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Abstract Background The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immuno-compromised patients. Recently developed high-throughput transcriptome and proteome technologies, such as microarrays, RNA deep-sequencing, and LC-MS/MS of peptide mixtures, are of enormous value for systematically investigating pathogenic organisms. In the field of infection biology, one of the priorities is to collect and standardise data, in order to generate datasets that can be used to investigate and compare pathways and gene responses involved in pathogenicity. The “omics” era provides a multitude of inputs that need to be integrated and assessed. We therefore evaluated the potential of paired-end mRNA-Seq for investigating the regulatory role of the central mitogen activated protein kinase (MpkA). This kinase is involved in the cell wall integrity signalling pathway of A. fumigatus and essential for maintaining an intact cell wall in response to stress. Results The comparison of the transcriptome and proteome of an A. fumigatus wild-type strain with an mpkA null mutant strain revealed that 70.4% of the genome was found to be expressed and that MpkA plays a significant role in the regulation of many genes involved in cell wall remodelling, oxidative stress and iron starvation response, and secondary metabolite biosynthesis. Moreover, absence of the mpkA gene also strongly affects the expression of genes involved in primary metabolism. The data were further processed to evaluate the potential of the mRNA-Seq technique. We comprehensively matched up our data to published transcriptome studies and were able to show an improved data comparability of mRNA-Seq experiments independently of the technique used. Analysis of transcriptome and proteome data revealed only a weak correlation between mRNA and protein abundance. Conclusions High-throughput analysis of MpkA-dependent gene expression confirmed many previous findings that this kinase is important for regulating many genes involved in metabolic pathways. Our analysis showed more than 2000 differentially regulated genes. RNA deep-sequencing is less error-prone than established microarray-based technologies. It also provides additional information in A. fumigatus studies and as a result is more suitable for the creation of extensive datasets.

Fusarium proliferatum, a cosmopolitan saprophytic species, is also known to be a pathogen of garlic. Rot of stored bulbs is an emerging problem that affects Allium sativum L. cultivations worldwide. The presence of the pathogen on plants during the growing season is hard to predict, as infected plants only show yellowing and deterioration of leaves, that can be easily confused with the normal senescence phenomenon. After harvest, during the conservation stage, bulbs undergo a slow deterioration process, caused by the pathogen. Bulbs appear emptied and softened, under the sheath cloves present brown necrotic polygonal spots with a spongy centre, possibly evolving in black depressed lesions. During 2012, we performed a mycological screening in order to evaluate the presence of F. proliferatum on asymptomatic garlic plants growing in the north-east of Italy. Fields were chosen for their different agronomic conditions. Field samples consisted in whole plants eradicated with roots and bulbs. Fungal colonies morphologically resembling F. proliferatum were recovered from all the bulbs examined. The morphological identification was confirmed by Translation Elongation Factor 1-alpha (TEF) gene sequencing. The high frequency of recovery of F. proliferatum suggests a systemic infection before the harvesting stage. Moreover, the presence of this fungus is clearly not correlated to the different agronomic practices; this finding suggest a possible role of the reproduction material in the pathogen spread. [ABSTRACT FROM AUTHOR]

