A Second Fungal Outbreak in Castañar Cave, Spain, Discloses the Fragility of Subsurface Ecosystems.

Castañar is a cave with strict visitor control measures since it was open to public visits in 2003. However, in recent years, the cave suffered two fungal outbreaks, the first in 2008 and controlled by cleaning the contaminated sediments and subsequent closure of the cave until 2014. The cave was reopened but limited to a maximum of 450 visitors/year. Despite these restrictions on visit, the cave experienced a second outbreak in 2021, originating from the installation of a steel grating walkway, aiming at protecting the ground sediments from the visitors' footsteps. Here, we conducted an analysis using Next-Generation Sequencing and culture-dependent techniques to investigate the fungal communities related to the second outbreak and compare with those present before the cave suffered the outbreak. The results show that the most abundant fungi involved in the 2021 outbreak were already detected in 2020, and even in 2008 and 2009, although the main species that originating both outbreaks were different, likely due to the different carbon sources introduced into the cave.

Total neoadjuvant therapy in rectal cancer: the evidence and expectations.

Current management of locally advanced rectal cancer achieves high cure rates, distant metastatic spread being the main cause of patients' death. Total neoadjuvant therapy (TNT) employs (chemo)radiotherapy and combined chemotherapy prior to surgery to improve the treatment outcomes. TNT has been shown to reduce significantly distant metastases, increase disease-free survival by 5 - 10% in 3 years, and finally also overall survival (≈ 5% in 7 years). It proved to double the rate of pathologic complete responses, making it an attractive strategy for non-operative management to avoid permanent colostomy in patients with distal tumors. In addition, it endorses adherence to the therapy due to better tolerance and, potentially, shortens its overall duration. A number of questions related to TNT remain currently unresolved including the indications, preferred radiotherapy and chemotherapy regimens, their sequence, timing of surgery, and role of adjuvant therapy. A stratified approach may be the optimal way to go.

Higher white-nose syndrome fungal isolate yields from UV-guided wing biopsies compared with skin swabs and optimal culture media.

BACKGROUND: North American bat populations have suffered severe declines over the last decade due to the Pseudogymnoascus destructans fungus infection. The skin disease associated with this causative agent, known as white-nose syndrome (WNS), is specific to bats hibernating in temperate regions. As cultured fungal isolates are required for epidemiological and phylogeographical studies, the purpose of the present work was to compare the efficacy and reliability of different culture approaches based on either skin swabs or wing membrane tissue biopsies for obtaining viable fungal isolates of P. destructans. RESULTS: In total, we collected and analysed 69 fungal and 65 bacterial skin swabs and 51 wing membrane tissue biopsies from three bat species in the Czech Republic, Poland and the Republic of Armenia. From these, we obtained 12 viable P. destructans culture isolates. CONCLUSIONS: Our results indicated that the efficacy of cultures based on wing membrane biopsies were significantly higher. Cultivable samples tended to be based on collections from bats with lower body surface temperature and higher counts of UV-visualised lesions. While cultures based on both skin swabs and wing membrane tissue biopsies can be utilised for monitoring and surveillance of P. destructans in bat populations, wing membrane biopsies guided by UV light for skin lesions proved higher efficacy. Interactions between bacteria on the host's skin also appear to play an important role.

Diversity of Microfungi in a High Radon Cave Ecosystem.

Castañar Cave is a clear example of an oligotrophic ecosystem with high hygrothermal stability both seasonal and interannual and the particularity of registering extraordinary levels of environmental radiation. These environmental conditions make the cave an ideal laboratory to evaluate both the responses of the subterranean environment to sudden changes in the matter and energy fluxes with the exterior and also any impact derived from its use as a tourist resource under a very restrictive access regime. In 2008, a fungal outbreak provoked by a vomit contaminated the sediments which were removed and subsequently treated with hydrogen peroxide. Fungal surveys were carried out in 2008 and 2009. The visits were resumed in 2014. Here, 12 years after the outbreak, we present an exhaustive study on the cave sediments in order to know the distribution of the different fungal taxa, as well as the prevalence and spatio-temporal evolution of the fungi caused by the vomit over the years under the conditions of relative isolation and high radiation that characterize this cave.

