Search Results (100)

The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein’s N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders. Full article

Background: Optical genome mapping (OGM) detects genome-wide structural variants (SVs), including balanced rearrangements and complex copy-number alterations beyond standard-of-care cytogenomic assays. In chronic lymphocytic leukemia (CLL), cytogenetic and genomic risk stratification is traditionally based on fluorescence in situ hybridization (FISH), karyotyping, targeted next-generation sequencing (NGS), and immunogenetic assessment of immunoglobulin heavy chain variable region (IGHV) somatic hypermutation status, each of which interrogates only a limited aspect of disease biology. Methods: We retrospectively evaluated fifty patients with CLL using OGM and integrated these findings with cytogenomics, targeted NGS, IGHV mutational status, and clinical time-to-first-treatment (TTFT) data. Structural variants were detected using OGM and pathogenic NGS variants were derived from a clinical heme malignancy panel. Clinical outcomes were extracted from the electronic medical record. Results: OGM identified reportable structural variants in 82% (41/50) of cases. The most frequent abnormality was del(13q), observed in 29/50 (58%) and comprising 73% (29/40) of all OGM-detected deletions with pathologic significance. Among these, 12/29 (42%) represented large RB1-spanning deletions, while 17/29 (58%) were focal deletions restricted to the miR15a/miR16-1 minimal region, mapping to the non-coding host gene DLEU2. Co-occurrence of adverse lesions, including deletion 11q/ATM, BIRC3 loss, trisomy 12, and deletion 17p/TP53, were recurrent and strongly associated with shorter TTFT. OGM also uncovered multiple cryptic rearrangements involving chromosomal loci that are not represented in the canonical CLL FISH probe panel, including IGL::CCND1, IGH::BCL2, IGH::BCL11A, IGH::BCL3, and multi-chromosomal copy-number complexity. IGHV data were available in 37/50 (74%) of patients; IGHV-unmutated status frequently co-segregated with OGM-defined high-risk profiles (del(11q), del(17p), trisomy 12 with secondary hits, and complex genomes whereas mutated IGHV predominated in OGM-negative or structurally simple del(13q) cases and aligned with indolent TTFT. Integration of OGM with NGS further improved genomic risk classification, particularly in cases with discordant or inconclusive routine testing. Conclusions: OGM provides a comprehensive, genome-wide view of structural variation in CLL, resolving deletion architecture, identifying cryptic translocations, and defining complex multi-hit genomic profiles that tracked closely with clinical behavior. Combining OGM and NGS analysis refined risk stratification beyond standard FISH panels and supports more precise, individualized management strategies in CLL. Prospective studies are warranted to evaluate the clinical utility of OGM-guided genomic profiling in contemporary treatment paradigms. Full article

A growing body of evidence indicates that artificial manipulation of transcriptional regulation is a powerful approach to activate cryptic biosynthetic gene clusters (BGCs) of secondary metabolites (SMs) in fungi. In this study, one mutant strain MNP-2-OE::veA was constructed by overexpressing the global transcription regulator veA in an Arctic-derived strain Aspergillus sydowii MNP-2. Chemical investigation of the mutant OE::veA resulted in the isolation of one novel polyhydroxy anthraquinone (1) together with nine known metabolites (2–10), which were unambiguously characterized by various spectroscopic methods including 1D and 2D NMR and HR-ESI-MS as well as via comparison with literature data. Biosynthetically, compounds 1 and 10 as new arising chemicals were, respectively, formed by type II polyketide synthase (T2PK) and non-ribosomal peptide synthetase (NRPS), which were silent in the wild-type (WT) strain MNP-2. A bioassay showed that only compound 3 had weak inhibitory effect on human pathogen Candida albicans, with a MIC value of 64 ug/mL, and 4 displayed in vitro weak cytotoxic activity against HCT116 cells (IC₅₀ = 44.47 μM). These results indicate that overexpression of veA effectively awakened the cryptic BGCs in fungal strains and enhanced their structural diversity in natural products. Full article

