Biology subjects

Wu, T.-H.

Publications and source records attributed to Wu, T.-H..

10 recordsLinked to original sources

bioRxiv2026

Adaptive sampling-based enrichment enables genome reconstruction of intracellular symbionts despite host background and reference divergence

Recovering genomes of intracellular microbes from host-dominated samples remains a major challenge in microbial genomics, due to low target abundance, overwhelming host DNA, and the inability to culture these organisms independently. Despite extensive interest in Wolbachia, efficient genome recovery directly from host tissues remains limited by the inefficiency of host-dominated sequencing and the constraints of existing enrichment strategies. Here, we demonstrate that Oxford Nanopore adaptive sampling (AS) enables efficient, real-time enrichment of target DNA directly from complex host tissues, providing a culture-free approach for genome recovery in such systems. To our knowledge, this represents the first application of enrichment-mode adaptive sampling to achieve de novo reconstruction of an intracellular endosymbiont genome in a mosquito system. Using Aedes aegypti mosquitoes infected with a locally derived wAlbB-like strain, we applied enrichment-mode AS to selectively sequence Wolbachia DNA. This resulted in an increase from <1% Wolbachia reads in conventional shotgun data to [~]90% under adaptive sampling. De novo assembly of AS-enriched long reads yielded a near-complete genome ([~]1.5 Mb) in two contigs with >96-99% completeness. Comparative analyses revealed multiple large-scale chromosomal rearrangements relative to the reference wAlbB genome, demonstrating that adaptive sampling does not impose reference-dependent genome structure. Annotation further identified three prophage-associated regions, including two strain-specific expansions absent from the reference genome. Notably, cytoplasmic incompatibility genes (cifA and cifB) were identified adjacent to one of these regions, consistent with their known genomic association with prophage elements. Importantly, adaptive sampling remained effective despite substantial structural divergence between the reference and target genomes, revealing an unexpectedly robust application of this approach beyond its presumed operating conditions. Together, these results establish enrichment-mode adaptive sampling as a robust and scalable strategy for genome-resolved analysis of intracellular bacteria in host-associated systems.

genomics↗

bioRxiv2025

Massive Chromosome Fission and Speciation in Asian River Chub: Integrating Kinetochore Reproduction Theory with Transposable Element Dynamics

Chromosomal speciation poses a paradox: if rearrangements cause postmating isolation, how do they persist and spread? We propose that centromere remodeling, driven by transposable element accumulation, provides a mechanistic solution. In Opsariichthys, a hyper-diverse lineage of Asian river chub with 2N = 76-78 chromosomes, we identify [~]15 fission events coinciding with TE-fueled centromeric DNA expansion, potentially linked to a mutation in the PIWI1 protein. These expansions enable neokinetochore formation, allowing fissioned chromosomes to segregate faithfully, while subsequent rearrangements disrupt pairing and reduce gene flow. Comparative genomics reveals that fissioned chromosomes exhibit lower migration and greater divergence, acting as reproductive barriers. Our findings show that TE-mediated kinetochore duplication can facilitate extensive chromosome fission without meiotic disruption, providing a viable and previously underappreciated path to rapid speciation.

genomics↗

bioRxiv2024

Label-Free Optical Mapping for Large-Area Biomechanical Dynamics of Multicellular Systems

Mapping cellular activities over large areas is crucial for understanding the collective behaviors of multicellular systems. Biomechanical properties, such as cellular traction force, serve as critical regulators of physiological states and molecular configurations. However, existing technologies for mapping large-area biomechanical dynamics are limited by the small field of view and scanning nature. To address this, we propose a novel platform that utilizes a vast number of optical diffractive elements for mapping large-area biomechanical dynamics. This platform achieves a field-of-view of 10.6 mm X 10.6 mm, a three-orders-of-magnitude improvement over traditional traction force microscopy. Transient mechanical waves generated by monolayer neonatal rat ventricular myocytes were captured with high spatiotemporal resolution (130 fps and 20 {micro}m for temporal and spatial resolution, respectively). Furthermore, its label-free nature allows for long-term observations extended to a week, with minimal disruption of cellular functions. Finally, simultaneous measurements of calcium ions concentrations and biomechanical dynamics are demonstrated.