Development of a new LC-MS method for accurate and sensitive determination of 33 pyrrolizidine and 21 tropane alkaloids in plant-based food matrices.

Setting of maximum limits for a number of plant alkaloids is under discussion in the EU. The novel method developed and optimized in this study enables simultaneous determination of 21 tropane alkaloids (TAs) and 33 pyrrolizidine (PAs) together with their N-oxides (PANOs). For analysis of aqueous-methanolic extract, reversed phase ultra-high-performance liquid chromatography and tandem mass spectrometry (RP-U-HPLC-MS/MS) was employed. The method was validated for frequently contaminated matrices (i) sorghum, (ii) oregano, and (iii) mixed herbal tea. The recoveries at two spiking levels were in the range of 82-115%, 80-106%, and 78-117%, respectively, and repeatabilities were less than 19% for all analyte/matrix combinations. As regards the achieved limits of quantification (LOQ), their values were in the range of 0.5-10 µg kg-1. The crucial problem encountered during method development, co-elution of multiple groups of isomeric alkaloids, was overcome by subsequent sample separation in the second chromatographic system, hydrophilic interaction liquid chromatography (HILIC), providing different separation selectivity. Lycopsamine, echinatine, and indicine (co-elution group 1) and N-oxides of indicine and intermedine (co-elution group 2), which could not be resolved on the commonly used RP column, were possible to separate fully by using the HILIC system.

Liquid chromatography-drift tube ion mobility-mass spectrometry as a new challenging tool for the separation and characterization of silymarin flavonolignans.

Silymarin, milk thistle (Silybum marianum) extract, contains a mixture of mostly isomeric bioactive flavonoids and flavonolignans that are extensively studied, especially for their possible liver-protective and anticancer effects. Because of the differing bioactivities of individual isomeric compounds, characterization of their proportion in a mixture is highly important for predicting its effect on health. However, because of silymarin's complexity, this is hardly feasible by common analytical techniques. In this work, ultraperformance liquid chromatography coupled with drift tube ion mobility spectrometry and quadrupole time-of-flight mass spectrometry was used. Eleven target silymarin compounds (taxifolin, isosilychristin, silychristins A and B, silydianin, silybins A and B, 2,3-cis-silybin B, isosilybins A and B and 2,3-dehydrosilybin) and five unknown flavonolignan isomers detected in the milk thistle extract were fully separated in a 14.5-min analysis run. All the compounds were characterized on the basis of their accurate mass, retention time, drift time, collision cross section and fragmentation spectra. The quantitative approach based on evaluation of the ion mobility data demonstrated lower detection limits, an extended linear range and total separation of interferences from the compounds of interest compared with the traditional approach based on evaluation of liquid chromatography-quadrupole time-of-flight mass spectrometry data. The following analysis of a batch of milk thistle-based food supplements revealed significant variability in the silymarin pattern, especially in the content of silychristin A and silybins A and B. This newly developed method might have high application potential, especially for the characterization of materials intended for bioactivity studies in which information on the exact silymarin composition plays a crucial role. Graphical Abstract.

Poor chemical and microbiological quality of the commercial milk thistle-based dietary supplements may account for their reported unsatisfactory and non-reproducible clinical outcomes.

