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Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens -mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of A. nidulans . Using suitably engineered binary vectors, gene-targeted ATMT of A. nidulans non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the Δ nkuA host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the wA locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted A. nidulans transformants.

Objectives: This study sought to clinically validate a novel 3-dimensional (3D) ultrafast cardiac magnetic resonance (CMR) protocol including cine (anatomy and function) and late gadolinium enhancement (LGE), each in a single breath-hold., Background: CMR is the reference tool for cardiac imaging but is time-consuming., Methods: A protocol comprising isotropic 3D cine (Enhanced sensitivity encoding [SENSE] by Static Outer volume Subtraction [ESSOS]) and isotropic 3D LGE sequences was compared with a standard cine+LGE protocol in a prospective study of 107 patients (age 58 ± 11 years; 24% female). Left ventricular (LV) mass, volumes, and LV and right ventricular (RV) ejection fraction (LVEF, RVEF) were assessed by 3D ESSOS and 2D cine CMR. LGE (% LV) was assessed using 3D and 2D sequences., Results: Three-dimensional and LGE acquisitions lasted 24 and 22 s, respectively. Three-dimensional and LGE images were of good quality and allowed quantification in all cases. Mean LVEF by 3D and 2D CMR were 51 ± 12% and 52 ± 12%, respectively, with excellent intermethod agreement (intraclass correlation coefficient [ICC]: 0.96; 95% confidence interval [CI]: 0.94 to 0.97) and insignificant bias. Mean RVEF 3D and 2D CMR were 60.4 ± 5.4% and 59.7 ± 5.2%, respectively, with acceptable intermethod agreement (ICC: 0.73; 95% CI: 0.63 to 0.81) and insignificant bias. Both 2D and 3D LGE showed excellent agreement, and intraobserver and interobserver agreement were excellent for 3D LGE., Conclusions: ESSOS single breath-hold 3D CMR allows accurate assessment of heart anatomy and function. Combining ESSOS with 3D LGE allows complete cardiac examination in <1 min of acquisition time. This protocol expands the indication for CMR, reduces costs, and increases patient comfort., Competing Interests: Funding Support and Author Disclosures Funding included Instituto de Salud Carlos III (ISCIII) and the European Regional Development Fund (ERDF) Grants DTS17/00136 to Dr. Ibáñez and PI19/01704 to Dr. Fernandez-Jimenez; Spanish Society of Cardiology Translational Research Grant 2016 to Dr. Ibáñez; European Research Council ERC-CoG 819775-MATRIX to Dr. Ibáñez; Comunidad de Madrid S2017/BMD-3867-RENIM-CM to Drs. Desco and Ibáñez; and Ministerio de Ciencia e Innovación (MICINN) RETOS2019-107332RB-I00 to Dr. Ibáñez. Dr. Fernandez-Jimenez received funding from the European Union Horizon 2020 research and innovation programme under Marie Skłodowska-Curie Hrant Agreement No. 707642. The CNIC is supported by the ISCIII, the MICINN, and the Pro CNIC Foundation. Drs. Fernandez-Jimenez, Nothnagel, Fuster, Ibáñez, and Javier Sánchez-González are inventors of a joint patent (Philips/CNIC) for the new cine imaging method here described and validated/protected under the IP #2014P00960EP. Drs. Nothnagel, Kouwenhoven, Clemence, and Javier Sánchez-González are Philips employees. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulose-degrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5'-CTGGGGTCTGGGG-3' CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A.

