Biology subjects

Loh, J.

Publications and source records attributed to Loh, J..

6 recordsLinked to original sources

bioRxiv2024

A rapid approach for linear epitope vaccine profiling reveals unexpected epitope tag immunogenicity

Antibody epitope profiling is essential for assessing the robustness of vaccine-induced immune responses, particularly while in development. Despite advancements in computational tools, high throughput experimental epitope validation remains an important step. Here, we describe a readily accessible method for rapid linear epitope profiling using phage-displayed oligo pools in combination with Nanopore deep sequencing. We applied this approach to TeeVax3, a Group A Streptococcus vaccine candidate, to investigate the antibody response generated in a pre-clinical rabbit model and assess antigen immunogenicity. Surprisingly, we found a strong bias in antibody binding response towards the N-terminal epitope tag used for purification. These tags are widely reported to have low immunogenicity and are frequently left uncleaved in pre-clinical studies. We further confirmed that the observed immune response against the epitope tag dominated even the conformational binding response and, using synthetic peptides, narrowed the epitope down to a set of 10 residues inclusive of the Histidine residues. Our findings highlight the importance of epitope-tag removal in pre-clinical studies and demonstrate the utility of rapid nanopore sequencing for early-stage vaccine evaluation.

immunology↗

bioRxiv2022

Past, present, and future of the Living Planet Index

As we enter the next phase of international policy commitments to halt biodiversity loss (e.g. Post-2020 Biodiversity Framework), biodiversity indicators will play an important role forming the robust basis upon which targeted, and time sensitive conservation actions are developed. Population trend indicators are perhaps the most powerful tool in biodiversity monitoring due to their responsiveness to changes over short timescales and their ability to aggregate species trends from global down to at a sub-national or even local scale. We consider how the project behind the foremost population level indicator - the Living Planet Index - has evolved over the last 25 years, its value to the field of biodiversity monitoring, and how its components have portrayed a compelling account of the changing status of global biodiversity through its application at policy, research and practice levels. We explore ways the project can develop to enhance our understanding of the state of biodiversity and share lessons learned to inform indicator development and mobilise action. O_TEXTBOXBox 1. The Living Planet Index Project The Living Planet Index project (the index, methodology, and database) and its secondary outputs (methods papers and R code, database and website, global index, and subset indices) have had wide-ranging applications within the fields of biodiversity monitoring and research, as well as across policy, education, and outreach. The Living Planet Index (LPI) is a biodiversity indicator which tracks trends in the relative abundance of wild vertebrate populations (where population is defined as to a single species in a defined location rather than the biological definition). Relative abundance captures how populations are changing over time on average in comparison to a reference point, or "baseline" (the LPI uses 1970). It is often described as analogous to a stock market index for species. The index is comprised of thousands of population time-series for vertebrate species from locations around the world; the trends from these populations are averaged to produce terrestrial, freshwater, and marine indices, which are further aggregated to a global LPI. The latest global LPI shows a decline of 68% between 1970 and 2016 globally 1. This is an average trend based on time-series data from 20,811 populations of 4,392 species of mammals, birds, reptiles, amphibians, and fish. The LPI database (LPD) can include population data for any species for which time-series population data could be found, regardless of threat status, or whether they show increasing or declining trends. These population time-series are sourced from scientific papers, online databases, government, and expert led published reports. They can be searched and downloaded from the project website (www.livingplanetindex.org). More technical information is available on the LPI stats website (http://stats.livingplanetindex.org/). C_TEXTBOX

ecology↗

bioRxiv2021

Dynamin-2 Phosphorylation as A Critical Regulatory Target of Bin1 and GSK3α for Endocytosis in Muscle

Tight regulation of endocytosis ensures accurate control of cellular signaling and membrane dynamics, which are crucial for tissue morphogenesis and functions. Mutations of Bin1 and dynamin-2 (Dyn2), proteins that generate membrane curvature and sever endocytic invaginations, respectively, cause progressive hereditary myopathy. Here, we show that Bin1 inhibits Dyn2 via direct interaction of its SRC Homology 3 (SH3) domain with the proline-rich domain (PRD) of Dyn2. Phosphorylation of S848 of Dyn2 by GSK3, a kinase downstream of insulin signaling, relieves Dyn2 from the inhibition of Bin1 and promotes endocytosis in muscle. Mutations of Bin1 associated with centronuclear myopathy disrupt its inhibition of Dyn2, thereby exaggerating Dyn2 fission activity and causing excessive fragmentation of T-tubules in the muscle cells. Our work reveals how Bin1-Dyn2 interaction fine-tunes membrane remodeling at the molecular level, and lay the foundation for future exploration of endocytic regulation and hereditary muscle diseases.

