Molecular prevalence and genotypes of Enterocytozoon bieneusi in cancer patients under chemotherapy in Aegean region of Türkiye.

BACKGROUND: Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye. METHODS: In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50). RESULTS: Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in Izmir in our previous study. CONCLUSIONS: High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats.

Single-molecule reconstruction of eukaryotic factor-dependent transcription termination.

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.

Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics.

Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared.

Validation of a qualitative real-time PCR assay for the detection of Candida auris in hospital inpatient screening.

Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1-2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings. IMPORTANCE: This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening.

Parvodontia relampaga sp. nov.: A Cystostereaceae fungal pathogen that is the causal agent of relampago blight of woody plants in Florida, USA.

Starting in the fall of 2019, mortality, blight symptoms, and signs of white fungal mycelia were observed on external host tissues of non-native landscape trees as well as numerous native trees, understory shrubs, and vines throughout northern and central Florida, USA. We determined that the fungus is an undescribed species of Basidiomycota based on morphological characteristics and DNA sequence analysis. Phylogenetic analyses of the internal transcribed spacer (ITS), large subunit (LSU), and translation elongation factor 1-alpha (tef1) regions revealed that this novel plant pathogen is an undescribed taxon of the genus Parvodontia (Cystostereaceae, Agaricales). We propose the name Parvodontia relampaga sp. nov. which describes its unique morphological features and phylogenetic placement. We confirmed the pathogenicity of P. relampaga in greenhouse inoculations on host plants from which strains of this novel pathogen were isolated, including the non-native gymnosperm Afrocarpus falcatus, the non-native and commercially important Ligustrum japonicum, and the native tree Quercus hemisphaerica. P. relampaga was also detected on a total of 27 different species of woody host plants, including such economically and ecologically important hosts as Fraxinus, Ilex, Magnolia, Persea, Prunus, Salix, Vitis, and Vaccinium. For this new plant disease, we propose the name "relampago blight," which refers to the lightning-like rhizomorph growth (relámpago means 'lightning' in Spanish). This study presents a newly discovered fungal taxon with a wide host range on both angiosperms and gymnosperms that may be an emerging pathogen of concern in Florida and the Gulf Coast region.

Mrakia polaris sp. nov. and Mrakia amundsenii sp. nov. (Mrakiaceae, Cystofilobasidiales), two novel psychrophilic yeast species isolated from Arctic habitats.

Four yeast strains belonging to the basidiomycetous yeast genus Mrakia were isolated from diverse habitats in the Ny-Ålesund region (Svalbard, High Arctic): two from vascular plants, one from seawater and one from freshwater. Phylogenetic analysis, based on the ITS region and the D1/D2 domain of the 28S rRNA gene, identified these four strains as representing two novel species within the genus Mrakia. The names Mrakia polaris sp. nov. (MycoBank number: MB 852063) and Mrakia amundsenii sp. nov. (MycoBank number: MB 852064) are proposed. These two new species show distinct psychrophilic adaptations, as they exhibit optimal growth at temperatures between 10 and 15°C, while being unable to grow at 25°C. The holotype of M. polaris sp. nov. is CPCC 300345T, and the holotype of M. amundsenii sp. nov. is CPCC 300572T.

Carbon nanoparticle-based lateral flow assay for the detection of specific double-tagged DNA amplicons of Paracoccidioides spp.

To overcome the limitations of current methods for diagnosing paracoccidioidomycosis (PCM), it is critical to develop novel diagnostic strategies that can be implemented in low-resource settings and dramatically improve turnaround times. This study focused on the development of a portable molecular test to screen for Paracoccidioides spp. The proposed approach integrated double-tagging polymerase chain reaction (PCR) and a paper-based lateral flow assay (LFA) for readout, using carbon nanoparticles as a signal generation system. Primers tagged with biotin and digoxigenin were employed to conduct the double-tagging PCR, which can be conveniently carried out on portable thermocyclers. This method can generate billions of tagged DNA copies from a single target molecule, which can be rapidly detected by the LFA platform, providing results within minutes. Avidin-modified carbon nanoparticles served as a signal generation system, enabling detection in the immunochromatographic assay. The LFA demonstrated the capability to detect double-tagged amplicons as low as 0.21 ng or 0.10 ng, depending on whether the results were assessed visually or with a smartphone equipped with an image processor. These findings suggest that the proposed approach holds great promise as a point-of-care diagnostic tool for the early and accurate detection of PCM in low-resource settings. The diagnostic test is rapid and inexpensive, requires minimal handling and can be easily introduced into the general practitioner's armoury for ambulatory screening of infection. This innovative approach has the potential to make a substantial contribution to PCM diagnosis, ultimately reducing morbidity and mortality associated with this disease.