l-rhamnose is found in nature mainly as a component of structural plant polysaccharides and can be used as a carbon source by certain microorganisms. Catabolism of this sugar in bacteria, archaea and fungi occurs by two routes involving either phosphorylated or non-phosphorylated intermediates. Unlike the corresponding pathway in yeasts, the metabolic details of the non-phosphorylated pathway in filamentous fungi are not fully defined. The first three genes (lraA, lraB and lraC) of the non-phosphorylated pathway in Aspergillus nidulans have recently been studied revealing dependence on lraA function for growth on l-rhamnose and α-l-rhamnosidase production. In the present work, two genes encoding the subsequent steps catalysed by l-2-keto-3-deoxyrhamnonate (l-KDR) aldolase (AN9425) and l-lactaldehyde dehydrogenase (AN0554) are identified. Loss-of-function mutations cause adverse growth effects on l-rhamnose. Akin to genes lraA-C and those encoding rhamnosidases (rhaA, rhaE), their expression is induced on l-rhamnose via the transcriptional activator RhaR. Interestingly, the aldolase belongs to the ftablamily of bacterial l-KDR aldolases (PF03328/COG3836) and not that of yeasts (PF00701/COG0329). In addition, AN0554 corresponds to the previously characterized aldA gene (encodes aldehyde dehydrogenase involved in ethanol utilization) thus revealing a previously unknown role for this gene in the catabolism of l-rhamnose., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

L-rhamnose (6-deoxy-mannose) occurs in nature mainly as a component of certain plant structural polysaccharides and bioactive metabolites but has also been found in some microorganisms and animals. The release of L-rhamnose from these substrates is catalysed by extracellular enzymes including α-L-rhamnosidases, the production of which is induced in its presence. The free sugar enters cells via specific uptake systems where it can be metabolized. Of two L-rhamnose catabolic pathways currently known in microorganisms a non-phosphorylated pathway has been identified in fungi and some bacteria but little is known of the regulatory mechanisms governing it in fungi. In this study two genes (lraA and lraB) are predicted to be involved in the catabolism of L-rhamnose, along with lraC, in the filamentous fungus Aspergillus nidulans. Transcription of all three is co-regulated with that of the genes encoding α-L-rhamnosidases, i.e. induction mediated by the L-rhamnose-responsive transcription factor RhaR and repression of induction in the presence of glucose via a CreA-independent mechanism. The participation of lraA/AN4186 (encoding L-rhamnose dehydrogenase) in L-rhamnose catabolism was revealed by the phenotypes of knock-out mutants and their complemented strains. lraA deletion negatively affects both growth on L-rhamnose and the synthesis of α-L-rhamnosidases, indicating not only the indispensability of this pathway for L-rhamnose utilization but also that a metabolite derived from this sugar is the true physiological inducer.

Background: Spain is the European country with the highest number of Trypanosoma cruzi infected patients. Due to the cardiac complications that these patients can develop, it is of paramount importance to evaluate the value of the different heart diagnostic tools., Method: In this observational study, we describe the main characteristics and data from electrocardiogram, chest X-ray, echocardiogram and cardiac magnetic resonance imaging (MRI) of 141 patients with Chagas' disease attended in a tertiary university hospital in Madrid from 2009 to 2018., Results: A total of 50 patients (35.4%) had at least one abnormal cardiac test: 34.2% altered electrocardiogram (40/117), 24.5% altered echocardiogram (27/110) and 9.2% abnormal cardiac MRI (13/41). Of those 13 with a pathological MRI, 53.8% had normal results for any other test. The most frequent alterations observed were hypokinesia with decreased LVEF (left ventricular ejection fraction), dilatation of cavities and cardiac fibrosis. Two thirds of patients with abnormal cardiac test were asymptomatic. Altered echocardiogram was found in 43.8% of patients ≥50 years compared to 16.6% under 50 years (p = 0.003)., Conclusions: A transthoracic echocardiogram and a MRI of the heart added a 23.8% increment in diagnosing cardiac pathological findings., (Copyright © 2020. Published by Elsevier Ltd.)