bioengineering↗

bioRxiv2022

Calcium-activated big conductance (BK) potassium channels traffic through nuclear envelopes into kinocilia in skate electrosensory cells

Electroreception through ampullae of Lorenzini in the little skate, Leucoraja erinacea, involves functional coupling between voltage-activated calcium channels (CaV1.3, cacna1d) and calcium-activated big conductance potassium (BK) channels (BK, kcnma1). Whole mount confocal microscopy was used to characterize expression of BK and CaV1.3 in intact ampullae. BK and CaV1.3 are present in electrosensory cell plasma membranes and colocalize with lamin B1 in nuclear envelopes and kinocilia. Predicted nuclear localization signals (NLS) were identified in BK and CaV1.3 by bioinformatic sequence analyses. The BK NLS is bipartite, occurs at an alternative splice site for the mammalian STREX exon and is modified by post-translational phosphorylation. Nuclear localization of skate BK channels was characterized in heterologously-transfected HEK293 cells. Double point mutations in the bipartite NLS (KR to AA, or SVLS to AVLA) independently attenuated BK channel nuclear localization, illustrating that BK trafficking into nuclei and electrosensory cell kinocilia is regulated through a newly identified bipartite NLS.

neuroscience↗

bioRxiv2021

De novo SNP calling reveals the candidate genes regulating days to flowering through interspecies GWAS of Amaranthus genus

Amaranths serve as pseudo cereals and also as traditional leafy vegetables worldwide. In addition to high vigor and richness in nutrients, drought and salinity tolerance of amaranth makes it a promising vegetable to acclimatize to the effects of global climate change. The World Vegetable Center genebank conserves about 1,000 amaranth accessions and various agronomic properties of these accessions were recorded during seed regeneration over decades. In this study, we verified the taxonomic annotation of the germplasm based on a 15K SNP set. Besides, in the assumption that the yield components of grain amaranths are different from those of leaf amaranths, we observed that grain amaranths presented larger inflorescences and earlier flowering than leaf amaranths. Dual-purpose amaranth showed larger leaves than leaf amaranths and later flowering than grain amaranths, which seemed reasonable because farmers can harvest more leaves during the prolonged vegetable stage, which also provides recovery time to enrich grain production. Considering frequent interspecies hybridization among grain amaranth complex, we proposed an interspecies GWAS for days to flowering, identifying a AGL20/SOC1 homolog. Meanwhile, another GWAS using only A. tricolor accessions revealed six candidate genes homologous to lba1, bri1, sgs1 and fca. These homologous genes were involved in the regulation of flowering time in Arabidopsis. This study revealed the usefulness of genotypes for species demarcation in the genus Amaranthus and the potential of interspecies GWAS to detect QTLs across different species, opening up the possibility of targeted introduction of specific genetic variants into different Amaranthus species.

genomics↗

bioRxiv2020

Stable transplantation of human mitochondrial DNA by high-throughput, pressurized mitochondrial delivery

Generating mammalian cells with specific mtDNA-nDNA combinations is desirable but difficult to achieve and would be enabling for studies of mitochondria-nucleus communication and coordination in controlling cell fates and functions. We developed MitoPunch, a force-actuated mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, an alternative force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.

cell biology↗

bioRxiv2018

Studies of Bending Effects of Microvilli of Leukocyte on Rolling Adhesion

It has been widely acknowledged that further understanding about the cell adhesion (e.g., leukocyte rolling adhesion) can help us gain more knowledge about the causes of relevant diseases and design more effective treatments and diagnoses. Although recent simulation studies considered the deformability of the leukocytes, most of them, however, did not consider the bending deformation of microvilli. In this paper, an advanced leukocyte model based on an immersed boundary lattice-Boltzmann lattice-spring model (LLM) and an adhesive dynamics (AD) is presented in details. The flexural stiffness of microvilli is introduced into the model for simulations of leukocyte rolling adhesion. This innovative model is applied to investigate the influences of bending deformation of microvilli on the process of leukocyte rolling adhesion and the underlying mechanism at different shear rates. It is demonstrated that the bending deformation of microvilli can be influenced by the flexural stiffness of microvilli and shear rates, resulting in the different rolling velocity of leukocytes, number of receptor-ligand bonds, and bond forces. The findings clearly indicate that the bending of microvilli plays a crucial role in the dynamics of leukocyte adhesion.

biophysics↗

bioRxiv2018

Further studies of ion channels in the electroreceptor of the skate through deep sequencing, cloning and cross species comparisons.