Background/Objectives: Krabbe disease (KD) is a hereditary lysosomal disorder whose hallmark is progressive demyelination, with variable involvement of the central nervous system. It is caused by pathogenic variants in the GALC gene that disrupt the function of its gene product, the lysosomal enzyme galactosylceramidase. We analyzed two unrelated cases (one early infantile and one adult) with a clinical suspicion of KD. Methods: We used a combination of biochemical techniques (high-performance liquid chromatography–tandem mass spectrometry), NGS (resequencing gene panels), splicing assays, and molecular modeling to identify and analyze the pathogenicity of the variants underlying the disorder. Results: The two probands were compound heterozygotes for disease-causing variants in the GALC gene, encoding the lysosomal hydrolase galactosylceramidase. Three of the variants were novel and caused aberrant splicing, either by exon skipping (c.908+5G>A and c.1034-1G>C) or by inclusion of a cryptic, deep intronic pseudoexon (c.621+772G>C). The fourth variant was a known missense change (c.956A>G, p.(Tyr319Cys)) with conflicting interpretations of pathogenicity in the databases. Conclusions: We demonstrated the pathogenicity of the three novel splicing variants, all with strong impact on galactosylceramidase function. We also concluded that the c.956A>G missense variant is a hypomorph usually underlying the later-onset, milder phenotypes of KD. Our results stress the importance of integrated approaches combining clinical, biochemical, and genetic testing to obtain a definitive diagnosis of lysosomal diseases. Full article

During the COVID-19 pandemic, the global focus on SARS-CoV-2 overshadowed the epidemiology of other respiratory pathogens. This study aimed to characterize the circulation of endemic human coronaviruses (HCoVs) in Brazil. We retrospectively analyzed results from 22,472 PCR tests for HCoVs (from 5183 patients) and 601,278 tests for SARS-CoV-2 (from 475,856 patients) between November 2019 and June 2021. HCoVs were detected in 160 patients (3.09%), with HCoV-NL63 as the most frequent species. HCoV circulation was intermittent, with positivity peaks up to 4% but also periods of up to six months with an absence of detections in 2020, contrasting with the sustained high positivity of SARS-CoV-2 (22.37%). Co-infections were frequent: 26.25% of HCoV-positive patients were co-infected with at least one other respiratory pathogen, most commonly Rhinovirus/Enterovirus, and cases involving up to five pathogens were observed, seven patients had co-infections between HCoVs and SARS-CoV-2. These findings reveal the persistent, often cryptic, circulation of HCoVs during the pandemic and highlight their role as key components in complex multi-pathogen infections. This underscores the critical importance of implementing comprehensive molecular diagnostic panels in routine respiratory surveillance to ensure accurate etiology, guide appropriate clinical management, and fully assess the public health burden of non-SARS-CoV-2 coronaviruses. Full article

Forest diseases threaten tree health, biodiversity, and ecosystem services, with impacts amplified by climate change and global trade. Understanding and managing these threats is difficult due to the longevity of trees, the size and inaccessibility of forests, and the often cryptic or delayed expression of symptoms. This review first introduces the field of forest pathology and the key challenges it faces, including multifactorial declines, root and vascular diseases, and emerging invasive pathogens. We then examine how artificial intelligence (AI) can be applied to biotic, abiotic, and decline-related diseases, integrating remote sensing, imaging, genomics, and ecological data across spatial and temporal scales. Lessons from agricultural systems are discussed, highlighting potential tools and pitfalls for forestry. Finally, we outline future directions, emphasizing the need for interpretable models, incorporation of ecological context, cross-species validation, and coordinated data infrastructures to ensure AI delivers actionable, scalable solutions for complex forest ecosystems. Full article

Background: Genome sequencing is transforming infectious-disease diagnostics, surveillance, and precision therapy by enabling rapid, high-resolution pathogen identification, transmission tracking, and genomic-informed antimicrobial stewardship. Methods: We review contemporary sequencing platforms (short- and long-read), targeted and metagenomic approaches, and operational workflows that connect laboratory outputs to clinical and public health decision-making. We highlight strengths and limitations of genomic AMR prediction, the role of plasmids and mobile elements in resistance and virulence, and practical steps for clinical translation, including validation, reporting standards, and integration with electronic health records. Results: Comparative and population genomics reveal virulence determinants and host–pathogen interactions that correlate with clinical outcomes, improving risk stratification for high-risk infections. Integrating sequencing with epidemiological and clinical metadata enhances surveillance, uncovers cryptic transmission pathways, and supports infection control policies. Despite these advances, clinical implementation faces technical and interpretative barriers, as well as challenges related to turnaround time, data quality, bioinformatic complexity, cost, and ethical considerations. These issues must be addressed to realize routine, point-of-care sequencing. Conclusions: Emerging solutions, including portable sequencing devices, standardized pipelines, and machine-learning models, promise faster, more actionable results and tighter integration with electronic health records. The widespread adoption of sequencing in clinical workflows has the potential to shift infectious disease management toward precision medicine, thereby improving diagnostics, treatment selection, and public health responses. Full article

