Biology subjects

Chan, C.-K.

Publications and source records attributed to Chan, C.-K..

5 recordsLinked to original sources

bioRxiv2026

Ion-Pair-Free Capillary HILIC-MS for Sensitive Nucleic Acid Analysis and RNA Modification Mapping

Mass spectrometry (MS)-based nucleic acid analysis provides direct chemical evidence for oligonucleotide sequence, composition, and modifications. However, oligonucleotide LC-MS analysis commonly relies on ion-pairing reversed-phase liquid chromatography (IP-RPLC). Although IP-RPLC provides strong retention and high-resolution separation of highly charged nucleic acids, ion-pairing reagents can contaminate LC-MS systems, suppress electrospray ionization, require extensive system cleaning, and limit the use of high-end MS platforms that are primarily dedicated to proteomics or metabolomics. Here, we developed and evaluated an ion-pair-free capillary hydrophilic interaction liquid chromatography mass spectrometry (capillary HILIC-MS) workflow for RNA modification mapping. To enable robust analysis of biologically relevant samples, we optimized sample preparation, high-organic loading conditions, chromatographic parameters, and MS source settings to overcome key challenges associated with capillary HILIC, including limited sample volume, solvent compatibility, and solvent breakthrough during injection. The optimized capillary HILIC-MS method provided effective separation of oligonucleotides below 30 nt and enabled sensitive detection of RNA modifications in the populations of tRNAs and rRNAs in biological samples. Importantly, the ion-pair-free workflow also allowed switching between nucleic acid analysis and proteomics on the same LC-MS platform without the need for extensive system decontamination. Together, this workflow provides a sensitive, robust, and MS-compatible approach for nucleic acid analysis, expanding the utility of high-end LC-MS systems for both therapeutic oligonucleotide characterization and biological RNA modification profiling.

biochemistry↗

bioRxiv2025

tRNA Modification Landscapes in Streptococci: Shared Losses and Clade-Specific Adaptations

tRNA modifications are central to bacterial translational control. Here, we integrated genetics, mass spectrometry, epitranscriptomics, and comparative genomics to map the tRNA modification genes of the Gram-positive pathogens Streptococcus mutans and Streptococcus pneumoniae. Both species show a marked loss of modifications dependent on Fe-S enzymes, consistent with a broader trend of Fe-S enzyme reduction in Streptococcus central metabolism. In addition, the D, m1A, m7G, t6A, and i6A modifications were mapped in S. pneumoniae tRNAs, and we confirmed that a unique DusB1 enzyme is responsible for the insertion of all the detectable D modifications. We uncovered differences in queuosine (Q) metabolism: while S. mutans synthesizes Q de novo, S. pneumoniae instead salvages preQ and accumulates the epoxy-Q precursor, a strategy shared with multiple other Streptococci as revealed by analysis of Q pathways in 1,599 sequenced streptococcal genomes. Comparative essentiality profiling of modification genes revealed notable differences, including the essentiality of the NLJ-threonylcarbamoyladenosine (tLJA) synthesis enzyme TsaE in S. pneumoniae but not in S. mutans, which was confirmed by genetic studies. We found that suppressor mutations in asnS encoding asparaginyl-tRNA synthetase (AsnRS) restored viability to {Delta}tsaE mutants, albeit with reduced growth. Our finding highlights the functional importance of modifications in the recognition of tRNAs by aminoacyl-tRNA synthetases.

microbiology↗

bioRxiv2024

Target-aware Molecule Generation for Drug Design Using a Chemical Language Model

Generative drug design facilitates the creation of compounds effective against pathogenic target proteins. This opens up the potential to discover novel compounds within the vast chemical space and fosters the development of innovative therapeutic strategies. However, the practicality of generated molecules is often limited, as many designs focus on a narrow set of drug-related properties, failing to improve the success rate of subsequent drug discovery process. To overcome these challenges, we develop TamGen, a method that employs a GPT-like chemical language model and enables target-aware molecule generation and compound refinement. We demonstrate that the compounds generated by TamGen have improved molecular quality and viability. Additionally, we have integrated TamGen into a drug discovery pipeline and identified 7 compounds showing compelling inhibitory activity against the Tuberculosis ClpP protease, with the most effective compound exhibiting a half maximal inhibitory concentration (IC50) of 1.9 M. Our findings underscore the practical potential and real-world applicability of generative drug design approaches, paving the way for future advancements in the field.