Protocol for studying topological DNA interactions by purified fission yeast condensin.

To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers. For complete details on the use and execution of this protocol, please refer to Tang et al.1.

Dynamics and prognostic value of plasma cell-free DNA PCR in patients with invasive aspergillosis and mucormycosis.

Aspergillus species and Mucorales agents are the primary etiologies of invasive fungal disease (IFD). Biomarkers that predict outcomes are needed to improve care. Patients diagnosed with invasive aspergillosis and mucormycosis using plasma cell-free DNA (cfDNA) PCR were retested weekly for 4 weeks. The primary outcome included all-cause mortality at 6 weeks and 6 months based on baseline cycle threshold (CT) values and results of follow-up cfDNA PCR testing. Forty-five patients with Aspergillus and 30 with invasive Mucorales infection were retested weekly for a total of 197 tests. Using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSG) criteria, 30.7% (23/75), 25.3% (19/75), and 38.7% (29/75) had proven, probable, and possible IFD, respectively. In addition, 97.3% (73/75) were immunocompromised. Baseline CT increased significantly starting at week 1 for Mucorales and week 2 for Aspergillus. Aspergillosis and mucormycosis patients with higher baseline CT (CT >40 and >35, respectively) had a nonsignificantly higher survival rate at 6 weeks, compared with patients with lower baseline CT. Mucormycosis patients with higher baseline CT had a significantly higher survival rate at 6 months. Mucormycosis, but not aspergillosis patients, with repeat positive cfDNA PCR results had a nonsignificantly lower survival rate at 6 weeks and 6 months compared with patients who reverted to negative. Aspergillosis patients with baseline serum Aspergillus galactomannan index <0.5 and <1.0 had significantly higher survival rates at 6 weeks when compared with those with index ≥0.5 and ≥1.0, respectively. Baseline plasma cfDNA PCR CT can potentially be used to prognosticate survival in patients with invasive Aspergillus and Mucorales infections. IMPORTANCE: We show that Aspergillus and Mucorales plasma cell-free DNA PCR can be used not only to noninvasively diagnose patients with invasive fungal disease but also to correlate the baseline cycle threshold with survival outcomes, thus potentially allowing the identification of patients at risk for poor outcomes, who may benefit from more targeted therapies.

Phylogenomic delineation of two new species of ascomycetous yeasts, Wickerhamiella koratensis sp. nov. and Wickerhamiella limtongiae sp. nov., and proposal of two synonyms, Wickerhamiella infanticola and Wickerhamiella tropicalis.

Two novel ascomycetous yeast species of the genus Wickerhamiella are proposed based on isolates obtained in Thailand from food waste and the fruiting body of a polypore fungus, and on a combination of conventional DNA-barcode sequence analyses and whole-genome phylogenies. We focus on a particular subclade of the genus Wickerhamiella that contains species found in anthropic environments and describe Wickerhamiella limtongiae sp. nov. (DMKU-FW31-5T=PYCC 9022T=TBRC 15055T), found on food waste samples. In an adjacent clade, we describe Wickerhamiella koratensis sp. nov. (DMKU-KO16T=PYCC 8908T=TBRC 14869T), which represents the closest relative of Wickerhamiella slavikovae and was isolated from the fruiting body of Bjerkandera sp. In the subclade of W. limtongiae sp. nov., we propose that Wickerhamiella infanticola should be regarded as a synonym of Wickerhamiella sorbophila and that Wickerhamiella tropicalis should be regarded as a synonym of Wickerhamiella verensis.

Performance of Mucorales spp. qPCR in bronchoalveolar lavage fluid for the diagnosis of pulmonary mucormycosis.