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus., Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli., Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Background/objectives: Current diagnostic criteria for left ventricular non-compaction (LVNC) may result in over-diagnosis of the disease. We evaluate the role of speckle imaging in differential diagnosis of LVNC., Methods and Results: We included all patients who, between January 2012 and May 2015, fulfilled currently accepted criteria for LVNC (28 patients). A control group of 28 healthy individuals and a third group of 13 patients with dilated cardiomyopathy (DCM) were created. Speckle-tracking echocardiography was performed in all groups. Thirteen patients with LVNC had an ejection fraction (EF) <50% (33.5%, SD 10). When compared to controls, patients with LVNC and EF<50% had a larger LV, larger left atrial diameter (LA), reduced e', and reduced global longitudinal strain (GLS). All but one patient with LVNC and EF<50% showed an abnormal LV rotation. This abnormal pattern was observed in 4 LVNC patients (27%) with EF≥50% and in none of the controls. In patients with LVNC, EF ≥50%, and abnormal rotation, GLS was lower than in controls, (-17 (SD 3) vs -21 (SD 3)). Rigid body rotation (RBR) was also observed in 2 DCM patients, with significant differences in EF, GLS, LV diameters relative to the rest of the DCM group., Conclusions: In patients who fulfil the morphologic criteria for LVNC, speckle myocardial imaging techniques could be useful in differentiating between healthy individuals (functionally normal LV) and patients with LVNC (with functional abnormalities in the myocardium in spite of a preserved EF)., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Decreased plasma vitamin D (VD) levels are linked to cardiovascular damage. However, clinical trials have not demonstrated a benefit of VD supplements on left ventricular (LV) remodelling. Anterior ST-elevation acute myocardial infarction (STEMI) is the best human model to study the effect of treatments on LV remodelling. We present a proof-of-concept study that aims to investigate whether VD improves LV remodelling in patients with anterior STEMI., Methods and Analysis: The VITamin D in Acute Myocardial Infarction (VITDAMI) trial is a multicentre, randomised, double-blind, placebo-controlled trial. 144 patients with anterior STEMI will be assigned to receive calcifediol 0.266 mg capsules (Hidroferol SGC)/15 days or placebo on a 2:1 basis during 12 months., Primary Objective: to evaluate the effect of calcifediol on LV remodelling defined as an increase in LV end-diastolic volume ≥10% (MRI)., Secondary Objectives: change in LV end-diastolic and end-systolic volumes, ejection fraction, LV mass, diastolic function, sphericity index and size of fibrotic area; endothelial function; plasma levels of aminoterminal fragment of B-type natriuretic peptide, galectin-3 and monocyte chemoattractant protein-1; levels of calcidiol (VD metabolite) and other components of mineral metabolism (fibroblast growth factor-23 (FGF-23), the soluble form of its receptor klotho, parathormone and phosphate). Differences in the effect of VD will be investigated according to the plasma levels of FGF-23 and klotho. Treatment safety and tolerability will be assessed. This is the first study to evaluate the effect of VD on cardiac remodelling in patients with STEMI., Ethics and Dissemination: This trial has been approved by the corresponding Institutional Review Board (IRB) and National Competent Authority (Agencia Española de Medicamentos y Productos Sanitarios (AEMPS)). It will be conducted in accordance with good clinical practice (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use - Good Clinical Practice (ICH-GCP)) requirements, ethical principles of the Declaration of Helsinki and national laws. The results will be submitted to indexed medical journals and national and international meetings., Trial Registration Number: NCT02548364; Pre-results., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

The β-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 β-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Monoterpenes are important contributors to grape and wine aroma. Moreover, certain monoterpenes have been shown to display health benefits with antimicrobial, anti-inflammatory, anticancer or hypotensive properties amongst others. The aim of this study was to construct self-aromatizing wine yeasts to overproduce de novo these plant metabolites in wines., Results: Expression of the Ocimum basilicum (sweet basil) geraniol synthase (GES) gene in a Saccharomyces cerevisiae wine strain substantially changed the terpene profile of wine produced from a non-aromatic grape variety. Under microvinification conditions, and without compromising other fermentative traits, the recombinant yeast excreted geraniol de novo at an amount (~750 μg/L) well exceeding (>10-fold) its threshold for olfactory perception and also exceeding the quantities present in wines obtained from highly aromatic Muscat grapes. Interestingly, geraniol was further metabolized by yeast enzymes to additional monoterpenes and esters: citronellol, linalool, nerol, citronellyl acetate and geranyl acetate, resulting in a total monoterpene concentration (~1,558 μg/L) 230-fold greater than that of the control. We also found that monoterpene profiles of wines derived from mixed fermentations were found to be determined by the composition of the initial yeast inocula suggesting the feasibility of producing 'à la carte' wines having predetermined monoterpene contents., Conclusions: Geraniol synthase-engineered yeasts demonstrate potential in the development of monoterpene enhanced wines.