Trichoderma atroviride is a mycoparasitic fungus with antagonistic activity against fungal pathogens and is used as a pathogen control agent alternative to synthetic fungicides. Sensing nutrient availability in the environment and adjusting metabolism for optimal growth, development and reproduction is essential for adaptability and is relevant to its mycoparasitic activity. During mycoparasitism, secondary metabolites are produced to weaken the fungal prey and support the attack. Are1-like proteins act as major GATA-type transcription factors in the activation of genes subject to nitrogen catabolite repression. Since the quality and quantity of nitrogen has been proven particularly relevant in remodeling the biosynthesis of secondary metabolites in fungi, we decided to functionally characterize Are1, the ortholog of Aspergillus nidulans AreA, in T. atroviride. We show that the growth of the T. atroviride ∆are1 mutant is impaired in comparison to the wild type on several nitrogen sources. Deletion of are1 enhanced sensitivity to oxidative and cell-wall stressors and altered the mycoparasitic activity. We were able to identify for the first time a link between Are1 and iron homeostasis via a regulatory mechanism that does not appear to be strictly linked to the nitrogen source, but rather to an independent role of the transcription factor., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Nucleoside diphosphate kinases (NDPKs) are encoded by nme genes and exist in various isoforms. Based on interactions with other proteins, they are involved in signal transduction, development and pathological processes such as tumorigenesis, metastasis and heart failure. In this study, we report a 1.25 Å resolution structure of human homohexameric NDPK-C bound to ADP and describe the yet unknown complexes formed with GDP, UDP and cAMP, all obtained at a high resolution via X-ray crystallography. Each nucleotide represents a distinct group of mono- or diphosphate purine or pyrimidine bases. We analyzed different NDPK-C nucleotide complexes in the presence and absence of Mg 2+ and explain how this ion plays an essential role in NDPKs' phosphotransferase activity. By analyzing a nucleotide-depleted NDPK-C structure, we detected conformational changes upon substrate binding and identify flexible regions in the substrate binding site. A comparison of NDPK-C with other human isoforms revealed a strong similarity in the overall composition with regard to the 3D structure, but significant differences in the charge and hydrophobicity of the isoforms' surfaces. This may play a role in isoform-specific NDPK interactions with ligands and/or important complex partners like other NDPK isoforms, as well as monomeric and heterotrimeric G proteins. Considering the recently discovered role of NDPK-C in different pathologies, these high-resolution structures thus might provide a basis for interaction studies with other proteins or small ligands, like activators or inhibitors.

Admissions of the elderly related to medication errors are frequent in hospital, more than half would be avoidable, but there is currently no validated method in French to identify them. The objective of this work was to validate the French version of the AT-HARM10 tool in order to use it for patients admitted in our healthcare facilities. The tool has 10 questions. A positive response to any of the first 3 questions identify admissions that are unlikely to be drug-related. A positive response to one of the following 7 questions identify possible medication-related admissions. For semantic and linguistic validation, we performed cross-validation with forward-backward translation. To clinically validate the method, we conducted a retrospective study including patients over 65 admitted to short-stay units (UHCD) and to orthopedic surgery units in two French hospitals. Two hundred and sixty-six (266) patients were included ; 166 patients admitted to UHCD (mean age 86.0±5.7 years; sex ratio 0.66; mean number of drugs prescribed 7.7±3.8) and 100 patients admitted to orthopedic units (mean age 85.2±6.1 years; sex ratio 0.43; mean number of prescribed drugs 6.4±3.6). We identified 55 % of admissions probably related to medication in UHCD and 76 % in orthopedic units (p<0.05). The most represented item was P5 in both groups (Might [side] effects of the medications the patient was taking [prescribed or not prescribed] prior to hospitalization have caused the admission [including over-treatment] ? The validated AT-HARM10 tool is now integrated into our clinical pharmacy practices and medication reviews are offered as a priority to patients admitted for iatrogenic reasons., (Copyright © 2023 Société française de pharmacologie et de thérapeutique. Published by Elsevier Masson SAS. All rights reserved.)

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth., (© 2024. The Author(s).)

The mycoparasitic fungus Trichoderma atroviride is applied in agriculture as a biostimulant and biologic control agent against fungal pathogens that infest crop plants. Secondary metabolites are among the main agents determining the strength and progress of the mycoparasitic attack. However, expression of most secondary metabolism-associated genes requires specific cues, as they are silent under routine laboratory conditions due to their maintenance in an inactive heterochromatin state. Therefore, histone modifications are crucial for the regulation of secondary metabolism. Here, we functionally investigated the role of the class II histone deacetylase encoding gene hda1 of T. atroviride by targeted gene deletion, phenotypic characterization, and multi-omics approaches. Deletion of hda1 did not result in obvious phenotypic alterations but led to an enhanced inhibitory activity of secreted metabolites and reduced mycoparasitic abilities of T. atroviride against the plant-pathogenic fungi Botrytis cinerea and Rhizoctonia solani . The ∆hda1 mutants emitted altered amounts of four volatile organic compounds along their development, produced different metabolite profiles upon growth in liquid culture, and showed a higher susceptibility to oxidative and osmotic stress. Moreover, hda1 deletion affected the expression of several notable gene categories such as polyketide synthases, transcription factors, and genes involved in the HOG MAPK pathway.IMPORTANCEHistone deacetylases play crucial roles in regulating chromatin structure and gene transcription. To date, classical-Zn 2+ dependent-fungal histone deacetylases are divided into two classes, of which each comprises orthologues of the two sub-groups Rpd3 and Hos2 and Hda1 and Hos3 of yeast, respectively. However, the role of these chromatin remodelers in mycoparasitic fungi is poorly understood. In this study, we provide evidence that Hda1, the class II histone deacetylases of the mycoparasitic fungus Trichoderma atroviride , regulates its mycoparasitic activity, secondary metabolite biosynthesis, and osmotic and oxidative stress tolerance. The function of Hda1 in regulating bioactive metabolite production and mycoparasitism reveals the importance of chromatin-dependent regulation in the ability of T. atroviride to successfully control fungal plant pathogens., Competing Interests: The authors declare no conflict of interest.