Herbal-based dietary supplements have become increasingly popular. The extract from milk thistle (Silybum marianum), is often used for the treatment of liver diseases. However, serious concerns exist regarding the efficacy, composition, as well as the safety of these over-the-counter preparations. Therefore, the aim of the present study was to investigate the composition as well as chemical and biological safety of 26 milk thistle-based dietary supplements purchased from both the U.S. and Czech markets between 2016 and 2017. The study was focused on a determination of the composition of active ingredients, as well as analyses of possible contaminants including: mycotoxins, plant alkaloids, and pesticide residues, as well as the microbial purity. High-throughput analyses were performed using advanced U-HPLC-HRMS techniques. Large differences in the silymarin content were observed among individual milk thistle preparations, often in contrast with the information provided by the manufacturers. In addition, substantial inter-batch differences in silymarin content were also demonstrated. In all milk thistle preparations tested, large numbers and high concentrations of mycotoxins and several pesticides, as well as the substantial presence of microbiological contamination were detected, pointing to serious safety issues. In conclusion, our results strongly indicate the need for strict controls of the composition, chemical contaminants, as well as the microbiological purity of commercial milk thistle extracts used for the treatment of liver diseases. Poor definition of these preparations together with contamination by biologically active substances may not only account for the inconsistency of clinical observations, but also be responsible for possible herbal-based dietary supplements-induced liver injury.

Polyphasic data support the splitting of Aspergillus candidus into two species; proposal of Aspergillus dobrogensis sp. nov.

Aspergillus candidus is a species frequently isolated from stored grain, food, indoor environments, soil and occasionally also from clinical material. Recent bioprospecting studies highlighted the potential of using A. candidus and its relatives in various industrial sectors as a result of their significant production of enzymes and bioactive compounds. A high genetic variability was observed among A. candidus isolates originating from various European countries and the USA, that were mostly isolated from indoor environments, caves and clinical material. The A. candidus sensu lato isolates were characterized by DNA sequencing of four genetic loci, and agreement between molecular species delimitation results, morphological characters and exometabolite spectra were studied. Classical phylogenetic methods (maximum likelihood, Bayesian inference) and species delimitation methods based on the multispecies coalescent model supported recognition of up to three species in A. candidus sensu lato. After evaluation of phenotypic data, a broader species concept was adopted, and only one new species, Aspergillus dobrogensis, was proposed. This species is represented by 22 strains originating from seven countries (ex-type strain CCF 4651T=NRRL 62821T=IBT 32697T=CBS 143370T) and its differentiation from A. candidus is relevant for bioprospecting studies because these species have different exometabolite profiles. Evaluation of the antifungal susceptibility of section Candidi members to six antifungals using the reference EUCAST method showed that all species have low minimum inhibitory concentrations for all tested antifungals. These results suggest applicability of a wide spectrum of antifungal agents for treatment of infections caused by species from section Candidi.

Cultivable microscopic fungi from an underground chemosynthesis-based ecosystem: a preliminary study.

Movile Cave, a unique groundwater ecosystem in southern Romania, was discovered in 1986. This chemoautotrophic cave contains an abundant and diverse fauna with terrestrial and aquatic invertebrate communities, including 33 endemic species. Since its discovery, studies have focused mainly on cave chemoautotrophic bacteria, while the microfungal community has been largely neglected. In this study, we determined the microfungal species living on various substrates in Movile Cave and compared this spectrum with the mycobiota detected outside the cave (outside air-borne and soil-borne microfungi). To investigate all of the niches, we collected samples for two consecutive years from the dry part of the cave (cave air and sediment, corroded limestone walls, isopod feces, and isopod and spider cadavers) and from the post-siphon part of the cave, i.e., Airbell II (sediment and floating microbial mat). A total of 123 microfungal species were identified from among several hundred isolates. Of these, 96 species were only detected in the cave environment and not outside of the cave, while 90 species were from the dry part of the cave and 28 were from Airbell II. The most diverse genera were Penicillium (at least 18 species) and Aspergillus (14 species), followed by Cladosporium (9 species). Surprisingly, high CFU counts of air-borne microfungi were found inside the cave; they were even higher than outside the cave during the first year of investigation.

A virtual clinical trial comparing static versus dynamic PET imaging in measuring response to breast cancer therapy.