Background: Various plant-derived substrates contain L-rhamnose that can be assimilated by many fungi and its liberation is catalyzed by α-L-rhamnosidases. Initial data obtained in our laboratory focussing on two Aspergillus nidulans α-L-rhamnosidase genes (rhaA and rhaE) showed α-L-rhamnosidase production to be tightly controlled at the level of transcription by the carbon source available. Whilst induction is effected by L-rhamnose, unlike many other glycosyl hydrolase genes repression by glucose and other carbon sources occurs in a manner independent of CreA. To date regulatory genes affecting L-rhamnose utilization and the production of enzymes that yield L-rhamnose as a product have not been identified in A. nidulans. The purpose of the present study is to characterize the corresponding α-L-rhamnosidase transactivator., Results: In this study we have identified the rhaR gene in A. nidulans and Neurospora crassa (AN5673, NCU9033) encoding a putative Zn(II)2Cys6 DNA-binding protein. Genetic evidence indicates that its product acts in a positive manner to induce transcription of the A. nidulans L-rhamnose regulon. rhaR-deleted mutants showed reduced ability to induce expression of the α-L-rhamnosidase genes rhaA and rhaE and concomitant reduction in α-L-rhamnosidase production. The rhaR deletion phenotype also results in a significant reduction in growth on L-rhamnose that correlates with reduced expression of the L-rhamnonate dehydratase catabolic gene lraC (AN5672). Gel mobility shift assays revealed RhaR to be a DNA binding protein recognizing a partially symmetrical CGG-X11-CCG sequence within the rhaA promoter. Expression of rhaR alone is insufficient for induction since its mRNA accumulates even in the absence of L-rhamnose, therefore the presence of both functional RhaR and L-rhamnose are absolutely required. In N. crassa, deletion of rhaR also impairs growth on L-rhamnose., Conclusions: To define key elements of the L-rhamnose regulatory circuit, we characterized a DNA-binding Zn(II)2Cys6 transcription factor (RhaR) that regulates L-rhamnose induction of α-L-rhamnosidases and the pathway for its catabolism in A. nidulans, thus extending the list of fungal regulators of genes encoding plant cell wall polysaccharide degrading enzymes. These findings can be expected to provide valuable information for modulating α-L-rhamnosidase production and L-rhamnose utilization in fungi and could eventually have implications in fungal pathogenesis and pectin biotechnology.

Isolated right ventricular (RV) infarction may occur during percutaneous coronary intervention resulting from selective occlusion of a ventricular branch of the right coronary artery (RCA). We present a case of a pure RV infarction without iatrogenic origin that at the initial electrocardiographic analysis was suggestive of a left anterior descending artery-related acute myocardial infarction. Coronary angiography results made us suspect thrombotic occlusion of a small branch of the ectatic RCA resulting from slow flow. Final diagnosis was confirmed by coronary computed tomographic angiography and cardiac magnetic resonance imaging, underlining the essential diagnostic role of these imaging modalities., (Copyright © 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T(73) was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.

Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type., (2010 Elsevier Inc. All rights reserved.)

The Aspergillus nidulans xlnR gene encodes a Zn(2)Cys(6) transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X(22), X(24) and X(34), and beta-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X(22)) and xlnB (encodes X(24)) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creA(d)30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.

Grape musts contain a variety of terpenols that significantly affect wine aroma. The amounts of these metabolites depend on the grape variety, and many cultivars are non-aromatic. Yeasts like Saccharomyces cerevisiae cannot produce and excrete monoterpenes efficiently, mainly due to their lack of monoterpene synthases. By metabolic engineering we have modified the isoprenoid biosynthesis pathway in a wine yeast strain of S. cerevisiae expressing the Clarkia breweri S-linalool synthase gene. Under microvinification conditions, without compromising other desirable and useful fermentative traits, the recombinant yeast efficiently excreted linalool to levels exceeding the threshold of human perception. Bearing in mind the possibility of (co-)expressing other genes that encode enzymes leading to the production of various aroma compounds and the feasibility of controlling the levels of their expression, the potential of this achievement for future genetic manipulation of wine varietal aroma or for use in other alcoholic drinks seems very promising.