Background/objectives: Bacterial infections (BIs) are well-recognized precipitants of hepatic encephalopathy (HE). Nevertheless, there is a paucity of data in patients with HE associated with BI. Our aim was to describe clinical characteristics, recurrence, and prognosis of HE in patients with BI., Methods: A prospective study with inclusion of hospitalized cirrhotic patients with BI, followed until discharge, death, or liver transplantation ., Results: 172 patients (age 57 ± 13, model of end-stage liver disease [MELD]-sodium 22 ± 8) were included. Infections were more commonly due to spontaneous bacterial peritonitis and cellulitis (22% and 23%), non-nosocomial (70%), and associated with systemic inflammatory response syndrome and septic shock in 40% and 9%, respectively. HE was diagnosed in 66 patients (grade ≥2 in 58%). In multivariate analysis, MELD-sodium, albumin, and prior HE were associated with HE at diagnosis of BI. Recurrence of HE was diagnosed in 30 patients (median 13 [interquartile range 5-22] days), more commonly manifested as overt HE (90% vs. 60% at first episode, P  = 0.012) and more frequently in patients with hyponatremia (54% vs. 27% for patients without, P < 0.001). In-hospital mortality was 34% and was more common for patients with HE (51% vs. 22%, P < 0.001), irrespective of grade, and for those with recurrence (63% vs. 42%, P < 0.001). In multivariate analysis, HE at diagnosis of infection and MELD-sodium were predictors of mortality., Conclusions: HE is frequent in cirrhotic patients with BI and is associated with severity of liver disease, but not with infection. These patients are at increased risk of short-term HE recurrence, especially those with hyponatremia. The presence and recurrence of HE, independent of severity, are associated with in-hospital mortality., (© 2023 Indian National Association for Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Sastré-Velásquez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Inducible promoters are indispensable elements when considering the possibility to modulate gene expression on demand. Desirable traits of conditional expression systems include their capacity for tight downregulation, high overexpression, and in some instances for fine-tuning, to achieve a desired product's stoichiometry. Although the number of inducible systems is slowly increasing, suitable promoters comprising these features are rare. To date, the concomitant use of multiple regulatable promoter platforms for controlled multigene expression has been poorly explored. This work provides pioneer work in the human pathogenic fungus Aspergillus fumigatus, wherein we investigated different inducible systems, elucidated three candidate promoters, and proved for the first time that up to three systems can be used simultaneously without interfering with each other. Proof of concept was obtained by conditionally expressing three antifungal drug targets within the ergosterol biosynthetic pathway under the control of the xylose-inducible PxylP system, the tetracycline-dependent Tet-On system, and the thiamine-repressible PthiA system. IMPORTANCE In recent years, inducible promoters have gained increasing interest for industrial or laboratory use and have become key instruments for protein expression, synthetic biology, and metabolic engineering. Constitutive, high-expressing promoters can be used to achieve high expression yields; however, the continuous overexpression of specific proteins can lead to an unpredictable metabolic burden. To prevent undesirable effects on the expression host's metabolism, the utilization of tunable systems that allow expression of a gene product on demand is indispensable. Here, we elucidated several excellent tunable promoter systems and verified that each can be independently induced in a single strain to ultimately develop a unique conditional multigene expression system. This highly efficient, modular toolbox has the potential to significantly advance applications in fundamental as well as applied research in which regulatable expression of several genes is a key requirement.