Sporothrix schenckii is a pathogenic fungus with both clinical and environmental origins that was traditionally described as a single species but is increasingly recognized as being genetically diverse. In this study, we analyzed multiple isolates recovered from human sporotrichosis cases and environmental sources across Latin America (Mexico, Guatemala, Colombia). We conducted a polyphasic analysis of 16 isolates, integrating morphological data with multilocus sequence analysis (MLSA) targeting the internal transcribed spacer (ITS), calmodulin (CAL), β-tubulin (BT2), and translation elongation factor 1-α (TEF) gene regions. Phylogenetic relationships were resolved via maximum likelihood, and genetic structure was corroborated via four independent clustering methods: minimum spanning tree, principal component analysis, multidimensional scaling, and self-organizing maps. MLSA reidentified six isolates as S. globosa and confirmed the absence of S. brasiliensis in the cohort. The remaining S. schenckii s. str. isolates were resolved into three clades (A, B, and C). Notably, clade B (EH748, EH194, and EH257) formed a genetically divergent cluster with the highest nucleotide diversity (π = 0.03556) and was consistently segregated by all clustering algorithms. Clinical and environmental isolates were phylogenetically intermingled, supporting an active environmental reservoir for human infections. Phenotypic data, including colony size and conidial and yeast dimensions, varied but did not clearly distinguish between clinical and environmental origins. Our study provides compelling molecular evidence for a previously unrecognized, highly divergent clade within S. schenckii s. str., indicative of ongoing cryptic speciation. These findings refine the taxonomy of medically important Sporothrix species and reveal a distinct epidemiological profile for sporotrichosis in the studied regions, separate from the S. brasiliensis-driven epizootic. This highlights the critical role of molecular surveillance for accurate diagnosis, treatment, and public health strategies. Full article

Fibrinogen (FIB), a key component of the coagulation cascade, is traditionally recognized for its role in hemostasis and tissue repair. However, due to its high plasma abundance and susceptibility to proteolytic cleavage during inflammation, it may also represent a previously unrecognized source of bioactive peptides. This study presents, for the first time, a comprehensive analysis of the antimicrobial, anti-inflammatory, and antiviral properties of six cationic antimicrobial peptides (AMPs) deriving from the C-terminal extremities of the three subunits of human fibrinogen (FIBα, FIBβ, and FIBγ), identified using a scoring function developed by our group. Antibacterial assays against Gram-positive and Gram-negative pathogens revealed different antimicrobial activity profile depending on their parent protein. Selected peptides displayed additive or synergistic effects when combined with conventional antibiotics or the thrombin-derived peptide (P)GKY20, highlighting their potential for combination therapies. Hemolytic assay confirmed the biocompatibility of fibrinogen-derived cryptic peptides with erythrocytes. Furthermore, the peptides significantly reduced LPS-induced nitric oxide release in murine macrophages Raw 264.7 cells, indicating anti-inflammatory activity. Notably, antiviral activity was observed against enveloped viruses (HCoV-229E and HSV-1) under various treatment conditions, while no activity was detected against the non-enveloped virus CVB3. Overall, these findings reveal human fibrinogen as a source of multifunctional cryptic peptides with broad-spectrum antimicrobial, antiviral, and immunomodulatory activities, supporting their potential as part of the innate immune system. Full article

