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Kwan, H. S.

Publications and source records attributed to Kwan, H. S..

11 recordsLinked to original sources

Fu Ling (Wolfiporia cocos) Fungal β-Glucan in Combination with Bacillus coagulans Improve Colitis-Related Outcomes and Cecal Fermentation in DSS-Treated Mice

Inflammatory bowel disease challenges modern gastroenterology due to high relapse rates. This study evaluated the effects of medicinal fungal sclerotium Fu Ling (Wolfiporia cocos), its derived {beta}-1,3-1,6-Glucan, Bacillus coagulans GBI-30, 6086, and their combinations in dextran sulfate sodium-induced colitis in male BALB/c mice. The {beta}-glucan-B. coagulans combination significantly improved body weight, disease activity index, colon length and architecture, mucus secretion, and cecal fermentation over single ingredient. Consistently, this combination increased total short-chain fatty acids production, particularly acetate, improved gut tight-junction organization and reduced myeloperoxidase accumulation. Shotgun metagenomic sequencing indicated that this combination reshaped the gut microbiota, notably enriching Anaeromassilibacillus sp., which associated with improved clinical feature. These findings highlight the potential of fungal-derived {beta}-glucan and probiotic combinations as functional food ingredients for mitigating intestinal inflammation and supporting barrier repair.

microbiology↗

A response-geometry framework separates microbiome movement magnitude from directional coherence in intervention studies

Dietary and lifestyle microbiome interventions often produce mild but heterogeneous remodeling rather than uniform community shifts. In this setting, scalar diversity or group-level summaries can appear weak or inconclusive even when participants move in organized but magnitude-limited directions, or move substantially in divergent directions. We developed a response-geometry framework that jointly describes baseline-referenced response magnitude and cross-participant directional coherence within a compositional feature space. The framework complements diversity, ordination, trajectory, PERMANOVA, PERMDISP, and beta-diversity analyses by asking whether paired responses differ in size, shared direction, or both. MethodsA response vector for each participant was defined as the follow-up minus baseline profile after adding a 0.5 pseudocount and applying centered log-ratio transformation in Aitchison-based response space. Response magnitude was the Euclidean length of this vector. Directional coherence was quantified as cosine alignment between participant-level response vectors and the mean group response vector, with sign-flip permutations as a paired-structure-preserving diagnostic null. We evaluated the framework using workflow-sensitive diversity comparisons, 198,000 logistic-normal compositional simulations with 100 or 500 features and small-to-large shared-direction effects, public-data-derived implementation stress tests, a synbiotic and dietary-intervention cohort, and a fiber/fermented-food application in 16S rRNA gene amplicon and shotgun-derived CAZyme gene-family feature spaces. A beta research-preview repository accompanying the preprint is available at https://github.com/carolyyszeto/microbiome-response-interpreter-beta as v6.5-beta, including documented scripts, a toy dataset, environment notes, output-interpretation guidance, and exploratory implementation utilities. ResultsWorkflow comparisons showed that richness-sensitive differences were concentrated in rare-tail and low-abundance structure, informing the analytical feature-space context for response interpretation. In simulations, null and magnitude-only random-direction scenarios showed near-null detection rates of 0.061 and 0.062, close to nominal alpha = 0.05, whereas shared-direction scenarios showed increasing coherence with stronger effects and larger sample sizes. Mixed-responder and opposing-subgroup scenarios attenuated or cancelled pooled coherence, supporting separation between response magnitude and directional organization. The synbiotic and dietary-intervention cohort showed modest, heterogeneous displacement with limited within-arm coherence, with permutation p values from 0.575 to 0.653. In the fiber/fermented-food application, fermented-food exposure showed stronger 16S response organization than the baseline-period reference, while CAZyme estimates used non-identical sampling endpoints and remained feature-space-specific. ConclusionsThis response-geometry framework helps distinguish paired microbiome movement size from shared response orientation. It is intended as an interpretively cautious response-organization descriptor for mild, heterogeneous intervention settings, not as a replacement for existing multivariate methods. Its interpretation depends on sample size, effect structure, endpoint alignment, zero handling, group-direction stability, and feature-space definition. The framework does not convert weak, null, endpoint-limited, or sensitivity-dependent findings into efficacy, predictive, or mechanistic claims.

microbiology↗

Protein kinase A gene knockout in Coprinopsis cinerea by CRISPR/Cas9 RNP complex system

