Biology subjects

Yeh, T.-Y.

Publications and source records attributed to Yeh, T.-Y..

4 recordsLinked to original sources

bioRxiv2025

The Filterprep: A Simple and Efficient Approach for High-Yield, High-Purity Plasmid DNA Purification

Plasmid DNA purification remains a fundamental yet resource-intensive step in molecular biology and biotechnology. Conventional protocols often yield plasmids with suboptimal purity, while commercial kits, though efficient, are costly and use proprietary formulations that limit transparency and customization. Here, we present Filterprep, a hybrid method combining classical ethanol precipitation with a single spin-column cleanup step, enabling rapid recovery of high-purity plasmid DNA with yields notably higher than those obtained using commercial kits, in approximately 40 minutes. Filterprep offers a transparent, cost-effective, and scalable alternative, simplifying workflows and reducing hands-on time without compromising DNA quality. The purified plasmids are compatible with a variety of downstream applications, including mammalian cell transfection, protein expression, and reporter assays. With its balance of efficiency and practicality, Filterprep is well suited for high-throughput laboratories and resource-limited settings.

biochemistry↗

bioRxiv2025

Recombination alters the furin cleavage site in novel bat-borne HKU5-CoV-2 coronavirus

HKU5-CoV-2 is a new bat-infecting coronavirus phylogenetically related to MERS-CoV. It has recently been confirmed that HKU5-CoV-2 can enter human cells and organoids in vitro via the ACE2 receptor, raising concerns about its pandemic potential due to zoonotic spillover. Whether recombination has an influence on HKU5-CoV-2 is completely unclear to date. Here we report the first evidence of HKU5-CoV-2 viral recombination, in association with mutations at the receptor binding domain (RBD) and S1/S2 furin cleavage site (FCS) of the spike protein. Using linkage disequilibrium and haploblock analysis, we identified that 167 recombination hotspots and 27 haploblocks. SNP23016/23043/23064/23156/23193/23285 at the RBD and SNP23833/23847 at the FCS are recombinant hotspots. Our results suggest that recombination may lead to the substitution at RBD residue 498 (Thr498Val/Val498Thr, Thr498Ile/Ile498Thr), which Thr498 directly contacts the ACE2 receptor. Recombination also causes Ser723 deletion/insertion and Ser729Ala substitution at the FCS. These mutations could affect host tropism and change furin cleavage activity. Our results indicate that recombination has played a critical role in HKU5-CoV-2 evolution and infectivity.

microbiology↗

bioRxiv2024

A packaging signal-binding protein regulates the assembly checkpoint of integrative filamentous phages

Many integrative filamentous phages not only lack Ff coliphage homologues essential for assembly but also have distinct packaging signals (PS). Their encapsidation remains completely uncharacterized to date. Here we report the first evidence of a PS-dependent checkpoint for integrative filamentous phage assembly. Suppressor screening of PS-deficient phages identified an unknown protein, PSB15 (PS-binding 15 kDa), crucial for encapsidation. The WAGFXF motif of the PSB15 N-terminus directly binds to PS DNA with conformational change, while suppressor mutations relieve DNA binding specificity constraints to rescue assembly arrest. PSB15 interacts with phospholipid cardiolipin via its basic helix and C-terminus, and recruits PS DNA to the inner membrane (IM). The PSB15-PS complex is released from the IM by interaction between its hydrophobic linker and thioredoxin (Trx), a host protein that is required for Ff assembly but whose mechanisms are still unclear. Live cell imaging shows that thioredoxin and DNA binding regulate the dwelling time of PSB15 at cell poles, suggesting that they both facilitate the dissociation of PSB15 from the IM. Loss of PSB15 or its PS-binding and IM-targeting/dissociation activity compromised virus egress, indicating that the PS/PSB15/Trx complex establishes a regulatory phage assembly checkpoint critical for integrative phage infection and life cycles.

microbiology↗

bioRxiv2023

The packaging signal of Xanthomonas integrative filamentous phages

Unlike Ff, the packaging signal (PS) and the mechanism of integrative filamentous phage assembly remain largely unknown. Here we revived two Inoviridae prophage sequences, {phi}Lf2 and {phi}Lf-UK, as infectious virions lysogenize black rot pathogen Xanthomonas campestris pv. campestris. {phi}Lf2 and {phi}Lf-UK genomes consist of 6,363 and 6,062 nucleotides and share 85.8% and 98.7% identity with {phi}Lf, respectively. To explore their assembly, we first identified 20-26- nucleotide long PS sequences of 10 Xanthomonas phages. These PS consist of a DNA hairpin with the consensus GGX(A/-)CCG(C/T)G sequence in the stem and C/T nucleotides in the loop, both of which are conserved and essential for PS activity. In contrast to Ff, the 5 to 3 orientation of PS sequence is not conserved or critical for their competence. This is the first report to offer insights into the structure and function of integrative phage PS, revealing the diversity of filamentous phage encapsidation.

molecular biology↗