The role of DNA regulatory elements mediating activation of the xylanase-encoding gene xyl4 by the transcription factor XlnR in the fungal pathogen Fusarium oxysporum, was studied by in vitro and in vivo functional analysis of the xyl4 promoter. Recombinant XlnR protein specifically bound the sequence GGCTAA in electrophoretic mobility shift assays. Experiments with xyl4 promoter fusions with the lacZ reporter gene showed that the GGCTAA sequence is required for xylan-induced transcriptional activation of xyl4 in F. oxysporum. The results support a model in which the interaction between the transcriptional activator XlnR and an unknown constitutive repressor regulates xylanase gene expression in F. oxysporum.

We present a summary of the most interesting works and commentaries by Dr. Norciso Serrallach Mauri, presented in Urological Records, a journal edited under his direction continuously from 1913 to 1935, annually or every six months. The state-of-the-art urological topics of the time rising the highest interest can be found in the journal, as well as in the publications and activities of the medical societies where he carried out his professional urological activity. We also emphasize the evaluation of his activity in relation to the estate of science and society at his time.

Atherothrombosis (i.e., atherosclerosis and its thrombotic complications) is a systemic disease with local manifestations. Basic understanding of the pathological processes involved in the development and progression of the disease makes it possible to adopt a general approach to early diagnosis with a view to timely treatment. Knowledge of an individual's overall atherosclerotic burden is extremely important, as it enables risk factors to be treated more aggressively. Non-invasive imaging provides a means of quantifying atherosclerotic burden in accessible areas such as the carotid arteries and the aorta. There is some evidence that there is a correlation between atherosclerotic burden in these areas and that in other less accessible areas, such as the coronary arteries. It may be possible, therefore, to obtain an estimate of overall atherosclerotic burden using non-invasive techniques. Novel imaging modalities, such as multidetector computed axial tomography, enable the coronary artery tree to be explored non-invasively, with highly promising results for both diagnosis and screening in high-risk patients. Molecular imaging techniques enable not only the anatomy but also the function of specific tissues and anatomical territories to be studied non-invasively. These new techniques provide highly promising tools for an early diagnosis in high-risk locations.

Acute bacterial prostatitis (ABP) (NIH Category I), has not undergone any modification in the update of prostatitis classification. ABP was diagnosed in 614 patients in our centre over 9 years (1993-2001). We analyse the clinical pattern of ABP and the role of bladder outlet obstruction in its etiology, as well as whether two different ABP sub-categories could be defined as a function of a history of previous manipulation of the lower urinary tract. The results of the study show that the clinical pattern of a patient suffering from ABP does not differ from the statements of previous publications. On the other hand, patients with ABP have been shown to present with no bladder outlet obstruction. Finally, this study has disclosed the fact that the cases of ABP elicited by previous manipulation of the lower urinary tract (10%) show a different pattern from those cases where no previous manipulation has occurred (90%). The patients with ABP secondary to manipulation are older, have a higher risk of prostate abscess and higher frequency of multiple infections and also infections by pathogens other than Escherichia coli. Due to all of these reasons, it would be advisable to subdivide category I within the classification of prostatitis.

End-diastolic volume and end-systolic volume were measured in 35 consecutive patients with cardiomyopathy using 2-dimensional (2-D) and 3-dimensional (3-D) echocardiography (2, 4, and 8 planes) and cardiac magnetic resonance imaging. Three-dimensional echocardiography correlates better with magnetic resonance imaging than does 2-D echocardiography. Its accuracy improves with the increase in the number of planes used. Two-dimensional echocardiography underestimates volumes, mainly in the subgroup with an ejection fraction of <50%, whereas 3-D echocardiography does not, if enough planes are used. However, in patients with an end-diastolic volume > or =150 ml, the underestimation of 3-D echocardiography is statistically significant. Increasing the number of planes to 8 reduces this bias. Conversely, patients with an end-diastolic volume <150 ml are accurately studied with just 4 planes.

