Metabolic engineering of Aspergillus niger to enhance production of ethanol.

This work describes the genetic transformation of a strain of Aspergillus niger with five different constructs containing 16 different heterologous genes, coding for four oxidoreductases, two cellobiohydrolases, one endoglucanase, one ß-glucosidase, six enzymes involved in xylose metabolism, and two enzymes involved in fermentation. The aim was to try and engineer a consolidated bioprocessing in A. niger. The fungus already contains most of these enzymes and we only enhanced endogenous activities. We recovered nine transformants containing all genes, as indicated by polymerase chain reaction (PCR). To confirm that the products of the genes were functional, we measured the activity of five different enzymes in all the strains, and they all showed enhanced activity over the wild-type (wt) strain. The strains were grown on carboxymethyl cellulose (CMC) and xylan as substrates, and they produced considerably more ethanol than the wt. The levels of ethanol production were comparable to those reported in the literature.

Effect of Shock Waves on the Growth of Aspergillus niger Conidia: Evaluation of Germination and Preliminary Study on Gene Expression.

Shock waves, as used in medicine, can induce cell permeabilization, genetically transforming filamentous fungi; however, little is known on the interaction of shock waves with the cell wall. Because of this, the selection of parameters has been empirical. We studied the influence of shock waves on the germination of Aspergillus niger, to understand their effect on the modulation of four genes related to the growth of conidia. Parameters were varied in the range reported in protocols for genetic transformation. Vials containing conidia in suspension were exposed to either 50, 100 or 200 single-pulse or tandem shock waves, with different peak pressures (approximately 42, 66 and 83 MPa). In the tandem mode, three delays were tested. To equalize the total energy, the number of tandem "events" was halved compared to the number of single-pulse shock waves. Our results demonstrate that shock waves do not generate severe cellular effects on the viability and germination of A. niger conidia. Nevertheless, increase in the aggressiveness of the treatment induced a modification in four tested genes. Scanning electron microscopy revealed significant changes to the cell wall of the conidia. Under optimized conditions, shock waves could be used for several biotechnological applications, surpassing conventional techniques.

Antifungal and Cytotoxic Evaluation of Photochemically Synthesized Heparin-Coated Gold and Silver Nanoparticles.

Heparin-based silver nanoparticles (AgHep-NPs) and gold nanoparticles (AuHep-NPs) were produced by a photochemical method using silver nitrate and chloroauric acid as metal precursors and UV light at 254 nm. UV-Vis spectroscopy graphs showed absorption for AgHep-NPs and AuHep-NPs at 420 nm and 530 nm, respectively. TEM revealed a pseudospherical morphology and a small size, corresponding to 10-25 nm for AgHep-NPs and 1.5-7.5 nm for AuHep-NPs. Their antifungal activity against Candida albicans, Issatchenkia orientalis (Candida krusei), and Candida parapsilosis was assessed by the microdilution method. We show that AgHep-NPs were effective in decreasing fungus density, whereas AuHep-NPs were not. Additionally, the viability of human gingival fibroblasts was preserved by both nanoparticle types at a level above 80%, indicating a slight cytotoxicity. These results are potentially useful for applications of the described NPs mainly in dentistry and, to a lesser extent, in other biomedical areas.

Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.

Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

Shock Wave Application Increases the Antineoplastic Effect of Molecular Iodine Supplement in Breast Cancer Xenografts.

We evaluated the effect of oral molecular iodine supplementation and shock wave application under three different conditions on human MDA-MB231 cancer cell xenografts. After tumor volume reached 1 cm3, mice were randomly assigned to groups and treated for 3 weeks. The results revealed that high-dose shock wave treatment (150 shock waves at a pressure of 21.7 MPa, SW150/21.7) generated tissue lesions without decreasing tumor growth, canceled the antineoplastic action of iodine and promoted pro-tumor conditions (increased hypoxia-induced factor [HIF] and vascular endothelial growth factor [VEGF]). In contrast, moderate (SW35/21.7) and low (SW35/9.9) doses of shock waves had significant antineoplastic effects and, in combination with iodine supplement, attenuated the aggressiveness of these cells by decreasing expression of the markers of stem cells (CD44 and Sox2) and invasion (HIF and VEGF). These results allow us to propose the combination of shock waves and iodine as a possible adjuvant in breast cancer therapy.