To estimate the diagnostic performance of Mucorales polymerase chain reaction (PCR) in Bronchoalveolar lavage fluid (BALF) in routine practice. This was a single-center retrospective study including all consecutive patients >18 years who underwent Mucorales PCR assay in BALF between January 2021 and May 2022. Index testing was prospectively performed using the MycoGENIE Aspergillus spp.-Mucorales spp. PCR. The reference was the diagnosis of pulmonary mucormycosis by the Adjudication Committee. Mucorales PCR in BALF was performed for 938 patients and was positive for 21 of 938 (2.2%). Eleven pulmonary mucormycosis (including one disseminated) were diagnosed. Among them, one (9.1%) was classified as proven mucormycosis, three (27.3%) as probable, and seven (63.6%) as possible according to the EORTC/MSGERC 2019 criteria. The main host factor was hematological malignancy (10 of 11, 90.9%). Mucorales PCR was positive in serum for eight patients (72.7%). Three patients had positive PCR in BALF, but negative in serum. The mean cycle threshold value was significantly lower in mucormycosis than false-positive cases. Sensitivity was 72.7% (95% confidence interval [CI], 43.4-90.3%), and specificity was 98.6% (95% CI, 97.6-99.2%). The positive and negative predictive values were 38.1% (95% CI, 20.8-59.1%) and 99.7% (95% CI, 99.1-99.9%), respectively. Mucorales PCR in BALF showed good diagnostic performance for mucormycosis, particularly in combination with serum PCR. A positive result should be interpreted with caution, given the possibility of carriage in the airway. However, its high negative predictive value and specificity suggest the utility of Mucorales PCR in BALF in the diagnosis of pulmonary mucormycosis.

Morphological and phylogenetic analyses reveal a new species of Anthracophyllum (Omphalotaceae, Agaricales) in Zhejiang Province, China.

During the investigations of macrofungi resources in Zhejiang Province, China, an interesting wood rot fungus was collected. Based on morphological and molecular phylogenetic studies, it is described as a new species, Anthracophyllum sinense. A. sinense is characterized by its sessile, charcoal black and pleurotoid pileus, sparse lamellae occasionally branching, clavate basidia with long sterigmata [(3-)6-7(-8) µm], and non-heteromorphous cystidia. A. sinense establishes a separate lineage close to A. archeri and A. lateritium in the phylogenetic tree.

Genetic analysis of emerging fungal pathogens: Trichosporon asahii.

Trichosporon asahii is an emerging opportunistic fungus that mainly causes fatal disseminated trichosporonosis, especially in immunocompromised patients. T. asahii infection has been reported in Thailand, but few studies of this fungus have been published. Therefore, this study investigated the genetic diversity of 51 clinical strains of T. asahii from urine samples in Thailand. We sequenced and characterized the beta-1-tubulin (TUB1), copper-exporting ATPase (ATP), phosphate carrier protein (PHCP), and topoisomerase-1 (TOP1) genes. In addition, intergenic spacer 1 (IGS1) sequences from our previous studies were investigated. The numbers of haplotypes were 3, 3, 2, 2, and 2 for IGS1, TUB1, ATP, PHCP, and TOP1, respectively. The results suggested a relatively low level of genetic diversity among the strains. The findings illustrated that IGS1, TUB1, ATP, PHCP, and TOP1 can be collectively used as an alternative molecular typing tool for investigating the population diversity and structure of T. asahii.

Taxonomic and phylogenetic contributions to Fuscoporia (Hymenochaetales, Basidiomycota): two new species from Hawaii with a key to North American species.

Fuscoporia is a cosmopolitan, poroid, wood-decaying genus, belonging to the Hymenochaetales. During a study of wood-inhabiting fungi in the USA, four unknown specimens were collected from Hawaii. Both morphological criteria and molecular genetic analyses based on the ITS+nLSU+EF1-α datasets and the nLSU dataset confirmed that these four specimens represent two new species of Fuscoporia, and they are described as F. hawaiiana and F. minutissima. Fuscoporia hawaiiana is characterized by pileate basidiocarps, the absence of cystidioles, hooked hymenial setae, broadly ellipsoid to subglobose basidiospores measuring 4-6 × 3.5-4.5 µm. Fuscoporia minutissima is distinguished by small pores (10-13 per mm) and basidiospores (3.4-4 × 2.4-3 µm). The taxonomic status of the two new species is briefly discussed. A key to the North American species of Fuscoporia is provided.