Granulocytic sarcoma, or chloroma, is an uncommon presentation of acute leukemia. Cardiac involvement is very rare and usually diagnosed at autopsy. We present a case that discloses the essential role of transesophageal echocardiography for its in vivo diagnosis. The principal features with this imaging technique are finely described.

Introduction and Objectives: Tako-tsubo-like transient left ventricular apical ballooning has been described in Japan, but few cases have been reported in Western countries. We report one of the first series outside Japan, which provides new information on the coronary anatomy of this disorder., Patients and Methods: From January 1998 to February 2003 we observed 11 patients with a clinical suspicion of acute myocardial infarction, normal coronary arteries, and transient tako-tsubo-like systolic left ventricular apical ballooning. We compared the coronary artery anatomy in these 11 patients with that in 44 controls matched for age and sex: 22 with normal coronary arteries and 22 with acute myocardial infarction related with an obstructive thrombus in the left anterior descending coronary artery., Results: As in Japanese patients, tako-tsubo syndrome in Caucasian patients frequently occurs in women in their seventh or eighth decades of life, and is usually preceded by emotional or physical stress. The left anterior descending in our patients with tako-tsubo syndrome was longer overall, and its recurrent diaphragmatic segment was longer, than in controls. To compare these groups we designed a measure termed recurrent segment index (left anterior descending recurrent segment length/total left anterior descending length x 100). In tako-tsubo syndrome this index was 22.3 (1.5)%, vs 10.9 (6.7)% in normal controls (P<.001), and 11.3 (7.7)% in acute myocardial infarction patients (P<.001). Patients with acute myocardial infarction and a high recurrent segment index (> or =16%) had ventriculographic findings of systolic apical ballooning identical to those in patients with tako-tsubo syndrome., Conclusions: All our patients with tako-tsubo syndrome had a left anterior descending with a long recurrent segment. The identical ventriculographic findings in patients with tako-tsubo syndrome and those with acute myocardial infarction with a long recurrent segment may be due to a common etiology.

The Aspergillus aculeatus rhaA gene encoding an alpha-L-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a beta-glucosidase, show increased content mainly of the aromatic compound linalool.

The Phycomyces blakesleeanus wild-type is yellow, because it accumulates beta-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, zeta-carotene and neurosporene, in decreasing amounts, and traces of beta-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase.

Background: Recurrent symptomatic pericardial effusion can complicate different cardiac and extracardiac diseases. When recurrent pericardial effusion after drainage with conventional catheter techniques occurred the creation of a pericardial window by open surgery used to be the unique treatment available until the recent development of percutaneous balloon pericardiotomy., Objective: The aim of this paper is to review our initial experience with percutaneous balloon pericardiotomy for the treatment of patients with recurrent pericardial effusion., Patients and Method: Five patients with recurrent pericardial effusion have been treated with percutaneous pericardiotomy until now. Four patients had malignant pericardial effusion secondary to metastasis of extracardiac tumors, in one patient recurrent pericardial effusion was idiopathic. In all patients percutaneous balloon pericardiotomy was performed with a pediatric valvuloplasty balloon catheter, through a subxiphoid approach., Results: Successful drainage and balloon pericardiotomy was achieved in all patients without severe complications. In all cases only one pericardial site was dilated. Minor complications were registered, which included mainly mild pleural effusion occurring in all patients with spontaneous resolution. During a mean follow-up period of 8.6 6.5 months (range 2 to 18 months) there were no recurrences of effusion or tamponade. Two patients died, 1 month and 9 months after the procedure, due to their malignant condition., Conclusions: Percutaneous balloon pericardiotomy is an easy and useful technique to manage patients with large recurrent pericardial effusion with a low r

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.

The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues approximately 252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA, is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creAd30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA.C1, that is responsible for direct CreA repression in vivo. Using the creAd30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.