Dynamic light scattering: A fast and reliable method to analyze bacterial growth during the lag phase.

A comparison between plate counting (PC) and dynamic light scattering (DLS) is reported. PC is the standard technique to determine bacterial population as a function of time; however, this method has drawbacks, such as the cumbersome preparation and handling of samples, as well as the long time required to obtain results. Alternative methods based on optical density are faster, but do not distinguish viable from non-viable cells. These inconveniences are overcome by using DLS. Two different bacteria strains were considered: Escherichia coli and Staphylococcus aureus. DLS was performed at two different illuminating conditions: continuous and intermittent. By the increment of particle size as a function of time, it was possible to observe cell division and the formation of aggregates containing very few bacteria. The scattered intensity profiles showed the lag phase and the transition to the exponential phase of growth, providing a quantity proportional to viable bacteria concentration. The results revealed a clear and linear correlation in both lag and exponential phase, between the Log10(colony-forming units/mL) from PC and the Log10 of the scattered intensity Is from DLS. These correlations provide a good support to use DLS as an alternative technique to determine bacterial population.

Shock Wave-Induced Damage and Poration in Eukaryotic Cell Membranes.

Shock waves are known to permeabilize eukaryotic cell membranes, which may be a powerful tool for a variety of drug delivery applications. However, the mechanisms involved in shock wave-mediated membrane permeabilization are still poorly understood. In this study, the effects on both the permeability and the ultrastructural features of two human cell lineages were investigated after the application of underwater shock waves in vitro. Scanning Electron Microscopy of cells derived from a human embryo kidney (HEK)-293 and Michigan Cancer Foundation (MCF)-7 cells, an immortalized culture derived from human breast adenocarcinoma, showed a small amount of microvilli (as compared to control cells), the presence of hole-like structures, and a decrease in cell size after shock wave exposure. Interestingly, these effects were accompanied by the permeabilization of acid and macromolecular dyes and gene transfection. Trypan blue exclusion assays indicated that cell membranes were porated during shock wave treatment but resealed after a few seconds. Deformations of the cell membrane lasted for at least 5 min, allowing their observation in fixed cells. For each cell line, different shock wave parameters were needed to achieve cell membrane poration. This difference was correlated to successful gene transfection by shock waves. Our results demonstrate, for the first time, that shock waves induce transient micro- and submicrosized deformations at the cell membrane, leading to cell transfection and cell survival. They also indicate that ultrastructural analyses of cell surfaces may constitute a useful way to match the use of shock waves to different cells and settings.

Shock waves and DNA-cationic lipid assemblies: a synergistic approach to express exogenous genes in human cells.

Cationic lipid/DNA complexes (lipoplexes) represent a powerful tool for cell transfection; however, their use is still limited by important concerns, including toxicity and poor internalization into deep tissues. In this work, we investigated the use of shock wave-induced acoustic cavitation in vitro for the transfection of lipoplexes in human embryo kidney 293 cells. We selected shock waves with the ability to internalize 10-kDa fluorescein isothiocyanate-dextran into cells while maintaining survival rates above 50%. Cell transfection was tested using the green fluorescent protein-encoding plasmid pCX::GFPGPI2. Confocal microscopy and fluorescence-assisted cell sorting analyses revealed successful transfection after treatments ranging from 1 to 3 min using 60 to 180 shock waves at peak amplitudes of 12.3 ± 1.5 MPa. Interestingly, the combination of shock waves and lipoplexes induced a 3.1- and 3.8-fold increase in the expression of the reporter gene compared with the use of lipoplexes or shock waves alone, respectively. These results indicate that cationic DNA assembly and shock waves act in a synergistic manner to promote transfection of human cells, revealing a potential approach for non-invasive site-specific gene therapy.

In situ TLR2 and TLR4 expression in a murine model of mycetoma caused by Nocardia brasiliensis.

