Biology subjects

Kuo, C.-W.

Publications and source records attributed to Kuo, C.-W..

6 recordsLinked to original sources

bioRxiv2025

Hyperglycemia-Driven Hepatic Immune Dysfunction Facilitates Microbial Dissemination Post-Myocardial Infarction

BackgroundThe gut microbiome is intimately connected to cardiovascular health through the gut-heart axis and plays a pivotal role in maintaining homeostasis. Myocardial infarction (MI) disrupts this homeostatic balance, leading to widespread adverse effects. Hyperglycemia, a hallmark of metabolic dysfunction, further exacerbates these disruptions, emphasizing the need to understand the underlying mechanisms to develop effective therapeutic strategies for mitigating the cascading complications along the gut-heart axis. This study aims to elucidate the dynamics of gut barrier disruption during MI, and explore the livers function as an immune sentinel in this process, with a focus on the impact of hyperglycemia on microbial dissemination, systemic inflammation, and liver immune function. MethodsA murine MI model was used to evaluate gut permeability, bacterial translocation, and hepatic immune responses. MI was induced via permanent left anterior descending artery ligation. Hyperglycemia was established through streptozotocin injections and a high-fat, high-sugar diet. Gut barrier integrity was assessed using FITC-dextran assays, and microbial translocation was tracked through intravital imaging and anaerobic bacterial cultures from multiple organs. Hepatic immune function was analyzed via flow cytometry, cytokine profiling, and phagocytosis assays. 16S rRNA sequencing characterized the composition of translocated bacteria. ResultsMI significantly increased intestinal permeability, with hyperglycemia further exacerbating gut barrier dysfunction. Intravital imaging revealed bacterial translocation through the portal vein to the liver, highlighting the livers role in microbial interception. Hyperglycemia impaired hepatic macrophage function by activating NLRP3 inflammasome signaling, reducing bacterial clearance and promoting persistent liver colonization. Systemic inflammatory cytokines, particularly TNF-, were elevated, further facilitating microbial dissemination. 16S rRNA sequencing demonstrated host-dependent stochastic variability in translocated bacterial composition. ConclusionThe liver serves as a key immune regulator in the gut-liver-heart axis but is functionally compromised under hyperglycemia, exacerbating systemic inflammation and microbial dissemination post-MI. Targeting NLRP3 signaling and restoring gut barrier integrity may mitigate post-MI complications, particularly in hyperglycemic conditions. These findings underscore the need for integrated therapeutic strategies incorporating metabolic control and microbiome-targeted interventions to improve post-MI outcomes.

immunology↗

bioRxiv2024

Lipid Nanoparticle Barcoding for Multiplexed Single-Cell RNA Sequencing

Sample multiplexing is an emerging method in single-cell RNA sequencing (scRNA-seq) that addresses high costs and batch effects. Current multiplexing schemes use DNA labels to barcode cell samples but are limited in their stability and extent of labeling across heterogeneous cell populations. Here, we introduce Nanocoding using lipid nanoparticles (LNPs) for high barcode labeling density in multiplexed scRNA-seq. LNPs reduce dependencies on cell surface labeling mechanisms due to multiple controllable means of cell uptake, amplifying barcode loading 10-100-fold and allowing both protection and efficient release by dissolution. In cultured cell lines and heterogeneous cells from tissue digests, Nanocoding occurs in 40 minutes with stability after sample mixing and requires only commercially available reagents without novel chemical modifications. In spleen digests, 6-plex barcoded samples show minimal unlabeled cells, with all barcodes giving bimodal count distributions. Challenging samples containing lipid-rich debris and heterogeneous cells from adipose tissue of obese rodents show more than 95% labeling with all known subtypes identified. Using Nanocoding, we investigate gene expression changes related to aging in adipose tissue, profiling cells that could not be readily identified with current direct conjugate methods using lipid or antibody conjugates. This ease of generating and tuning these constructs may afford efficient and robust whole-sample multiplexing with minimal sample crosstalk.

bioengineering↗

bioRxiv2021

Cancer malignancy is correlated with up-regulation of PCYT2-mediated glycerol phosphate modification of α-dystroglycan

The dystrophin-glycoprotein complex connects the cytoskeleton with base membrane components such as laminin through unique O-glycans displayed on -dystroglycan (-DG). Genetic impairment of elongation of these glycans causes congenital muscular dystrophies. We previously identified that glycerol phosphate (GroP) can cap the core part of the -DG O-glycans and terminate their further elongation. This study examined the possible roles of the GroP modification in cancer malignancy, focusing on colorectal cancer. We found that the GroP modification critically depends on PCYT2, which serves as CDP-Gro synthase. Furthermore, we identified a significant positive correlation between cancer progression and GroP modification, which also correlated positively with PCYT2 expression. Moreover, we demonstrate that GroP modification promotes the migration of cancer cells. Based on these findings, we propose that the GroP modification by PCYT2 disrupts the glycan-mediated cell adhesion to the extracellular matrix and thereby enhances cancer metastasis. Thus, the present study suggests the possibility of novel approaches for cancer treatment by targeting the PCYT2-mediated GroP modification.

biochemistry↗

bioRxiv2021

Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants

Extensive glycosylation of the spike protein of SARS-CoV-2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the COVID-19 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain that make it refractory to most neutralizing antibodies directed against this domain. Despite maintaining an overall similar structural conformation, our mass spectrometry-based site-specific glycosylation analyses of similarly produced spike proteins with and without the D614G and Alpha variant mutations reveal a significant shift in the processing state of N-glycans on one specific N-terminal domain site. Its conversion to a higher proportion of complex type structures is indicative of altered spatial accessibility attributable to mutations specific to the Alpha variant that may impact its transmissibility. This and other more subtle changes in glycosylation features detected at other sites provide crucial missing information otherwise not apparent in the available cryogenic electron microscopy-derived structures of the spike protein variants.

biochemistry↗

bioRxiv2021

Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as self, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4GlcNAc and Neu5Ac2-3Gal{beta}1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Ac2-3Gal{beta}1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.

immunology↗

bioRxiv2021

COVID-19 dominant D614G mutation in the SARS-CoV-2 spike protein desensitizes its temperature-dependent denaturation

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, making it the dominant form in the COVID-19 pandemic. Here we demonstrated by cryo-electron microscopy that the D614G mutation does not significantly perturb the structure of the spike protein, but multiple receptor binding domains are in an upward conformation poised for host receptor binding. The impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics, and to significantly increase the thermal stability under physiological pH. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation, and suggest an approach for better design of spike-protein based conjugates for vaccine development.

biochemistry↗