The Aspergillus nidulans IPNS gene, encoding isopenicillin N synthetase, is a secondary metabolism gene. It is contiguous to, but divergently transcribed from, the ACVS gene at the penicillin gene cluster. The untranslated region between both ORFs is 872bp long. Here we present the physical and functional characterization of the IPNS transcriptional unit. Transcriptional start point (tsp) mapping reveals heterogeneity at the 5'-end of the mRNA, with a major start at -106 relative to the initiation codon. This indicates that the actual length of the non-transcribed intergenic region is 525bp. Functional elements in the IPNS upstream region have been defined by assaying beta-galactosidase activity in extracts from recombinant strains carrying deletion derivatives of the IPNS promoter fused to lacZ, integrated in single copy at the argB locus. Strains were grown in penicillin production broth under carbon catabolite repressing or derepressing conditions. The results of deletion analysis indicate that: (i) the IPNS promoter is mostly regulated by negative controls that act upon a high basal activity; (ii) sequential deletion of three of the negative cis-acting elements results in a mutated promoter that is 40 times (sucrose broth) or 12 times (lactose broth) more active than the wild type; (iii) one of these negative cis-acting elements is involved in sucrose repression. Strikingly, it is located outside the non-transcribed 525bp intergenic region and maps to the coding region of the divergently transcribed ACVS gene; (iv) a 5'-deletion up to -56 (relative to the major tsp) contains information to provide almost half of the maximal promoter activity and allows initiation of transcription at the correct site. By using total-protein extracts from mycelia grown under penicillin producing conditions we have detected a DNA-binding activity that specifically shifts a promoter fragment located between -654 and -455 (relative to IPNS tsp). Deletions covering this region partially abolish IPNS promoter activity. The fragment in question overlaps the ACVS tsp.

The organization of the quinic acid (qa) genes in Neurospora crassa has been compared to that in several other Neurospora species. This gene cluster was found to be highly conserved in all species examined. However, there are numberous restriction fragment length polymorphisms that distinguish the heterothallic and homothallic species. Catabolic dehydroquinase assays indicated that qa-2 gene expression in the homothallic species is subject to induction by quinic acid, as is the case in N. crassa. The qa-x-qa-2 intergenic region of the homothallic species N. africana was cloned and sequenced. Conserved qa activator (qa-1F) binding sites have been identified in this region. When the qa-x-qa-2 intergenic region of N. crassa was replaced with its N. africana counterpart, qa-2 gene expression was reduced; however repression by glucose appeared normal. Furthermore, the N. africana start site for qa-2 transcription (which differs from the N. crassa start site) was utilized in the transformant. The overall evidence suggests that a weakening of the -120 activator binding site in the qa-x-qa-2 intergenic region may be responsible for these differences.

When sporangiophores of the fungus Phycomyces blakesleeanus adapt from high to low fluence rate, dark adaptation (sensitivity recovery) can be accelerated by dim subliminal light [Galland et al. (1989) Photochem. Photobiol. 49, 485-491]. We measured fluence rate-response curves for this acceleration under the following conditions. After sporangiophores were initially adapted symmetrically to a fluence rate of 1 W m-2 (447 nm), they were exposed to unilateral subliminal light (subthreshold for phototropism) of variable wavelength and fluence rate, and then to unilateral test light (447 nm) of fluence rate either 10(-3) or 10(-5) W m-2. The duration of the subliminal light was chosen so that phototropism would not occur during this period. Phototropic latencies could be shortened by subliminal light that was less intense than the test light by several orders of magnitude. In experiments with the final unilateral light of fluence rate 10(-3) W m-2, the 447 nm subliminal light had a threshold (for the acceleration effect) of about 10(-11) W m-2. Yellow light of wavelength 575 nm, which itself is extremely ineffective for phototropism was extremely effective in shortening phototropic latencies in response in response to the test light. At 575 nm, the threshold was about 2 x 10(-12) W m-2. Conversely, near-UV light of wavelength 347 nm, which is highly effective for phototropism, was relatively ineffective (threshold approximately 7 x 10(-8) W m-2) in shortening the phototropic latency. Our results suggest the presence of a novel yellow-light absorbing pigment in Phycomyces that specifically regulates dark adaptation.(ABSTRACT TRUNCATED AT 250 WORDS)

Complementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.