Coprinopsis cinerea is a model mushroom for studying the developmental processes of homobasidiomycetous fungi. The development of C. cinerea depends on the sensing of environmental conditions, such as light, temperature, humidity and nutrients. The signal transduction pathway from the environmental condition sensors to the regulators and reproduction is an important research topic as the knowledge on it is still fragmentary. Protein kinases not only are crucial to the signal transduction, but also can be master switches of development. However, information on their role in basidiomycetous fungi is still limited. Using the effective gene-targeting of the current gene modification, a system of CRISPR/ Cas9 RNP with double strain template donor that contained a selective mark hygromycin cassette was developed in this study. In this system, CcPka1 gene knockout was successfully generated through homologues repair together with Cas9 RNP complexes. This was the first report to use CRISPR/ Cas9 RNP approach with C. cinerea. {Delta}CcPka1 mutants showed slow mycelial growth and rapid fruiting body induction compared to the wild type even when under high glucose condition. In addition, {Delta}CcPka1 mutants had significantly increased intracellular glucose level and decreased glycogen level. Moreover, {Delta}CcPka1 mutants regulated the gene expression of components in cAMP-PKA pathway. All these results demonstrated that PKA played an important role in the nutrient signal transduction and negatively regulated the fruiting body formation. This study also confirmed that GSK3 could be the downstream target of PKA, but their interaction requires further investigation.

genetics↗

The Phylotranscriptomic Hourglass Pattern in Fungi: An Updated Model

The "developmental hourglass" describes the morphological convergence to a conserved form at mid-stages of animal embryogenesis. The molecular hourglass pattern during embryogenesis was also identified across kingdoms. Previously, we reported young fruiting body as the conserved "waist" in mushroom-forming "developmental hourglass". However, its robustness is doubted because of the fungal diversity. Additionally, fungi lack embryogenesis, and develop directly from spore to hyphae with morphological similarities during the transition. Here, we updated the "developmental hourglass" model in the life cycle of fungi, namely, spore germination, vegetative growth, and sexual reproduction. Germinating spores, both sexual and asexual, showed the strongest transcriptomic conservation signals across the phyla Mucoromycota, Ascomycota and Basidiomycota. Cross kingdom comparisons revealed high expression levels of "information storage and processing" genes at the waist stages of embryonic and non-embryonic developments in animals, plants, and fungi. The "developmental hourglass" might reflect the mutual transcriptome switches on developmental transitions in eukaryotes that are additional to embryonic organogenesis. HighlightsO_LIUpdated fungal molecular "developmental hourglass" model in the life cycle of fungi C_LIO_LIGerminating spores are the evolutionarily conserved "waist" across fungal phyla C_LIO_LIHigh expression levels of "information storage and processing" genes at the waist stages in the embryonic and non-embryonic hourglasses across kingdoms C_LIO_LI"Developmental hourglass" may reflect the mutual transcriptome switches on developmental transitions in eukaryotes C_LI

evolutionary biology↗

Chromosome-level de novo assembly of Coprinopsis cinerea A43mut B43mut pab1-1 #326 and genetic variant identification of mutants using Nanopore MinION sequencing

The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform. HighlightO_LIA chromosome-level genome assembly of C. cinerea #326 C_LIO_LIA fast and efficient high-molecular-weight fungal genomic DNA isolation protocol C_LIO_LIStructural variant and single nucleotide variant calling using Nanopore reads C_LIO_LIA series of solutions and reference parameters for fungal genomic analysis on MinION C_LI

genetics↗

Horseshoe crab genomes reveal the evolutionary fates of genes and microRNAs after three rounds (3R) of whole genome duplication

Whole genome duplication (WGD) has occurred in relatively few sexually reproducing invertebrates. Consequently, the WGD that occurred in the common ancestor of horseshoe crabs ~135 million years ago provides a rare opportunity to decipher the evolutionary consequences of a duplicated invertebrate genome. Here, we present a high-quality genome assembly for the mangrove horseshoe crab Carcinoscorpius rotundicauda (1.7Gb, N50 = 90.2Mb, with 89.8% sequences anchored to 16 pseudomolecules, 2n = 32), and a resequenced genome of the tri-spine horseshoe crab Tachypleus tridentatus (1.7Gb, N50 = 109.7Mb). Analyses of gene families, microRNAs, and synteny show that horseshoe crabs have undergone three rounds (3R) of WGD, and that these WGD events are shared with spiders. Comparison of the genomes of C. rotundicauda and T. tridentatus populations from several geographic locations further elucidates the diverse fates of both coding and noncoding genes. Together, the present study represents a cornerstone for a better understanding of the consequences of invertebrate WGD events on evolutionary fates of genes and microRNAs at individual and population levels, and highlights the genetic diversity with practical values for breeding programs and conservation of horseshoe crabs.