Background and Aims: Patients with decompensated cirrhosis are at increased risk of mortality, even in absence of ACLF. The CLIF-C AD score (CLIF-C ADs) was proposed as a prognostic score but lacks sufficient validation. Our aim was to describe clinical characteristics and hospital evolution according to score groups and evaluate prognostic capability of CLIF-C ADs alone or in combination with other scores., Methods: Two hundred and sixty-six patients (55 ± 14 years, ascites in 63%, MELD 14 ± 5) were included, and classified as high, intermediate and low CLIF-C ADs in 13, 60 and 27% of cases. Development of new complications of cirrhosis during hospitalization and survival at 3 months were evaluated., Results: Patients with high CLIF-C ADs had more severe systemic inflammation parameters and higher frequency of organ dysfunction. CLIF-C ADs ≥ 60, when compared to intermediate and low groups, was associated with higher incidence of complications of cirrhosis (90% vs 70% and 49%, p < 0.001) and lower survival (93%, 80% and 50%, p < 0.0001). In multivariate analysis, CLIF-C ADs, ascites and MELD were predictors of survival [(AUROC 0.76 (95% CI 0.69-0.83)]. Absence of ascites or MELD < 14 identified patients with intermediate CLIF-C ADs and good survival (89 and 84%, respectively)., Conclusion: CLIF-C ADs predicts survival in cirrhotic patients with AD. High CLIF-C ADs is associated with higher frequency of organ dysfunction, increased risk of new complications of cirrhosis and high short-term mortality. On the contrary, individuals with low CLIF-C ADs, as well as those with intermediate score without ascites or with low MELD have excellent prognoses., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

The airborne fungus Aspergillus fumigatus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine 161 , sumoylation of lysine 242 and phosphorylation of threonine 319 . All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of hapX caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in A . fumigatus .

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus &nbsp; fumigatus , which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum . This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

The role of liver stiffness measurement (LSM) after sustained virological response (SVR) in HCV patients treated by direct-acting antivirals (DAAs) remains unclear. We aimed to evaluate LSM regression value after SVR and to identify risk factors associated with liver related complications (LRC) or death. This retrospective study analyzed patients with LSM ≥ 10 kPa with LSM by transient elastography pre-DAAs and post-SVR. Patients with previous hepatic decompensation were excluded. Medical records were reviewed to identify primary outcomes. Kaplan-Meier curves and time-to-event Cox proportional-hazard models were performed. 456 patients [65% female, 62 years (IQR 57-68)] were included. During a follow-up of 2.3 years (IQR 1.6-2.7), 28 patients developed 37 outcomes [rate = 29.0 (95% CI 20.0-42.0) per 1000 person-years]. The cumulative incidence of outcomes was significantly lower in patients who regressed LSM ≥ 20% [3.4% (95% CI 1.8-7.0) vs. 9.0% (5.5-14.5), p = 0.028]. In a multivariate Cox-model [HR(95% CI)], male gender [HR = 3.00 (1.30-6.95), p = 0.010], baseline albumin < 3.5 mg/dL [HR = 4.49 (1.95-10.34), p < 0.001] and baseline unfavorable Baveno-VI [HR = 4.72 (1.32-16.83), p = 0.017] were independently associated and LSM regression ≥ 20% after SVR had a trend to reduce the risk of LRC or death [HR = 0.45 (0.21-1.02), p = 0.058]. The use of simple parameters before DAAs and repetition of LSM post-SVR can identify patients with different risks for severe outcome after HCV eradication.

Background: Although recently challenged, systemic inflammatory response syndrome (SIRS) criteria are still commonly used in daily practice to define sepsis. However, several factors in liver cirrhosis may negatively impact its prognostic ability. Goals . To investigate the factors associated with the presence of SIRS, the characteristics of SIRS related to infection, and its prognostic value among patients hospitalized for acute decompensation of cirrhosis. Study . In this cohort study from two tertiary hospitals, 543 patients were followed up, up to 90 days. Data collection, including the prognostic models, was within 48 hours of admission., Results: SIRS was present in 42.7% of the sample and was independently associated with upper gastrointestinal bleeding (UGB), ACLF, infection, and negatively related to beta-blockers. SIRS was associated with mortality in univariate analysis, but not in multiple Cox regression analysis. The Kaplan-Meier survival probability of patients without SIRS was 73.0% and for those with SIRS was 64.7%. The presence of SIRS was not significantly associated with mortality when considering patients with or without infection, separately. Infection in SIRS patients was independently associated with Child-Pugh C and inversely related to UGB. Among subjects with SIRS, mortality was independently related to the presence of infection, ACLF, and Child-Pugh C., Conclusions: SIRS was common in hospitalized patients with cirrhosis and was of no prognostic value, even in the presence of infection., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Ariane Borgonovo et al.)