Actinomycetoma caused by Nocardia brasiliensis is a common disease in tropical regions. This ailment is characterized by a localized chronic inflammation that mainly affects the lower limbs. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, inducing the production of proinflammatory mediators. The role of TLRs in the immune response against N. brasiliensis is unknown. The aim of this work was to locate and quantify in a murine model the expression of TLR2 and TLR4 in the infection site using reverse transcription-PCR and immunohistochemistry. The results showed that TLR2 expression increased in the infected tissue, whereas TLR4 expression decreased. The presence of TLR2 and TLR4 was demonstrated in different cell populations throughout the chronic infectious process. In the early stages of this process, TLR2 was expressed in neutrophils and macrophages in direct contact with the inoculum, whereas TLR4 was observed in mast cells. In the advanced stages of the infection, TLR2 was expressed in foam cells and fibroblasts and was likely associated with bacterial containment, while TLR4 was downregulated, probably resulting in an imbalance between the host immune response and the bacterial load that favoured chronic disease.

[Onychomycosis-causing yeasts in four Mexican dermatology centers and their antifungal susceptibility to azolic compounds]. / Levaduras causantes de onicomicosis en cuatro centros dermatológicos mexicanos y su sensibilidad antifúngica a compuestos azólicos.

BACKGROUND: Yeasts represent the second cause of nail fungal infection in the world, and Candida albicans and Candida parapsilosis are the two most common species. OBJECTIVES: To determine the yeast species frequency and their in vitro antifungal susceptibility, obtained from patients with clinical features suggestive of onychomycosis. METHODS: A prospective study was carried out in four dermatological care centers in Mexico from 2004 to 2007. Clinical diagnosis was corroborated by direct examination and culture. The yeast species was determined by morphological and biochemical tests. An antifungal susceptibility test to ketoconazole, itraconazole and fluconazole by the broth microdilution method was performed on each isolate (document M27-A2). RESULTS: One hundred sixty-six yeast isolates were obtained; the most frequently found species were C. parapsilosis (31.9%), C. albicans (22.4%) and Candida guilliermondii (12.7%). Of all isolates, 51 showed resistance to one or several of the azole compounds: 33 to itraconazole, 12 to ketoconazole and 6 to fluconazole. It was remarkable that the four Candida glabrata isolates were resistant to the three azole compounds; C. guilliermondii and Candida famata were resistant to itraconazole in 42.9% and 54.5%, respectively. CONCLUSION: The results obtained show the importance of identifying the aetiological agent and antifungal susceptibility testing in order to avoid therapeutic failures in onychomycosis.

[Effectiveness of imazalil to control the effect of fungal deterioration on mummies at the Mexico City Museum "El Carmen"]. / Efectividad del imazalil en el control del deterioro por hongos de momias del museo de El Carmen, Ciudad de México.

We present a study on the control and elimination of the fungi affecting the mummies specifically at the museum "El Carmen", in San Angel, Mexico City. Twelve analysed mummies presented an important deterioration attributed to colonizing fungi. The degree of fungal contamination and the efficacy of imazalil were evaluated. Two samplings were performed in order to isolate and identify the fungal genera, one for control and the other after the treatment. Isolation was done by the carpet-square technique and identification was performed by morphological features. Each sampling gave a total of 100 samples as follows: 17 from the air, 23 from the walls and 60 from the mummies. Samples were cultured on Sabouraud dextrose agar. From the first sampling a total of 649 colonies corresponding to 24 genera were obtained being the most frequent Penicillium, Cladophialophora and Aspergillus. From the second sampling, after the imazalil treatment, which was applied by means of lit candles containing the antifungal drug, 57 colonies were recovered, representing a 91.2% fungal reduction; 18 genera were eliminated. In spite of resistance showed by many Penicillium strains, the imazalil is an alternative drug for the control of fungal colonization on these studied materials.

[Resistance to azolic compounds in clinical Trichophyton spp. strains]. / Resistencia a compuestos azólicos de aislamientos clínicos de Trichophyton spp.

The increase of dermathophytosis in patients with poor therapeutic response leads us to study the antifungal susceptibility of 36 clinical isolates to itraconazole, ketoconazole and fluconazole by the E-test method. According to established parameters by the Clinical Laboratory Standards Institute, the resistance to one or more antifungal drugs was demonstrated in seven isolates (19.4%) as follows: three Trichophyton rubrum, three T. mentagrophytes and one T. tonsurans. A T. rubrum isolate was resistant to the three azolic drugs; the other six only to fluconazole. It is important to establish the antifungal susceptibility as part of the study procedures in patients with dermatophytosis and a poor antifungal response.