evolutionary biology↗

Carbon metabolism, transcriptome and RNA editome in developmental paths differentiation of Coprinopsis cinerea

The balance and interplay between sexual and asexual reproduction is one of the most attractive mysteries in fungi. The choice of developmental strategy reflects the ability of fungi to adapt to the changing environment. However, the evolution of developmental paths and the metabolic regulation during differentiation and morphogenesis are poorly understood. Here, we monitor the carbohydrate metabolism and gene expression regulation during the early differentiation process from the \"fungal stem cell\", vegetative mycelium, to the highly differentiated tissue/cells, fruiting body, oidia or sclerotia, of a homokaryotic fruiting Coprinopsis cinerea strain A43mut B43mut pab1-1 #326, uncovering the systematic changes during morphogenesis and the evolutionary process of developmental strategies. Conversion between glucose and glycogen and conversion between glucose and beta-glucan are the main carbon flows in the differentiation processes. Genes related to carbohydrate transport and metabolism are significantly differentially expressed among paths. RNA editing, a novel layer of gene expression regulation, occurs in all four developmental paths and enriched in cytoskeleton and carbohydrate metabolic genes. It is developmentally regulated and evolutionarily conserved in basidiomycetes. Evolutionary transcriptomic analysis on four developmental paths showed that all transcriptomes are under purifying selection, and the more stressful the environment, the younger the transcriptome age. Oidiation has the lowest value of transcriptome age index (TAI) and transcriptome divergence index (TDI), while fruiting process has the highest of both indexes. These findings provide new insight to the regulations of carbon metabolism and gene expressions during fungal developmental paths differentiation.\n\nImportanceFungi is a group of species with high diversity and plays essential roles to the ecosystem. The life cycle of fungi is complex in structure and delicate in function. Choice of developmental strategies and internal changes within the organism are both important for the fungus to fulfill their ecological functions, reflecting the relationship between environment and the population. This study put the developmental process of vegetative growth, sexual and asexual reproduction, resistant structure formation of a classical model basidiomycetes fungus, C. cinerea, together for the first time to view the developmental paths differentiation process with physiology, transcriptomics and evolutionary prospects. Carbohydrate assays and RNA-seq showed the changes of the fungus. Our results fill the gaps on gene expression regulation during the early stage of developmental paths differentiation, and expand our understanding of the evolutionary process of life history and reproductive strategy in fungi.

microbiology↗

Modified recipe to inhibit GSK-3 for the living fungal biomaterial manufacture

Living fungal mycelium with suppressed or abolished fruit-forming ability is a self-healing substance particularly valuable biomaterial for further engineering and development in applications such as monitoring/sensing environmental changes and secreting signals. The ability to suppress fungal fruiting is also a useful tool for maintaining stability (e.g., shape, form) of a mycelium-based biomaterial with ease and lower cost.\n\nThe objective of this present study is to provide a biochemical solution to regulate the fruiting body formation to replace heat killing of mycelium during production. We discovered that GSK-3 activity directly correlates with the development of fruiting bodies in fungi, especially mushroom forming fungi such as Coprinopsis cinerea. By regulating GSK-3 expression and activity, one can control the fungal fruiting body development.\n\nWe successfully demonstrated that treatment of an inhibitor of GSK-3 kinase activity resulted in acceleration in mycelium growth rate, absence of fruiting body and general decrease in GSK-3 gene expression. Therefore, GSK-3 inhibitor is suggested to be included in the mycelium cultivation recipes for regulating the growth of fungal mycelium and for inhibiting the development of fruiting bodies. This is the first report of using a GSK-3 inhibitor, such as lithium or any other GSK-3 inhibitor, to suppress or abolish fruiting body formation in living fungal mycelium-based biomaterial. It also provides an innovative strategy for easy, reliable, and low cost maintenance of biomaterial containing live fungal mycelium.

developmental biology↗

eQTL network analysis reveals that regulatory genes are evolutionarily older and bearing more types of PTM sites in Coprinopsis cinerea

