DESIGN: Analysis of immunoglobulin G (IgG) in BAL fluid by electrophoresis followed by immunoblotting and characterization of immunoglobulins as antilipid autoantibodies. SETTING: Intensive care unit of a university hospital and two research university laboratories. SUBJECTS: Twenty-seven mechanically ventilated patients in total, including nine patients with ARDS and two control groups. INTERVENTIONS: Patients were ventilated with a mechanical ventilation mode. Six aliquots of 20-mL sterile normal saline at 37 degrees C were infused through the working channel of the bronchoscope. MEASUREMENTS: Total protein, detection of IgG by electrophoresis followed by immunoblotting, and characterization of IgG by enzyme-linked immunosorbent assay using different lipids as target antigens. MAIN RESULTS: Antiphospholipid autoantibodies are present in BAL fluid of ARDS patients. Among the phospholipids tested, phosphatidic acid and phosphatidylserine gave the most significant activity. The IgG fraction, purified from BAL fluids by affinity chromatography, gave the same pattern of binding as that of the BAL fluid. CONCLUSION: The presence of antiphospholipid autoantibodies in BAL fluid suggests involvement of autoimmune mechanisms in the pathogenesis of ARDS.

Acyl-CoA elongase expression during seed development in Brassica napus.

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.

Taxonomy of gymnospermae: multivariate analyses of leaf fatty acid composition.

The fatty acid composition of photosynthetic tissues from 137 species of gymnosperms belonging to 14 families was determined by gas chromatography. Statistical analysis clearly discriminated four groups. Ginkgoaceae, Cycadaceae, Stangeriaceae, Zamiaceae, Sciadopityaceae, Podocarpaceae, Cephalotaxaceae, Taxaceae, Ephedraceae and Welwitschiaceae are in the first group, while Cupressaceae and Araucariaceae are mainly in the second one. The third and the fourth groups composed of Pinaceae species are characterized by the genera Larix, and Abies and Cedrus, respectively. Principal component and discriminant analyses and divisive hierarchical clustering analysis of the 43 Pinaceae species were also performed. A clear-cut separation of the genera Abies, Larix, and Cedrus from the other Pinaceae was evidenced. In addition, a mass analysis of the two main chloroplastic lipids from 14 gymnosperms was performed. The results point to a great originality in gymnosperms since in several species and contrary to the angiosperms, the amount of digalactosyldiacylglycerol exceeds that of monogalactosyldiacylglycerol.

Phosphatidylserine delivery to endoplasmic reticulum-derived vesicles of plant cells depends on two biosynthetic pathways.

Vesicles formed from endoplasmic reticulum (ER) by a cell-free system of leek cells (Allium porrum) are enriched in phosphatidylserine (PS), especially species containing very long chain fatty acids (VLCFA, at least 20 carbon atoms). In plant cells, PS is formed either by PS synthase or the serine exchange enzyme, although it is not known which pathway(s) contribute(s) to PS delivery in the ER-derived vesicles (EV), nor to what extent this occurs. Taking advantage of a cell-free system, we have shown that PS enrichment originates mainly from the serine exchange enzyme which is the only pathway that synthesizes the VLCFA-PS species. On the other hand, both enzymes synthesize PS with long chain fatty acids (up to 18 carbon atoms), but these species are given to the EV by PS synthase.

The characteristics of bronchoalveolar lavage from a patient with antiphospholipid syndrome who developed acute respiratory distress syndrome.