We developed a method to evaluate variations in the PET imaging process in order to characterize the relative ability of static and dynamic metrics to measure breast cancer response to therapy in a clinical trial setting. We performed a virtual clinical trial by generating 540 independent and identically distributed PET imaging study realizations for each of 22 original dynamic fluorodeoxyglucose (18F-FDG) breast cancer patient studies pre- and post-therapy. Each noise realization accounted for known sources of uncertainty in the imaging process, such as biological variability and SUV uptake time. Four definitions of SUV were analyzed, which were SUVmax, SUVmean, SUVpeak, and SUV50%. We performed a ROC analysis on the resulting SUV and kinetic parameter uncertainty distributions to assess the impact of the variability on the measurement capabilities of each metric. The kinetic macro parameter, K i , showed more variability than SUV (mean CV K i = 17%, SUV = 13%), but K i pre- and post-therapy distributions also showed increased separation compared to the SUV pre- and post-therapy distributions (mean normalized difference K i = 0.54, SUV = 0.27). For the patients who did not show perfect separation between the pre- and post-therapy parameter uncertainty distributions (ROC AUC < 1), dynamic imaging outperformed SUV in distinguishing metabolic change in response to therapy, ranging from 12 to 14 of 16 patients over all SUV definitions and uptake time scenarios (p < 0.05). For the patient cohort in this study, which is comprised of non-high-grade ER+ tumors, K i outperformed SUV in an ROC analysis of the parameter uncertainty distributions pre- and post-therapy. This methodology can be applied to different scenarios with the ability to inform the design of clinical trials using PET imaging.

White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America.

A striking feature of white-nose syndrome, a fungal infection of hibernating bats, is the difference in infection outcome between North America and Europe. Here we show high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. Our data provides evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established.

Separation and Identification of Odd Chain Triacylglycerols of the Protozoan Khawkinea quartana and the Mold Mortierella alpina Using LC-MS.

Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-MS/APCI) with reversed- and chiral phases was used for separation of triacylglycerols (TAG) from protozoan and mold. This study describes the separation and identification of odd numbered chains of regioisomers and enantiomers of triacylglycerols from different natural sources, i.e., the protozoan Khawkinea quartana and the mold Mortierella alpina. Using the above-mentioned separation methods and the synthesis of appropriate standards of TAG, we identified regioisomers and enantiomers of both even and odd numbered TAG. The biosynthesis of odd numbered TAG was found to be strictly stereospecific and to depend on the production microorganism, one enantiomer predominating in the protozoan and the other in the mold. It was proved that even numbered TAG are synthesized in a higher optical purity, which can be explained by a higher affinity of acyltransferases to the respective substrate, i.e., to even chain PUFA.

Wax Ester Analysis of Bats Suffering from White Nose Syndrome in Europe.

The composition of wax esters (WE) in the fur of adult greater mouse-eared bats (Myotis myotis), either healthy or suffering from white nose syndrome (WNS) caused by the psychrophilic fungus Pseudogymnoascus destructans, was investigated by high-resolution mass spectrometry analysis in the positive ion mode. Profiling of lipid classes showed that WE are the most abundant lipid class, followed by cholesterol esters, and other lipid classes, e.g., triacylglycerols and phospholipids. WE abundance in non-polar lipids was gender-related, being higher in males than in females; in individuals suffering from WNS, both male and female, it was higher than in healthy counterparts. WE were dominated by species containing 18:1 fatty acids. Fatty alcohols were fully saturated, dominated by species containing 24, 25, or 26 carbon atoms. Two WE species, 18:1/18:0 and 18:1/20:0, were more abundant in healthy bats than in infected ones.

Effect of 18F-FDG uptake time on lesion detectability in PET imaging of early stage breast cancer.