[Micosis among five highly underprivileged Mexican communities]. / Micosis observadas en cinco comunidades mexicanas con alto grado de marginación.

BACKGROUND: In many small rural communities in Mexico, medical care is deficient, empirical or absent. OBJECTIVE: In order to improve health coverage in rural areas, the Mexican Institute of Social Security organizes Medical and Surgical Meetings of various specialties including Dermatology and Mycology (MSDM). These include visits to rural hospitals by dermatologists and a mycologist to care for underprivileged communities. In addition to taking samples, they establish the clinical diagnosis and indicate medical and/or surgical treatment, with follow-up visits when needed. MATERIAL AND METHODS: In 2004 and 2005, five MSDM in Chiapas (two), Puebla (one), Michoacán (one) and Oaxaca (one) were organized. Mycoses were within the first four skin pathologies detected. RESULTS: Direct examination with potassium hydroxide led to the diagnosis of mycosis and other skin diseases such as scabies, pediculosis or hair disorders. The sample cultures showed, in addition to common fungi as dermatophytes (Trichophyton rubrum, 19 cases), other uncommon fungal agents such as Trichosporon spp, Chrysosporium spp, Cryptococcus, Geotrichum spp and Aspergillus spp. Most of the candidiasis cases were caused by Candida parapsilosis (nine cases) followed by C. albicans (three cases).

Micosis observadas en cinco comunidades mexicanas con alto grado de marginación / Micosis among five highly underprivileged Mexican communities

Antecedentes. En las pequeñas comunidades rurales de México la atención médica es deficiente, empírica o inexistente. Objetivo. Para mejorar la cobertura de salud en esas zonas, el Instituto Mexicano del Seguro Social (IMSS) organiza Encuentros Médico- Quirúrgicos de varias especialidades, entre ellos, los encuentros Médico- Quirúrgicos en Dermatología (EM-QD). Estos consisten en visitas de trabajo a hospitales rurales por parte de un grupo de especialistas en dermatología y micología durante los cuales se atiende a personas de comunidades marginadas. Además del diagnóstico clínico y toma de muestras se proporciona el tratamiento médico o quirúrgico. Posteriormente se hacen visitas de seguimiento a los pacientes que lo necesiten. Material y métodos. Durante 2004 y 2005 se realizaron cinco EM-QD: Chiapas (dos), Puebla, Michoacán y Oaxaca. Las micosis estuvieron entre las primeras cuatro causas de dermatosis. Resultados. El examen directo con hidróxido de potasio permitió diagnosticar micosis y otras patologías como escabiosis, pediculosis o alteraciones de pelo. El cultivo de las muestras demostró, además de los hongos habituales como los dermatofitos (Trichophyton rubrum 19 aislamientos), otros hongos poco habituales como causa de micosis cutáneas, entre ellos: Trichosporon spp., Chrysosporium spp., Cryptococcus spp., Geotrichum spp., y Aspergillus spp. Las candidosis en su mayoría fueron causadas por C. parapsilosis (9 casos) seguida en frecuencia por C. albicans (3 pacientes).

BACKGROUND: In many small rural communities in Mexico, medical care is deficient, empirical or absent. OBJECTIVE: In order to improve health coverage in rural areas, the Mexican Institute of Social Security organizes Medical and Surgical Meetings of various specialties including Dermatology and Mycology (MSDM). These include visits to rural hospitals by dermatologists and a mycologist to care for underprivileged communities. In addition to taking samples, they establish the clinical diagnosis and indicate medical and/or surgical treatment, with follow-up visits when needed. MATERIAL AND METHODS: In 2004 and 2005, five MSDM in Chiapas (two), Puebla (one), Michoacán (one) and Oaxaca (one) were organized. Mycoses were within the first four skin pathologies detected. RESULTS: Direct examination with potassium hydroxide led to the diagnosis of mycosis and other skin diseases such as scabies, pediculosis or hair disorders. The sample cultures showed, in addition to common fungi as dermatophytes (Trichophyton rubrum, 19 cases), other uncommon fungal agents such as Trichosporon spp, Chrysosporium spp, Cryptococcus, Geotrichum spp and Aspergillus spp. Most of the candidiasis cases were caused by Candida parapsilosis (nine cases) followed by C. albicans (three cases).