Biology subjects

Ooi, A.

Publications and source records attributed to Ooi, A..

4 recordsLinked to original sources

bioRxiv2024

Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

SummaryThrough the Astrocyte Neuron Lactate Shuttle, astrocyte-derived lactate fuels the high-energy demands of neurons and acts as a signaling molecule, promoting synaptic plasticity and memory consolidation. Lactate regulates neuronal excitability and modulates the expression of genes related to synaptic plasticity and neuroprotection, but the molecular mode for these signaling actions is uncertain. Using patch-clamp recordings in cultured cortical neurons, we found that lactate enhances both the amplitude and the inactivation time constant of NMDA receptor currents (INMDAR) evoked by brief applications of glutamate and glycine. Not reproduced by HCAR1 agonists, this modulation depends on monocarboxylate transporters and lactate dehydrogenase, indicating the requirement for lactate entry and metabolic conversion into pyruvate and NADH formation within neurons. Disruption of intracellular calcium dynamics or inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a NMDAR-associated kinase linking Ca2+ signal to long-term potentiation (LTP), significantly diminishes the effects of lactate on INMDAR. We identified two redox-sensitive cysteine- containing sequences in the intrinsically disordered intracellular C-terminal domain of the GluN2B subunit that play a role in the potentiation of NMDAR by lactate. In a compelling set of experiments using HEK cells, we observed that the presence of functional CaMKII and GluN2B-containing NMDARs is necessary for the lactate-enhancing effects. Mutations in GluN2B that prevent CaMKII binding or redox regulation via cysteines abrogate the modulatory action of lactate. Immunoprecipitation experiments in neurons attest that lactate increases the association between CaMKII and GluN2B. This interaction is crucial for the potentiation of INMDAR amplitude by lactate. Proximity ligation assays between GluN2B and the postsynaptic density marker PSD-95 revealed that lactate induced an accumulation of GluN2B in dendritic spines, an effect that was prevented by a CaMKII peptide inhibitor. These results highlight a mechanistic pathway whereby lactate boosts NMDAR function through intracellular metabolic conversion and redox-sensitive interactions requiring CaMKII, establishing a link between astrocyte metabolism and synaptic modulation in neurons.

neuroscience↗

bioRxiv2023

Proteome profiling of nasopharynx reveals pathophysiological signature of COVID-19 disease severity

An aberrant innate immune system caused by the beta coronavirus SARS-CoV-2 is a characteristic manifestation of severe coronavirus disease 2019 (COVID-19). Here, we performed proteome profiling of nasopharyngeal (NP) swabs from 273 hospitalized patients with mild and severe COVID-19 symptoms, including non-survivors. We identified depletion in STAT1-mediated type I interferon response, retinol metabolism and NRF2 antioxidant system that are associated with disease severity in our patient demography. We found that the dysregulation of glucocorticoid signaling and renin-angiotensin-aldosterone system (RAAS) contribute to the pathophysiology of COVID-19 fatality. Hyperactivation of host innate immune system was observed in severe patients, marked by elevated proteins involved in neutrophil degranulation and platelet aggregation. Our study using high-throughput proteomics on the nasopharynx of COVID-19 patients provides additional evidence on the SARS-CoV-2-induced pathophysiological signatures of disease severity and fatality.

immunology↗

bioRxiv2020

Single cell mutational profiling delineates clonal trajectories in myeloid malignancies

Myeloid malignancies, including acute myeloid leukemia (AML), arise from the proliferation and expansion of hematopoietic stem and progenitor cells which acquire somatic mutations. Bulk molecular profiling studies on patient samples have suggested that somatic mutations are obtained in a step-wise fashion, where mutant genes with high variant allele frequencies (VAFs) are proposed to occur early in disease development and mutations with lower VAFs are thought to be acquired later in disease progression1-3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity and clone size, or offer definitive evidence of mutational order. To elucidate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is most commonly comprised of a small number of dominant clones, which in many cases harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We also used these data to map the clonal trajectory of each patient and found that specific mutation combinations (FLT3-ITD + NPM1c) synergize to promote clonal expansion and dominance. We combined cell surface protein expression with single cell mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of clonal architecture at a single cell level provide novel insights into the pathogenesis of myeloid transformation and how clonal complexity contributes to disease progression.

cancer biology↗

bioRxiv2019

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in-depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project.

genomics↗