biochemistry↗

bioRxiv2022

Thermococci-to-Clostridia Pathway for the Evolution of the Bacteria Domain

With the identification of an archaeal Last Universal Common Ancestor phylogenetically related to Methanopyrus, the origin of Bacteria becomes a choice between independent emergence versus descent from Archaea. Recently, use of the similarity between paralogous valyl-tRNA synthetase (VARS) and isoleucyl-tRNA synthetase (IARS) as a measure of the ages of bacteria indicated that an Ancestral Bacterial Cluster centred at clostridial Mahella australiensis (Mau) were the oldest bacteria. Clostridial Thermincola potens (Tpo) also displayed an elevated similarity VARS-IARS bitscore. Overall, the high-bitscore bacteria dominated by Clostridia comprised a number of hydrogen producers. A search for archaea capable of hydrogen production that might be ancestral to the Bacteria domain yielded candidates led by Thermococci which, like Clostridia, form hydrogen through dark fermentation. A two-domain VARS tree based on Mahella, Thermincola, a broad spectrum of archaea together with both well known and newly reported species of Thermococci allocated these two Clostridia to a minor-Thermococcal division on the tree. The kinship between Thermoccoci and Clostridia suggested by this allocation was substantiated by conserved oligopeptide segments on their VARS sequences. It was therefore suggested that a Thermococci-to-Clostridia evolutionary pathway brought about the emergence of the Bacteria domain.

genomics↗

bioRxiv2019

Astrocytic mechanism of glutamate modulation underlying synchronous bursting in cortical cultures

Synchronous bursting (SB) is ubiquitous in neuronal networks. It is known for a long time that SB is driven by glutamatergic neurotransmissions but its underlying mechanism is still unclear. Recent studies show that local glutamate recycle by astrocytes can affect neuronal activities nearby. Since SB is independent of network structure, it is conceivable that the local dynamics might also be the origin of SB in networks. We investigated the effects of local glutamate dynamics on SBs in both cultures developed on multi-electrode array (MEA) systems and a tripartite synapse simulation model. In our experiments, local glutamate recycle dynamics are studied by pharmacologically targeting the astrocytic glutamate transporters (GLT-1), while neuronal firing activities and synaptic glutamate level are simultaneously monitored with MEA and glutamate sensor (iGluSnFR) expressed on surface of astrocytes respectively. We found SBs to be synchronized with glutamate transients and the manipulation of local glutamate dynamics can indeed alter the global properties of the SBs. Detailed simulation of a network with astrocytic glutamate uptake and recycle mechanisms conforming with the experimental observations revealed that astrocytes function as a slow negative feedback for the neuronal activities in the network. With this model, SB can be understood as the alternation between the positive and negative feedback in the neurons and astrocytes in the network respectively. An understanding of this glutamate trafficking dynamics is of general application to explain disordered phenomena in neuronal systems, and therefore can provide new insights into the origin of fatal seizure-like behavior. SignificanceSynchronous bursting (SB) is a hallmark of neuronal circuits. Contrary to the common belief that the SB is governed mainly by neuron-neuron interactions, this study shows that SBs are orchestrated through a generic neuron-astrocyte tripartite interactions. These interactions, identified as glutamate uptake and recycle processes in astrocytes, control the excitability of neuronal networks and shape the overall SB patterns. Our simulation results suggest that astrocytes traffic more glutamate than neurons and actively regulating glutamate proceedings around synapses. A bipartite synapse is a good approximation of a tripartite synapse provided that astrocyte-dependent glutamate content is taken into account. Our findings provide key insights into the ubiquity of SB and the origin of fatal seizure-like behavior in brain arising from astrocytic malfunction.

neuroscience↗