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We demonstrate that Mucorales fungi produce a toxin, which plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro and prevent hypovolemic shock, organ necrosis and death in mice with mucormycosis. Inhibition of the toxin in Rhizopus delemar through RNA interference compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin ricin, including the ability to inhibit protein synthesis through its N-glycosylase activity, the existence of a motif that mediates vascular leak and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name 'mucoricin' for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin-like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

Background: Few studies have evaluated whether combination and sequential evaluation of ACLF (acute-on-chronic liver failure) and hyponatremia aids prognosis., Aims: Describe clinical course and determine prognostic capability of assessing ACLF and hyponatremia at specific time-points., Methods: Prospective study with inclusion of 376 patients. ACLF and hyponatremia were evaluated at days 1 and 7 and classified as persistent, transient, de novo or absent. Follow-up was 90 days., Results: At inclusion, ACLF was diagnosed in 99 patients. Reversal was observed in 57 patients and was associated with lower creatinine and ACLF grade. De novo ACLF developed in 19 patients, and MELD (model of end-stage liver disease) score and lower albumin were predictive factors. Hyponatremia was present in 76 patients (persistent, transient and de novo in 27, 24 and 25 respectively). ACLF at D7 had the lowest survival compared to transient or no ACLF (21, 57 and 80%, p < 0.0001). Hyponatremia at admission was associated with low survival (35%) whereas survival was higher for de novo or absent cases (70%), p < 0.001. In multivariate analysis ACLF at D7 and hyponatremia at D1 were predictors of survival., Conclusion: ACLF and hyponatremia are dynamic and evaluation of both conditions at different time-points identifies patients at higher risk of short-term mortality., (Copyright © 2019 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

The original version of this Article contained an error in the spelling of the author Emilien Etienne, which was incorrectly given as Emilien Ettiene. These errors have now been corrected in both the PDF and HTML versions of the Article.

Mucormycosis is an aggressive, life-threatening infection caused by fungi in the order Mucorales. The current diagnosis of mucormycosis relies on mycological cultures, radiology and histopathology. These methods lack sensitivity and are most definitive later in the course of infection, resulting in the prevention of timely intervention. PCR-based approaches have shown promising potential in rapidly diagnosing mucormycosis. The spore coating protein homolog encoding CotH genes are uniquely and universally present among Mucorales. Thus, CotH genes are potential targets for the rapid diagnosis of mucormycosis. We infected mice with different Mucorales known to cause human mucormycosis and investigated whether CotH could be PCR amplified from biological fluids. Uninfected mice and those with aspergillosis were used to determine the specificity of the assay. CotH was detected as early as 24 h postinfection in plasma, urine, and bronchoalveolar lavage (BAL) samples from mice infected intratracheally with Rhizopus delemar , Rhizopus oryzae , Mucor circinelloides , Lichtheimia corymbifera , or Cunninghamella bertholletiae but not from samples taken from uninfected mice or mice infected with Aspergillus fumigatus Detection of CotH from urine samples was more reliable than from plasma or BAL fluid. Using the receiver operating characteristic method, the sensitivity and the specificity of the assay were found to be 90 and 100%, respectively. Finally, CotH was PCR amplified from urine samples of patients with proven mucormycosis. Thus, PCR amplification of CotH is a promising target for the development of a reliable, sensitive, and simple method of early diagnosis of mucormycosis., (Copyright © 2018 American Society for Microbiology.)

Mucormycosis is a life-threatening respiratory fungal infection predominantly caused by Rhizopus species. Mucormycosis has incompletely understood pathogenesis, particularly how abnormalities in iron metabolism compromise immune responses. Here we show how, as opposed to other filamentous fungi, Rhizopus spp. establish intracellular persistence inside alveolar macrophages (AMs). Mechanistically, lack of intracellular swelling of Rhizopus conidia results in surface retention of melanin, which induces phagosome maturation arrest through inhibition of LC3-associated phagocytosis. Intracellular inhibition of Rhizopus is an important effector mechanism, as infection of immunocompetent mice with swollen conidia, which evade phagocytosis, results in acute lethality. Concordantly, AM depletion markedly increases susceptibility to mucormycosis. Host and pathogen transcriptomics, iron supplementation studies, and genetic manipulation of iron assimilation of fungal pathways demonstrate that iron restriction inside macrophages regulates immunity against Rhizopus. Our findings shed light on the pathogenetic mechanisms of mucormycosis and reveal the role of macrophage-mediated nutritional immunity against filamentous fungi.