Our comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel, whose subunit is Kcnma1, a voltage-dependent calcium channel, Cacna1d, and a relatively uncharacterized K channel which interacts with the calcium channel to produce fast (20 Hz) oscillations. Large conductance calcium-activated K channels (BK) are comprised of four subunits, encoded by Kcnma1 and modulatory {beta} subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 derived from using purified mRNA of homogenized isolated electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the the Bellono et al RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent potassium channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel Kv1.1, which is expressed at appreciable levels and seems likely to explain the 20 Hz oscillations believed to occur in vivo. The second was more similar to Kv1.5 than to Kv1.1 but was somewhat atypical. We also found a beta subunit sequence (Kcnab2) which appears not to cause fast inactivation due to specific structural features. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of the calcium activated potassium channel, Kcnma1, and found a computer generated assembly that contained a complete sequence of its beta subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 (beta1) is absent. The evolutionary origin of the newly described channels and subunits was studied by aligning skate sequences with human sequences and those found in related fish: the whale shark (R. typus) an elasmobranch, and ghost shark (C.milii). There is also homology with the lamprey, which has electroreceptors. An evolutionary tree is presented. Further research should include focusing on the subcellular locations of these channels in the receptor cells, their gating behavior, and the effects of accessory subunits on gating.

neuroscience↗

bioRxiv2018

Inducible expression of immediate early genes is regulated through dynamic chromatin association by NF45/ILF2 and NF90/ILF3

Immediate early gene (IEG) transcription is rapidly activated by diverse stimuli without requiring new protein synthesis. This transcriptional regulation is assumed to involve constitutively expressed nuclear factors that are targets of signaling cascades initiated at the cell membrane. NF45 and its heterodimeric partner NF90 are chromatin-interacting proteins that are constitutively expressed and localized predominantly in the nucleus. Previously, NF90 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in K562 erythroleukemia cells revealed its enriched association with chromatin at active promoters and strong enhancers. NF90 specifically occupied the promoters of IEGs. Here, ChIP in serum-starved HEK293 cells demonstrated that NF45 and NF90 pre-exist and specifically occupy the promoters of IEG transcription factors EGR1, FOS and JUN. Cellular stimulation with phorbol myristyl acetate increased NF90 occupancy, while decreasing NF45 occupancy at promoters of EGR1, FOS and JUN. In HEK293 cells stably transfected with doxycycline-inducible shRNA vectors targeting NF90 or NF45, doxycycline-mediated knockdown of NF90 or NF45 attenuated the inducible expression of EGR1, FOS, and JUN at the levels of mRNA and protein. NF90 and NF45 operate as constitutively-expressed transcriptional regulators of IEGs. Dynamic chromatin association of NF45 and NF90 at IEG promoters are observed upon stimulation, and NF45 and NF90 contribute to inducible expression of IEGs. NF45 and NF90 operate as chromatin regulators of the immediate early response.

cell biology↗

bioRxiv2017

NF90/ILF3 is a transcription factor that promotes proliferation over differentiation by hierarchical regulation in K562 erythroleukemia cells

NF90 and splice variant NF110 are DNA- and RNA-binding proteins encoded by the Interleukin enhancer-binding factor 3 (ILF3) gene that regulate RNA splicing, stabilization and export. The role of NF90 in regulating transcription as a DNA-binding protein has not been comprehensively characterized. Here, ENCODE ChIP-seq identified 9,081 genomic sites specifically bound by NF90/110 in K562 cells. One third of binding sites occurred at promoters of annotated genes. NF90/110 binding colocalized with chromatin marks associated with active promoters and strong enhancers. Comparison with 150 ENCODE ChIP-seq experiments revealed that NF90 clustered with transcription factors exhibiting preference for promoters over enhancers (POLR2A, MYC, YY1). Differential gene expression analysis following shRNA knockdown of NF90 in K562 cells revealed that NF90 directly activates transcription factors that drive growth and proliferation (EGR1, MYC), while attenuating differentiation along erythroid lineage (KLF1). NF90/110 binds chromatin to hierarchically regulate transcription factors to promote proliferation and suppress differentiation.

genetics↗