Understanding the DNA variation in regulation of carbohydrate-active enzymes (CAZymes) is fundamental to the use of wood-decaying basidiomycetes in lignocellulose conversion into renewable energy. Our goal is to identify the regulators of lignocellulolytic enzymes in Coprinopsis cinerea, of which the genome harbors high number of Auxiliary Activities enzymes.\n\nThe DNA sequence of C. cinerea family including 46 single spore isolates (SSIs) from crosses of two homozygous strains are used to develop a panel of SNP markers. Then the RNA sequence were used to characterize the gene expression profiles. The RNA were extracted from cultures grown on softwood-enriched sawdust to induce lignocellulolytic enzymes and CCR de-repression genes. To assess the genetic contribution to enzyme expression variations among the 46 SSIs, associations between SNPs and gene expressions were examined genome-widely. 5148 local eQTLs and 7738 distant eQTLs were obtained. By analyzing these eQTLs, the potential regulatory factors of the CAZymes expression and the de-repression of Carbon Catabolism Repression (CCR) were identified.\n\nThe eQTL network is characterized in terms of hotspots, evolutionary age and post-translational modifications (PTMs). In the eQTL network of C. cinerea, the non-regulatory genes are younger than the regulatory genes. The proteins regulated by combinational multiple types of PTMs are more likely to function as super regulatory hotspots in protein-protein interactions. The evolutionary age analysis and the PTMome analysis could serve as alternative methods to identify master regulators from genomic data.\n\nThis work demonstrates a comprehensive bioinformatics approach to identify regulatory factors with next-generation sequencing data. The results provide candidate genes for bioengineering to increase the enzyme production, which will practically benefit the bioethanol production from lignocellulose.\n\nSignificanceThis eQTL analysis is designed to study the fungal CAZymes and carbon catabolism repression, especially during the mycelium stage.\n\nO_LIIn Coprinopsis cinerea, only the regions near two ends of the chromosomes have high recombination rate, and suitable for family based eQTL analysis.\nC_LIO_LIA sugar transporter is a hotspot controlling many CCR genes.\nC_LIO_LICAZymes are not regulated by a master regulator, but by individual regulators. This indicates that CAZymes are under specific regulatory pathways, so can response to specific conditions.\nC_LIO_LIIn the eQTL network, the rGenes are evolutionarily older, with more types of PTM sites than eGenes.\nC_LIO_LIIn the eQTL network, the proteins with more types of PTM sites are more likely associated with Information Storage and Processing, and act as super-hub in the network.\nC_LI

bioinformatics↗

Discovery of microRNA-like RNAs during early fruiting body development in the model mushroom Coprinopsis cinerea

Coprinopsis cinerea is a model mushroom particularly suited to study fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) are extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Besides, essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), two for Argonaute-like (AGO-like) and one for quelling deficient-2 (QDE-2) proteins, were identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO-like proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to animals and plants. Taken together, our findings provided the first evidence of milRNAs in the model mushroom and their potential roles in regulating fruiting body development. Information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights into further functional and evolutionary studies of miRNAs.

genetics↗

Draft genomes of the fungal pathogen Phellinus noxius in Hong Kong

The fungal pathogen Phellinus noxius is the underlying cause of brown root rot, a disease with causing tree mortality globally, causing extensive damage in urban areas and crop plants. This disease currently has no cure, and despite the global epidemic, little is known about the pathogenesis and virulence of this pathogen.\n\nUsing Ion Torrent PGM, Illumina MiSeq and PacBio RSII sequencing platforms with various genome assembly methods, we produced the draft genome sequences of four P. noxius strains isolated from infected trees in Hong Kong to further understand the pathogen and identify the mechanisms behind the aggressive nature and virulence of this fungus. The resulting genomes ranged from 30.8Mb to 31.8Mb in size, and of the four sequences, the YTM97 strain was chosen to produce a high-quality Hong Kong strain genome sequence, resulting in a 31Mb final assembly with 457 scaffolds, an N50 length of 275,889 bp and 96.2% genome completeness. RNA-seq of YTM97 using Illumina HiSeq400 was performed for improved gene prediction. AUGUSTUS and Genemark-ES prediction programs predicted 9,887 protein-coding genes which were annotated using GO and Pfam databases. The encoded carbohydrate active enzymes revealed large numbers of lignolytic enzymes present, comparable to those of other white-rot plant pathogens. In addition, P. noxius also possessed larger numbers of cellulose, xylan and hemicellulose degrading enzymes than other plant pathogens. Searches for virulence genes was also performed using PHI-Base and DFVF databases revealing a host of virulence-related genes and effectors. The combination of non-specific host range, unique carbohydrate active enzyme profile and large amount of putative virulence genes could explain the reasons behind the aggressive nature and increased virulence of this plant pathogen.\n\nThe draft genome sequences presented here will provide references for strains found in Hong Kong. Together with emerging research, this information could be used for genetic diversity and epidemiology research on a global scale as well as expediting our efforts towards discovering the mechanisms of pathogenicity of this devastating pathogen.

genomics↗