We assessed prophylactic or continuous therapy of isavuconazole, posaconazole, or voriconazole in treating pulmonary murine mucormycosis. In the prophylaxis studies, only isavuconazole treatment resulted in significantly improved survival and lowered tissue fungal burden of immunosuppressed mice infected with Rhizopus delemar. In the continuous treatment studies, isavuconazole and posaconazole, but not voriconazole, equally prolonged survival time and lowered tissue fungal burden compared to placebo-treated mice. These results support the use of isavuconazole and posaconazole in prophylaxis treatment., (Copyright © 2017 American Society for Microbiology.)

Melanins play a crucial role in defending organisms against external stressors. In several pathogenic fungi, including the human pathogen Aspergillus fumigatus, melanin production was shown to contribute to virulence. A. fumigatus produces two different types of melanins, i.e., pyomelanin and dihydroxynaphthalene (DHN)-melanin. DHN-melanin forms the gray-green pigment characteristic for conidia, playing an important role in immune evasion of conidia and thus for fungal virulence. The DHN-melanin biosynthesis pathway is encoded by six genes organized in a cluster with the polyketide synthase gene pksP as a core element. Here, cross-species promoter analysis identified specific DNA binding sites in the DHN-melanin biosynthesis genes pksP-arp1 intergenic region that can be recognized by bHLH and MADS-box transcriptional regulators. Independent deletion of two genes coding for the transcription factors DevR (bHLH) and RlmA (MADS-box) interfered with sporulation and reduced the expression of the DHN-melanin gene cluster. In vitro and in vivo experiments proved that these transcription factors cooperatively regulate pksP expression acting both as repressors and activators in a mutually exclusive manner. The dual role executed by each regulator depends on specific DNA motifs recognized in the pksP promoter region., (© 2016 John Wiley & Sons Ltd.)

Mitogen activated protein kinases (MAPKs) are highly conserved in eukaryotic organisms. In pathogenic fungi, their activities were assigned to different physiological functions including drug adaptation and resistance. Aspergillus fumigatus is a human pathogenic fungus, which causes life-threatening invasive infections. Therapeutic options against invasive mycoses are still limited. One of the clinically used drugs is caspofungin, which specifically targets the fungal cell wall biosynthesis. A systems biology approach, based on comprehensive transcriptome data sets and mathematical modeling, was employed to infer a regulatory network and identify key interactions during adaptation to caspofungin stress in A. fumigatus. Mathematical modeling and experimental validations confirmed an intimate cross talk occurring between the cell wall-integrity and the high osmolarity-glycerol signaling pathways. Specifically, increased concentrations of caspofungin promoted activation of these signalings. Moreover, caspofungin affected the intracellular transport, which caused an additional osmotic stress that is independent of glucan inhibition. High concentrations of caspofungin reduced this osmotic stress, and thus decreased its toxic activity. Our results demonstrated that MAPK signaling pathways play a key role during caspofungin adaptation and are contributing to the paradoxical effect exerted by this drug.

The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To study whether N-terminal probrain natriuretic peptide (NT-proBNP) is a short-term independent predictor of both all-cause and cardiovascular (CV) mortality in type 2 diabetic patients and to establish whether albuminuria and C-reactive protein (CRP) affect this relationship., Research Design and Methods: The prospective study included 1,825 type 2 diabetic patients from the population-based cohort of the Casale Monferrato study. CV risk factors, preexisting CVD, and NT-proBNP levels were evaluated at baseline. All-cause and CV mortality were assessed 5.5 years after baseline examination. Multivariate Cox proportional hazards modeling was used to estimate mortality hazard ratios (HRs)., Results: During the follow-up period, 390 people died (175 for CVD) out of 9,101 person-years of observations. A significantly increased mortality risk by quartiles of NT-proBNP was observed (test for trend, P < 0.001). NT-proBN P values >91 pg/mL conferred HRs of 2.05 (95% CI 1.47-2.86) for all-cause and 4.47 (2.38-8.39) for CV mortality, independently of CV risk factors, including CRP and albumin excretion rate (AER). The association was also significant for modest rises in NT-proBNP levels and in patients without microalbuminuria and CVD at baseline (upper quartiles HRs 3.82 [95% CI 1.24-13.75]) and 3.14 [1.00-9.94]). Albuminuria and NT-proBNP had an additive effect on mortality, though the association was stronger for NT-proBNP., Conclusions: NT-proBNP is a strong independent predictor of short-term CV mortality risk in elderly people with type 2 diabetes, including those without preexisting CVD. This association is evident even in people with slightly increased values, is not modified by CRP, and is additive to that provided by AER.