Phylogeny and diversity of Rigidoporus (Hymenochaetales, Basidiomycota), including three new species from Asia.

Phylogenetic and morphological analyses on Rigidoporus were carried out. The genus Rigidoporus (Hymenochaetales, Basidiomycota), typified by R. microporus (Fr.) Overeem. (synonym Polyporus micromegas Mont.), was established by Murrill in 1905. The genus is mainly characterized by annual to perennial, resupinate, effused-reflexed to pileate or stipitate basidiomata with azonate or concentrically zonate and sulcate upper surface, a monomitic to pseudo-dimitic hyphal structure, simple-septate generative hyphae, and ellipsoid to globose basidiospores. Phylogeny on species of the genus is reconstructed with two loci DNA sequences including the internal transcribed spacer regions and the large subunit. Three new species in Rigidoporus are described and illustrated from Asia, and one new combination in the genus is proposed. The main morphological characteristics of the currently accepted species of Rigidoporus are provided.

Two new species of Scytinostroma (Russulales, Basidiomycota) in Southwest China.

Two new species of Scytinostroma viz. S. acystidiatum and S. macrospermum, are described from southwest China. Phylogeny based on ITS + nLSU dataset demonstrates that samples of the two species form two independent lineages and are different in morphology from the existing species of Scytinostroma. Scytinostroma acystidiatum is characterized by resupinate, coriaceous basidiomata with cream to pale yellow hymenophore, a dimitic hyphal structure with generative hyphae bearing simple septa, the absence of cystidia, and amyloid, broadly ellipsoid basidiospores measuring 4.7-7 × 3.5-4.7 µm. Scytinostroma macrospermum is characterized by resupinate, coriaceous basidiomata with cream to straw yellow hymenophore, a dimitic hyphal structure with generative hyphae bearing simple septa, numerous cystidia embedded or projecting from hymenium, and inamyloid, ellipsoid basidiospores measuring 9-11 × 4.5-5.5 µm. The differences between the new species and morphologically similar and phylogenetically related species are discussed.

Cystofilobasidium josepaulonis sp. nov., a novel basidiomycetous yeast species.

A yeast strain belonging to the basidiomycetous yeast genus Cystofilobasidium was isolated from a marine sediment sample collected in an intertidal zone in Shandong province, PR China. The results of phylogenetic analyses based on sequences of the D1/D2 domain of the 26S ribosomal RNA gene and the internal transcribed spacer (ITS) region indicate that this strain, together with three other strains isolated from basal ice collected in Norway, the gut of an insect and an alga collected in Russia, represent a novel species of the genus, for which the name Cystofilobasidium josepaulonis sp. nov. (holotype strain CGMCC 2.6672T) is proposed. The novel species differs from the known species of the genus Cystofilobasidium by 1.7 %-4.1 and 11.3 %-17.1 % mismatches in the D1/D2 domain and the ITS region, respectively. This species forms teliospores on potato dextrose agar (PDA) and 10 % V8 juice agar, but teliospore germination with basidia was not observed.

The Smc5/6 complex is a DNA loop-extruding motor.

Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes1. Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown2. Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes.

Two Novel Species of Mesophoma gen. nov. from China.

During an investigation of the fungal pathogens associated with the invasive weed Ageratina adenophora from China, some interesting isolates were obtained from healthy leaf, leaf spot, and roots of this weed. Among them, a novel genus Mesophoma, containing two novel species M. speciosa and M. ageratinae, was found. Phylogenetic analysis of the combined, the internal transcribed spacer (ITS), large nuclear subunit ribosomal DNA (LSU), the RNA polymerase II second largest subunit (rpb2), and the partial ß-tubulin (tub2) sequences, showed that M. speciosa and M. ageratinae formed a distinct clade far from all genera previously described in the family Didymellaceae. Combined distinctive morphological characters, including smaller and aseptate conidia when comparing with nearby genera Stagonosporopsis, Boeremia, and Heterphoma, allowed us to describe them as novel species belonging to a novel genus Mesophoma. The full descriptions, illustrations, and a phylogenetic tree showing the position of both M. speciosa and M. ageratinae are provided in this paper. Moreover, the potential for two strains belonging to these two species to be developed into a biocontrol for the spread of the invasive weed Ag. adenophora is also discussed.