Prior reports have suggested that delayed FDG-PET oncology imaging can improve the contrast-to-noise ratio (CNR) for known lesions. Our goal was to estimate realistic bounds for lesion detectability for static measurements with one to four hours between FDG injection and image acquisition. Tumor and normal tissue kinetic model parameters were estimated from dynamic PET studies of patients with early stage breast cancer. These were used to generate time-activity curves (TACs) out to four hours, for which we assumed both nonreversible and reversible models with different rates of FDG dephosphorylation (k4). For each pair of tumor and normal tissue TACs, 600 PET sinogram realizations were generated, and images were reconstructed using OSEM. Test statistics for each tumor and normal tissue region of interest were output from the computer model observers and evaluated using an ROC analysis with the calculated AUC providing a measure of lesion detectability. For the nonreversible model (k4 = 0), the AUC increased in 11/23 (48%) of patients for one to two hours after the current standard post-radiotracer injection imaging window of one hour. This improvement was driven by increased tumor/normal tissue contrast before the impact of increased noise due to radiotracer decay began to dominate the imaging signal. As k4 was increased from 0 to 0.01 min-1, the time of maximum detectability shifted earlier, as the decreasing FDG concentration in the tumor lowered the CNR. These results imply that delayed PET imaging may reveal low-conspicuity lesions that would have otherwise gone undetected.

Revision of Aspergillus section Flavipedes: seven new species and proposal of section Jani sect. nov.

Aspergillus section Flavipedes contains species found worldwide in soils and rhizospheres, indoor and cave environments, as endophytes, food contaminants and occasionally as human pathogens. They produce many extensively studied bioactive secondary metabolites and biotechnologically relevant enzymes. The taxa were revised based on phylogenetic analysis of sequences from four loci (ß-tubulin, calmodulin, RPB2, ITS rDNA), two PCR fingerprinting methods, micro- and macromorphology and physiology. Section Flavipedes includes three known and seven new species: A. ardalensis, A. frequens, A. luppii, A. mangaliensis, A. movilensis, A. polyporicola and A. spelaeus. The name A. neoflavipes was proposed for Fennellia flavipes a distinct species from its supposed asexual state A. flavipes. Aspergillus iizukae, A. frequens and A. mangaliensis are the most common and widely distributed species, whereas A. flavipes s. str. is rare. A dichotomous key based on the combination of morphology and physiology is provided for all recognized species. Aspergillus section Jani is established to contain A. janus and A. brevijanus, species previously classified as members of sect. Versicolores, Terrei or Flavipedes. This new section is strongly supported by phylogenetic data and morphology. Section Jani species produce three types of conidiophores and conidia, and colonies have green and white sectors making them distinctive. Accessory conidia found in pathogenic A. terreus were found in all members of sects. Flavipedes and Jani. Our data indicated that A. frequens is a clinically relevant and produces accessory conidia during infection.

Molecular characterization of a heterothallic mating system in Pseudogymnoascus destructans, the Fungus causing white-nose syndrome of bats.

White-nose syndrome (WNS) of bats has devastated bat populations in eastern North America since its discovery in 2006. WNS, caused by the fungus Pseudogymnoascus destructans, has spread quickly in North America and has become one of the most severe wildlife epidemics of our time. While P. destructans is spreading rapidly in North America, nothing is known about the sexual capacity of this fungus. To gain insight into the genes involved in sexual reproduction, we characterized the mating-type locus (MAT) of two Pseudogymnoascus spp. that are closely related to P. destructans and homothallic (self-fertile). As with other homothallic Ascomycota, the MAT locus of these two species encodes a conserved α-box protein (MAT1-1-1) as well as two high-mobility group (HMG) box proteins (MAT1-1-3 and MAT1-2-1). Comparisons with the MAT locus of the North American isolate of P. destructans (the ex-type isolate) revealed that this isolate of P. destructans was missing a clear homolog of the conserved HMG box protein (MAT1-2-1). These data prompted the discovery and molecular characterization of a heterothallic mating system in isolates of P. destructans from the Czech Republic. Both mating types of P. destructans were found to coexist within hibernacula, suggesting the presence of mating populations in Europe. Although populations of P. destructans in North America are thought to be clonal and of one mating type, the potential for sexual recombination indicates that continued vigilance is needed regarding introductions of additional isolates of this pathogen.