cell biology↗

bioRxiv2021

Insulin Signaling Attenuates GLUT4 Endocytosis in Muscle Cells via GSK3a-Dyn2-Bin1 Interplay

Insulin-induced translocation of glucose transporter 4 (GLUT4) to the plasma membrane of skeletal muscle is critical for postprandial glucose uptake; however, whether the internalization of GLUT4 into cells is also regulated by insulin signaling remains unclear. Here, we discover that the activity of dynamin-2 (Dyn2), pivotal GTPase catalyzing GLUT4 internalization, is regulated by insulin signaling in muscle cells. The membrane fission activity of Dyn2 is inhibited in muscle cells through binding with the SH3 domain-containing protein Bin1. Phosphorylation of Serine848 on Dyn2 by GSK3 or the mutations of Bin1-SH3 in patients with centronuclear myopathy, elevate the activity of Dyn2 due to reduced binding affinity toward Bin1. The augmented Dyn2 fission activity in muscle cells leads to GLUT4 internalization and Bin1-tubule vesiculation. Together, our findings reveal a new role of insulin signaling in glucose metabolism and muscle physiology via attenuating Dyn2 activity thus regulating GLUT4 endocytosis in muscle cell.

cell biology↗

bioRxiv2019

Acute decrease in plasma membrane tension induces macropinocytosis via PLD2 activation

Internalization of macromolecules and membrane into cells through endocytosis is critical for cellular growth, signaling, and membrane tension homeostasis. Although endocytosis is responsive to both biochemical and physical stimuli, how physical cues modulate endocytic pathways is less understood. In contrary to the accumulating discoveries on effects of increased membrane tension on endocytosis, little is known about how a drop of tension impacts membrane trafficking. Here we reveal that acute reduction of plasma membrane tension results in phosphatidic acid, F-actin and dynamin 2-enriched dorsal membrane ruffling and subsequent macropinocytosis in myoblast. The membrane flaccidity-induced local phosphatidic acid production depends on phospholipase D2 (PLD2) that is activated via lipid raft disruption. Furthermore, the \"membrane flaccidity-PLD2-macropinocytosis\" pathway is dominant in myotube, reflecting a potential mechanism of membrane tension homeostasis upon intensive muscle stretching and relaxation. Together, we identify a new mechanotransduction pathway which converts acute tension drop into PA production and subsequently initiates macropinocytosis via actin and dynamin activities.\n\nSummaryWe reveal a mechanical induction of macropinocytosis that is elicited by acute decrease of plasma membrane tension, followed by lipid raft destabilization, PLD2 activation and PA production.

cell biology↗

bioRxiv2019

Comparison Between UMAP and t-SNE for Multiplex-Immunofluorescence Derived Single-Cell Data from Tissue Sections

Using human hepatocellular carcinoma (HCC) tissue samples stained with seven immune markers including one nuclear counterstain, we compared and evaluated the use of a new dimensionality reduction technique called Uniform Manifold Approximation and Projection (UMAP), as an alternative to t-Distributed Stochastic Neighbor Embedding (t-SNE) in analysing multiplex-immunofluorescence (mIF) derived single-cell data. We adopted an unsupervised clustering algorithm called FlowSOM to identify eight major cell types present in human HCC tissues. UMAP and t-SNE were ran independently on the dataset to qualitatively compare the distribution of clustered cell types in both reduced dimensions. Our comparison shows that UMAP is superior in runtime. Both techniques provide similar arrangements of cell clusters, with the key difference being UMAPs extensive characteristic branching. Most interestingly, UMAPs branching was able to highlight biological lineages, especially in identifying potential hybrid tumour cells (HTC). Survival analysis shows patients with higher proportion of HTC have a worse prognosis (p-value = 0.019). We conclude that both techniques are similar in their visualisation capabilities, but UMAP has a clear advantage over t-SNE in runtime, making it highly plausible to employ UMAP as an alternative to t-SNE in mIF data analysis.

bioinformatics↗