Invasive Phytophthora species are increasingly impacting UK landscapes. Most recently, cryptic outbreaks of P. pluvialis Reeser, Sutton & E. Hansen have occurred on western hemlock and Douglas fir at several forest sites across Britain. To better understand the ubiquity and life cycle of this pathogen in British forests and the assemblages of co-inhabiting Phytophthora species, metabarcoding and baiting methodologies were applied to soil, stream water, and rainwater samples collected over a full calendar year from seventeen sites across Britain. Thirty-five Phytophthora species were detected across all sites, substrates, and detection methods, with most detections occurring in stream water by metabarcoding. The three most frequently detected species were (1) P. pluvialis, (2) P. gonapodyides H.E. Petersen & Buisman and (3) P. ramorum Werres, De Cock & Man in ‘t’ veld. Other species detected included the regulated pathogens P. austrocedri Greslen & Hansen, P. kernoviae Brasier, Beales & S.A. Kirk and P. lateralis Tucker & Milbraith, as well as P. ornamentata Scanu, Linald & T. Jung, a new species record for the UK. Phytophthora pluvialis was most frequently detected in March, with rainfall trap metabarcoding data suggesting that aerial dissemination occurs predominantly in late winter/early spring. Consistent detections of P. pluvialis in soil by metabarcoding indicate the potential for soilborne transfer of this pathogen by animal or human vectors, including equipment or machinery in forest operations. The study’s findings are discussed in relation to understanding how P. pluvialis spreads and the approaches needed to address key knowledge gaps in relation to inoculum sources. The results provide a baseline for Phytophthora diversity in British commercial forests, facilitating a greater understanding of typical and unusual trends in species assemblages. This study also consolidates the value of metabarcoding as an effective surveillance tool for Phytophthora in commercial forests. Full article

Fruit bats in the genus Pteropus are the natural reservoirs for zoonotic paramyxoviruses, notably henipaviruses and pararubulaviruses, which are found across Southeast Asia and Oceania. The genetic and antigenic diversity of viruses in both genera, and region specificity, are ill-defined, limiting health security measures aimed at minimizing spillover. For example, Nipah virus has been isolated from bats in the Battambang province of western Cambodia, and surveys suggest bat foraging behaviors occur in close proximity to human settlements. However, there have been no historical cases of Nipah virus in Cambodia. Here, we use a multiplex microsphere immunoassay to identify cryptic human exposure to selected henipaviruses and pararubulaviruses in Cambodia. Convalescent human sera from persons presenting with acute respiratory illness were screened to detect the presence or absence of antibodies reactive with attachment glycoprotein antigens from Nipah virus, Hendra virus, Cedar virus, and Ghana virus, and a hemagglutinin-neuraminidase antigen from Menangle virus. In this sero-survey, we detected antibodies that were specifically reactive with Cedar virus and Menangle virus, including one serum sample that neutralized a recombinant Cedar virus. Additionally, we detected a pattern of cross-reactivity with Hendra virus, Cedar virus, and Ghana virus, suggesting previous infection by an antigenically-related henipavirus. We did not detect high antibody reactivity with the NiV glycoprotein. Future studies should expand serological surveillance for these transboundary pathogens, including genetic surveillance to aid in henipavirus discovery, and focused biosurveillance where interfaces with livestock and humans occur. Full article

Early and accurate detection of plant diseases is critical for ensuring global food security and agricultural resilience. Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is among the most economically significant diseases of sugarcane worldwide. Its cryptic nature—characterized by an absence of visible symptoms—renders timely diagnosis particularly difficult, contributing to substantial undetected yield losses across major sugar-producing regions. Here, we report the development of a potential-induced electrochemical (EC) nanobiosensor platform for the rapid, low-cost, and field-deployable detection of Lxx DNA directly from crude sugarcane sap. This method eliminates the need for conventional nucleic acid extraction and thermal cycling by integrating the following: (i) a boiling lysis-based DNA release from xylem sap; (ii) sequence-specific magnetic bead-based purification of Lxx DNA using immobilized capture probes; and (iii) label-free electrochemical detection using a potential-driven DNA adsorption sensing platform. The biosensor shows exceptional analytical performance, achieving a detection limit of 10 cells/µL with a broad dynamic range spanning from 10⁵ to 1 copy/µL (r = 0.99) and high reproducibility (SD < 5%, n = 3). Field validation using genetically diverse sugarcane cultivars from an inoculated trial demonstrated a strong correlation between biosensor signals and known disease resistance ratings. Quantitative results from the EC biosensor also showed a robust correlation with qPCR data (r = 0.84, n = 10, p < 0.001), confirming diagnostic accuracy. This first-in-class EC nanobiosensor for RSD represents a major technological advance over existing methods by offering a cost-effective, equipment-free, and scalable solution suitable for on-site deployment by non-specialist users. Beyond sugarcane, the modular nature of this detection platform opens up opportunities for multiplexed detection of plant pathogens, making it a transformative tool for early disease surveillance, precision agriculture, and biosecurity monitoring. This work lays the foundation for the development of a universal point-of-care platform for managing plant and crop diseases, supporting sustainable agriculture and global food resilience in the face of climate and pathogen threats. Full article

