Biology subjects

Chan, S.-H.

Publications and source records attributed to Chan, S.-H..

8 recordsLinked to original sources

bioRxiv2025

Muscle Activation of Upper Body in Different-Angle Suspension Push-Ups: An Analysis of Angle-Specific Muscle Engagement

The aim of this study was to understand the effect on upper body muscle activation of bodily angle during push-ups performed with TRX suspension training. Nineteen men (age: 21.1 {+/-} 1.2 years; height: 174.1 {+/-} 4.9 cm; weight: 70.7 {+/-} 7.2 kg) with resistance training experience participated in this study. The participants were required to perform five push-ups on a stable surface and with TRX at five angles (+30{degrees}, +15{degrees}, 0{degrees}, -15{degrees}, and -30{degrees}, where 0{degrees} indicates that the shoulder joints were at the same height as the ankle joints when the arms were extended). The recovery period between the sets at each angle was 3-5 minutes. Stable-surface and TRX push-ups were separated by at least 48 hours. During push-ups, electromyography (EMG) data from the pectoralis major (PM), anterior deltoids (AD), triceps brachii (TRI), upper trapezius (UT), and serratus anterior (SA) were recorded. Muscle activation was indicated by the percentage of maximum voluntary isometric contraction (%MVIC). The %MVIC of each muscle group was then categorized. Repeated-measured two-way analysis of variance was used to determine differences in the muscle group activation between the push-up surface types and five angles. Statistical significance was set at p < .05. The activation of the PM, AD, and TRI during TRX push-ups was categorized as medium to extremely high. Compared with that for stable-surface push-ups, the activation of the PM during TRX push-ups was significantly higher (p < .05). Furthermore, +30{degrees}, +15{degrees}, and 0{degrees} push-ups produced greater PM activation than push-ups at lower angles (p < .05). Both TRX and stable-surface push-ups resulted in greater anterior deltoids, TRI, UT, and SA activation during -30{degrees} push-ups. This study indicates the appropriate push-up practices for different muscle groups, as determined by quantifying the muscle activation.

physiology↗

bioRxiv2024

Structural Insights into the Assembly and Regulation of 2'-O RNA Methylation by SARS-CoV-2 nsp16/nsp10

2-O-ribose methylation of the first transcribed base (adenine or A1 in SARS-CoV-2) of viral RNA mimics the host RNAs and subverts the innate immune response. How nsp16, with its obligate partner nsp10, assembles on the 5-end of SARS-CoV-2 mRNA to methylate the A1 has not been fully understood. We present a [~] 2.4 [A] crystal structure of the heterotetrameric complex formed by the cooperative assembly of two nsp16/nsp10 heterodimers with one 10-mer Cap-1 RNA (product) bound to each. An aromatic zipper-like motif in nsp16 and the N-terminal regions of nsp10 and nsp16 orchestrate an oligomeric assembly for efficient methylation. The front catalytic pocket of nsp16 stabilizes the upstream portion of the RNA while the downstream RNA remains unresolved, likely due to its flexibility. An inverted nsp16 dimer extends the positively charged surface area for longer RNA to influence the catalysis. Additionally, a non-specific nucleotide-binding pocket on the backside of nsp16 plays a critical role in catalysis, further contributing to its enzymatic activity.

microbiology↗

bioRxiv2024

CD24a knockout transforms the tumor microenvironment from cold to hot by promoting tumor-killing immune cell infiltration in a murine triple-negative breast cancer model

BackgroundCD24 plays a crucial role not only in promoting tumor progression and metastasis but also in modulating macrophage-mediated anti-tumor immunity. However, the impact of tumor CD24 on the immune landscape of the tumor microenvironment (TME) remains poorly explored. Here, we investigated the role of CD24a, murine CD24 gene, in the progression and immune dynamics of the tumor microenvironment (TME) in the 4T1 murine model of triple-negative breast cancer (TNBC). Methods: We employed Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 technology to perform a gene knockout of Cd24a in 4T1 cells. Flow cytometry was utilized to analyze the distribution and number of immune cells, including myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells, T cells, and macrophages, within tumors, spleens, and bone marrow. Immunofluorescence (IF) staining was used to detect these immune cells in tumor sections. Additionally, ANDOR Dragonfly High-Speed Confocal was used to perform three-dimensional (3D) mapping of mouse tumors. ResultsOur study showed that knocking out CD24a significantly impeded tumor progression and prolonged mouse survival. Flow cytometry and IF analysis revealed that the loss of CD24a transformed tumor microenvironment from cold to hot by promoting the infiltration of M1 macrophages, cytotoxic CD8+ T cells, and CD49b+ natural killer (NK) cells while reducing the recruitment and expansion of granulocytic myeloid-derived suppressor cells (gMDSCs) in the TME. Additionally, the 3D mapping of TME further validated the "hot state" of CD24a knockout tumors. ConclusionsOur study provides the first evidence that targeting CD24a could effectively reprogram the TME, enhancing its immunogenicity, and transforming immune cold tumors into hot tumors. This strategy may offer a promising therapeutic strategy for enhancing the immune response against poorly immunogenic tumors.

cancer biology↗

bioRxiv2022

RNase H-based analysis of synthetic mRNA 5-prime cap incorporation

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5 cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5 cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of pre-defined short fragments from the 5 end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5 cleavage products, and LC-MS intact mass analysis. In this communication, we describe 1) a framework to design site-specific RNA cleavage using RNase H; 2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; 3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5 cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

biochemistry↗

bioRxiv2022

RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely regulated by structured RNA elements prevalent in long noncoding and other cellular RNAs.

biochemistry↗

bioRxiv2021

A metal ion orients mRNA to ensure accurate 2'-O ribosyl methylation of the first nucleotide of the SARS-CoV-2 genome

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in the host cell. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

biochemistry↗

bioRxiv2020

Structural Basis of RNA Cap Modification by SARS-CoV-2 Coronavirus

The novel severe acute respiratory syndrome coronoavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused over 2 million infections worldwide in four months. In SARS coronaviruses, the non-structural protein 16 (nsp16) methylates the 5-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of full-length nsp16 and nsp10 of SARS-CoV-2 in the presence of cognate RNA substrate and a methyl donor, S-adenosyl methionine. The nsp16/nsp10 heterodimer was captured in the act of 2-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We reveal large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This structure provides new mechanistic insights into the 2-O methylation of the viral mRNA cap. We also discovered a distantly located ligand-binding site unique to SARS-CoV-2 that may serve as an alternative target site for antiviral development.

biophysics↗

bioRxiv2019

In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme

Eukaryotic mRNAs are modified at their 5 end early during transcription by the addition of N7-methylguanosine (m7G), which forms the \"cap\" on the first 5 nucleotide. Identification of the 5 nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS (yDcpS) and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that yDcpS efficiently decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5 end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.

molecular biology↗