The purpose of this study was to investigate the biochemical characteristics as well as the occurrence and specificity of antiphospholipid antibodies in the bronchoalveolar lavage (BAL) fluid from a patient with both antiphospholipid antibodies syndrome (APS) and acute respiratory distress syndrome (ARDS). Proteins, lipids, cells and autoantibodies were determined. Immunoglobulins were purified with affinity chromatography. Autoantibody identification was assessed with enzyme-linked immunosorbent assay (ELISA) and with electrophoresis, followed by immunoblotting and revelation with antihuman IgG-peroxidase conjugate. Antiphospholipid antibodies were found to be present in the BAL fluid as well as in the serum from a patient with APS. Specifically, antiphosphatidylserine and antiphosphatidic acid IgG antibodies in the BAL fluid and antiphosphatidylcholine and anticardiolipin IgG antibodies in the serum were detected at high levels. BAL fluid protein and the percentage of neutrophils were found to be increased. A quantitative as well as qualitative deficiency of surfactant phospholipids was also observed. Antibodies directed against surfactant phospholipids could cause surfactant abnormalities and an inflammatory reaction. These disorders may be one of the causes of the ARDS or a factor in the perpetuation of the inflammation.

Fatty acid composition of tomato leaves as biomarkers of metal-contaminated soils.

The aim of this study was to determine whether fatty acid composition of leaves, cotyledons or roots could be used as an indicator of the bioavailability and of the adverse effects of heavy metals on plants. Tomato seedlings were grown on soils obtained by mixing increasing amounts of a highly metal-contaminated soil with an uncontaminated sandy soil, and the fatty acid composition of plant tissues was analyzed. The fatty acid composition of roots and cotyledons of plants grown on contaminated soils was mostly the same as in the control plants. In contrast, significant changes in the fatty acid composition of primary leaves occurred. Our results clearly indicate a relationship between metal accumulation (Cd, Pb, Zn, and Cu) and the fatty acid composition of primary leaves, with the contribution of 18 C atom fatty acids (as 18:3 fatty acid and precursors) being more closely correlated with the availability of heavy metals in soils.

Zinc finger proteins in the peripheral nerve of adult normal and Trembler mice.

By using monoclonal antibodies directed against the conserved zinc binding site of zinc finger proteins, we detected 2 prominent zinc finger proteins in rat peripheral nervous system (PNS) during development, and in adult normal and Trembler mice sciatic nerves. The protein of 55 kDa is abundant in adult normal mice and rats, but is weakly expressed in adult Trembler mice. The 29 kDa protein is expressed in neonatal rats and in the Trembler mouse, but is absent in adult rats and mice. These results suggest that the Schwann cell proliferation stage may be associated with the 29 kDa protein expression, and the 55 kDa protein may be implicated in the PNS myelination process.

Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase.

Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20:0-CoA and (E) 2,3-20:1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 microM 18:1-CoA and 30 microM malonyl-CoA, and that 18:0-CoA and 18:1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 microM Triton X-100 and 25 microg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.

Peroxisomal beta-oxidation enzyme gene expression in the developing mouse brain.

Using the northern blot technique, the steady-state levels for the mRNAs encoding acyl-CoA oxidase, pristanoyl-CoA oxidase, trans2, 3enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional enzyme type 2, 3-ketoacyl-CoA thiolase and sterol-carrier-protein x during postnatal brain development were measured. The developmental patterns obtained for each mRNA species studied were similar, with an increase in the mRNA level between birth and postnatal day 5, followed by a gradual decrease to 34-55% of the maximal value at postnatal day 30. These results are in agreement with a coordinately controlled expression of the genes involved in VLCFA beta-oxidation during brain development. Moreover, comparison of these developmental profiles with that obtained for ceramide galactosyltransferase showed that the set-up of the very-long-chain fatty acids beta-oxidation system is independent of the myelinating signal in the central nervous system.

Preparation, evaluation and application of new pseudo-affinity chromatographic supports for penicillin acylase purification.

New pseudo-affinity chromatographic supports for penicillin acylase were prepared and evaluated with three different samples: pure penicillin acylase, industrial clarified feedstock and crude extract. The different gels were studied for their purification fold (three to six) and their recovery power (80-100%). The best support was characterized by its dynamic capacity, (20 mg/ml) and its recovery power was tested at five flow-rates (30, 150, 300 and 750 cm/h) to determine the optimal flow-rate (300 cm/h). In addition we used cleaning in place to test the resistance to hard conditions of sanitization by 1 M NaOH (90% of recovery for 12 h of contact). These gels may therefore be used on an industrial scale.