Aquatic fungi serve as core ecological engines in freshwater ecosystems, driving organic matter decomposition and energy flow to sustain environmental balance. Wetlands, with their distinct hydrological dynamics and nutrient-rich matrices, serve as critical habitats for these microorganisms. As an internationally designated Ramsar Site, Yunnan Dashanbao Black-Necked Crane National Nature Reserve in China not only sustains endangered black-necked cranes but also harbors a cryptic reservoir of aquatic fungi within its peat marshes and alpine lakes. This study employed high-throughput sequencing to characterize fungal diversity and community structure across 12 understudied wetland sites in the reserve, while analyzing key environmental parameters (dissolved oxygen, pH, total nitrogen, and total phosphorus). A total of 5829 fungal operational taxonomic units (OTUs) spanning 649 genera and 15 phyla were identified, with Tausonia (4.17%) and Cladosporium (1.89%) as dominant genera. Environmental correlations revealed 19 genera significantly linked to abiotic factors. FUNGuild functional profiling highlighted saprotrophs (organic decomposers) and pathogens as predominant trophic guilds. Saprotrophs exhibited strong associations with pH, total nitrogen, and phosphorus, whereas pathogens correlated primarily with pH. These findings unveil the hidden diversity and ecological roles of aquatic fungi in alpine wetlands, emphasizing their sensitivity to environmental gradients. By establishing baseline data on fungal community dynamics, this work advances the understanding of wetland microbial ecology and informs conservation strategies for Ramsar sites. Full article

Anseriformes (waterfowl) and Charadriiformes (shorebirds) are well-recognized natural reservoirs of low pathogenic (LP) influenza A viruses (IAVs). Historically, LP IAVs circulate among healthy individuals during seasonal, and often transcontinental, migrations. However, following the introduction of clade 2.3.4.4b highly pathogenic (HP) A/Goose/Guangdong/1/1996 lineage H5 IAV to North America in 2021, countless wild birds succumbed to fatal infections across the Western Hemisphere. Due to their small size and cryptic plumage patterns, opportunities for carcass recovery and postmortem evaluation in sanderlings (Calidris alba) and other shorebirds are rare. A multispecies mortality event in coastal Virginia, USA, in March–April 2024 included sanderlings among other wild bird species. Nine sanderlings underwent postmortem evaluation and clade 2.3.4.4b H5 IAV RNA was detected in pooled oropharyngeal-cloacal swabs from 11/11 individuals by real-time reverse transcription polymerase chain reaction. Histopathology was similar to that in waterfowl and included necrosis in the pancreas and brain and less commonly in the gonad, adrenal gland, spleen, liver, and intestine. Immunohistochemistry revealed IAV antigen labeling in necrotic neurons of the brain (neurotropism) and epithelial cells of the pancreas, gonad, and adrenal gland (epitheliotropism). Describing HP IAV-attributed pathology in shorebirds is key to understanding ecoepidemiology and population health threats in order to further document and compare pathogenesis among avian species. Full article

This review summarizes recent findings on the diverse roles of bacterial sialidases in microbial biology. Bacterial sialidases, also known as neuraminidases, are exog α-lycosidases that cleave terminal sialic acid residues from a number of complex compounds designated as sialoglycoconjugates (glycoproteins, glycolipids and oligosaccharides). Metabolically, they are involved in sialic acid catabolism, providing energy, carbon and nitrogen sources. Catabolic degradation of sialic acids is a physiological feature that can be considered an important virulence factor in pathogenic microorganisms. Sialidases play a pivotal role in host–pathogen interactions and promotion of bacterial colonization. The activity of these enzymes enables bacterial adhesion, biofilm formation, tissue invasion, and also provides immune evasion by exposing cryptic receptors and modifying immune components. Many different perspectives are being developed for the potential application of sialidases. In the field of medicine, they are being explored as appropriate targets for antimicrobials, vaccines, diagnostic preparations and in tumor immunotherapy. In the field of enzymatic synthesis, they are used for the regioselective production of oligosaccharide analogs, enzymatic separation of isoenzymes and as a tool for structural analysis of sialylated glycans, among other applications. Full article