Six species of Anacroneuria are recorded from Parque Nacional do Itatiaia and Bacia do Rio Macaé, Rio de Janeiro State, Brazil including the description of a new species, A. itatiaiensis. Anacroneuria debilis (Pictet) is redescribed from newly collected specimens. Comments on other Anacroneuria species from Rio de Janeiro State, Brazil are also presented.

A semi-intensive wildlife boars farm presented a clinical history of high mortality in 70 - 90 days-old pigs (> 50 %). Two 90 days-old animals with weight loss and wasting were necropsied and the samples tested for PCV2 by polymerase chain reaction (PCR). The genetic material of PCV2 was sequenced and classified into the PCV2a genotype together with PCV2 sequences obtained from samples of Poland, Brazil, Slovenia and Greece wild boars.

Purpose: In renal disease therapy (RDT) patients, high plasma homocysteine (tHcy) is common and high C-reactive protein(CRP) levels can be observed, attributed to the inflammatory process caused by the dialysis itself. Hyperhomocysteinemia and bioincompatibility are considered independent vascular risk factors. This study evaluated the behavior of these parameters in patients undergoing on-line hemodiafiltration (OL-HDF)., Methods: In 56 patients, HDF was performed using high permeability polyamide membranes, exchanging in the post-dilution mode 16-18 L/session of a reinfusate obtained by the on-line system (triple filtration AK200, Gambro). CRP was measured by an immunological method at the start and the end of the session in patients without comorbidities (group 1, n=30)and with inflammatory diseases (group 2, n=26). In 23 of the 56 patients, tHCY was measured (by high performance liquid chromatography (HPLC)) before and after the mid-week session on different schedule of folinic acid, vitamin B12 and vitamin B6., Results: Pre-dialytic CRP was in the normal range in group 1 patients, whereas it was higher in group 2 patients; dialysis did not induce a significant change in either group. The intradialytic percentage tHcy decrease was approximately 50% regardless of the pre-dialytic value, which was significantly different according to the vitamin supplements administered., Conclusions: HDF, as performed in this study, demonstrated biocompatibility and efficient Hcy removal; therefore, it can prevent cardiovascular disease (CVD) in patients on regular extracorporeal dialysis.

Electrolyte and acid-base balance was evaluated in 14 high UF (124 +/- 7 ml/min) hemodiafiltration sessions. The dialysate contained (in mEq/l): Na 138-140, K 2-3, Ca 3.5, Mg 0.5-0.7, Cl 106-110, acetate 38 or acetate 3 and bicarbonate 35-38. The fluid, infused in postdilutional mode, was 23.5 +/- 21 per session (session length 203 +/- 22 minuti), 80% containing Na 138, K 2, Ca 3.5, Mg 1, Cl 109.5, acetate 35 and 20% Na 145, HCO3 100, Cl 45. The balance was: negative for Na (-255 +/- 220 mEq), for K (-74 +/- 22 mEq) and for Mg (-166 +/- 141 mg), positive for Ca (215 +/- 147 mg) and for acetate (590 +/- 15 and 966 +/- 412 mmol); the electrolytes and bicarbonate plasma values were within of close to normal limits during the session. An unphysiological feature was the positive balance of acetate which, though, was metabolized during the interdialytic period as to return to normal predialytic values. Therefore, in high UF HDF, the above combination of dialysate and reinfusate allows a reasonable electrolyte and acid-base balance; however, bicarbonate should be the only buffer in order to avoid unphysiological levels of other buffers in the biological fluids.