Soil food web changes during spontaneous succession at post mining sites: a possible ecosystem engineering effect on food web organization?

Parameters characterizing the structure of the decomposer food web, biomass of the soil microflora (bacteria and fungi) and soil micro-, meso- and macrofauna were studied at 14 non-reclaimed 1- 41-year-old post-mining sites near the town of Sokolov (Czech Republic). These observations on the decomposer food webs were compared with knowledge of vegetation and soil microstructure development from previous studies. The amount of carbon entering the food web increased with succession age in a similar way as the total amount of C in food web biomass and the number of functional groups in the food web. Connectance did not show any significant changes with succession age, however. In early stages of the succession, the bacterial channel dominated the food web. Later on, in shrub-dominated stands, the fungal channel took over. Even later, in the forest stage, the bacterial channel prevailed again. The best predictor of fungal bacterial ratio is thickness of fermentation layer. We argue that these changes correspond with changes in topsoil microstructure driven by a combination of plant organic matter input and engineering effects of earthworms. In early stages, soil is alkaline, and a discontinuous litter layer on the soil surface promotes bacterial biomass growth, so the bacterial food web channel can dominate. Litter accumulation on the soil surface supports the development of the fungal channel. In older stages, earthworms arrive, mix litter into the mineral soil and form an organo-mineral topsoil, which is beneficial for bacteria and enhances the bacterial food web channel.

Two new species of the genus Ochroconis, O. lascauxensis and O. anomala isolated from black stains in Lascaux Cave, France.

In the year 2001, some conspicuous black stains appeared on the walls of Lascaux Cave in France, which progressively disseminated throughout the cave. These black stains were so evident by 2007 that they have become one of the cave's major problems. In a mycological study of the black stains, Ochroconis strains were abundant among the isolates and constituted the major group of melanised fungi. Two new species of the genus Ochroconis, O. lascauxensis and O. anomala, were isolated and described. The description is based on the morphology of the fungi and the phylogenetic relationships of two of its gene regions internal transcribed spacer (ITS) and RNA polymerase II subunit B (RPB2). In addition, data on their physiology and cellular fatty acid profiles are reported. The development of these species was likely linked to the presence of unusual carbon and nitrogen organic sources provided by the intensive biocide treatments.

Aspergillus baeticus sp. nov. and Aspergillus thesauricus sp. nov., two species in section Usti from Spanish caves.

Two novel species of Aspergillus that are clearly distinct from all known species in section Usti were revealed during a study of microfungal communities in Spanish caves. The novel species identified in this study and additional species of Aspergillus section Usti are associated with places and substrates related to human activities in caves. Novel species are described using data from four loci (ITS, benA, caM and rpb2), morphology and basic chemical and physiological analyses. Members of the species Aspergillus thesauricus sp. nov. were isolated from various substrates, including decaying organic matter, cave air and cave sediment of the Cueva del Tesoro Cave (the Treasure cave); the species is represented by twelve isolates and is most closely related to the recently described Aspergillus germanicus. Members of the species Aspergillus baeticus sp. nov. were isolated from cave sediment in the Gruta de las Maravillas Cave (the Grotto of the Marvels); the species is represented by two isolates. An additional isolate was found in the Cueva del Tesoro Cave and in the Demänovská Peace Cave (Slovakia), suggesting a potentially wide distribution of this micro-organism. The species is related to Aspergillus ustus and Aspergillus pseudoustus. Both species were unable to grow at 37 °C, and a weakly positive, light greenish yellow Ehrlich reaction was observed in A. thesauricus. Unique morphological features alone are sufficient to distinguish both species from related taxa.