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Biology subjects

Nguyen, H. C.

Publications and source records attributed to Nguyen, H. C..

13 recordsLinked to original sources

Low-cost monophasic transcranial magnetic stimulator

Transcranial magnetic stimulation (TMS) excites neurons noninvasively by electromagnetic induction and is used in neurophysiology research and in approved therapy for depression. Commercial stimulators cost tens of thousands of dollars. Existing open-source designs are either low-energy and unvalidated or rely on expensive switches and laboratory infrastructure. We present a monophasic, fixed-pulse-shape TMS device built at a parts cost of ~USD 700 which, under specific modeling assumptions, can exceed average human motor thresholds. Our design assumes access to basic, off-the-shelf equipment such as a 24 V power supply unit, an oscilloscope, and a few basic tools. The device charges a 230 F film-capacitor bank and discharges it through a self-wound figure-of-eight coil using a thyristor, producing a fixed pulse with a positive lobe lasting approximately 90 s. A Zero-Voltage Switching (ZVS) driver-based charging circuit charges the capacitor bank up to 1460 V from a 24 V bench supply. Three galvanically isolated voltage domains, redundant interlocks, and passive and active discharge paths help mitigate the safety risks involved with handling lethal energy levels. We also present a low-cost way to characterize the device by reconstructing coil di/dt from pickup-coil dB/dt maps to estimate the induced cortical E-fields. At the maximum capacitor voltage, the recovered maximal di/dt is 110.86 A/s, giving estimated 99.9th percentile cortical E-fields of 159 V/m at Oz and 196 V/m at C3 on an example anatomy. Although not yet approved for clinical trials and routine stimulation, the device demonstrated the possibility of a cost-effective TMS unit.

bioengineering↗

Title - ATG7 vs. ATG5: Distinct Autophagy Pathways Shaping TGF-β Signaling and Endothelial Function

Autophagy and transforming growth factor-beta (TGF-{beta}) signalling are critical cellular processes that maintain homeostasis and regulate various physiological functions, including endothelial cell function. This study explores the distinct roles of two essential autophagy regulators, autophagy-related gene 5 (ATG5) and ATG7, in modulating TGF-{beta} signalling and endothelial function. To this end, ATG5 and ATG7 were selectively silenced in endothelial cells, with knockdown efficiency confirmed via RT-qPCR and Western blot analyses. Gene expression profiling revealed differential regulation of TGF-{beta} signalling components following ATG5 or ATG7 silencing. Functional assays demonstrated that ATG5 knockdown enhanced endothelial cell proliferation and migration, whereas ATG7 silencing produced distinct, less pronounced effects. Furthermore, downstream effectors of the TGF-{beta} pathway exhibited gene-specific modulation, underscoring divergent roles of ATG5 and ATG7 in this signalling cascade. Collectively, these findings highlight the non-redundant functions of ATG5 and ATG7 in coordinating TGF-{beta} signalling pathways, offering new insights into their contribution to endothelial physiology and potential as therapeutic targets in vascular pathologies.

cell biology↗

BRCA2-deficiency Causes Global Transcriptomic Alterations in Endothelial Cells

This study aims to explore the alterations in gene expression following BRCA2 loss in endothelial cells and to investigate the potential contribution of BRCA2 in regulating the endothelial protein-coding transcriptome. Cultured human umbilical vein endothelial cells (HUVECs) were transfected with siBRCA2 or non-targeting scrambled RNA as control. Total RNA was extracted and used for RNA sequencing following purification and quality check. Transcription profiles and differentially expressed genes (DEGs) were identified. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In total, 3196 DEGs with (cut-off 1.5-fold) were identified, of which 1852 genes were upregulated, and 1344 genes were downregulated in BRCA2-deficient HUVECs. Cell cycle exit and neuronal differentiation 1 (CEND1) and mannose receptor C-type 1 (MRC1) were the most significantly up- and downregulated genes, respectively, in BRCA2-deficient endothelial cells. The expression of top DEGs were further validated by RT-qPCR in HUVECs and in human coronary artery endothelial cells. The GO and KEGG analysis suggest that cell adhesion molecules and cytokine-cytokine receptor interaction may play an important role in BRCA2-deficient endothelial cells. Overall, these findings contribute to a greater understanding of the mechanisms involved in BRCA2-mediated endothelial function.

genomics↗

The effect of fatty acid binding protein-3 exposure on endothelial transcriptomics

BackgroundFatty acid binding protein-3 (FABP3) is released in circulation following myocardial infarction and an increased level of circulatory FABP3 is also reported in peripheral artery disease patients exposing endothelial cells to higher level of FABP3. Recently, loss of endothelial FABP3 was shown to protect endothelial cells against inflammation-induced endothelial dysfunction, however, the effect of FABP3-exposure on endothelial cells is unknown. Accordingly, to study the effect of FABP3-exposure on endothelial cells we performed transcriptomic profiling following recombinant human FABP3 (rhFABP3) treatment to endothelial cells. MethodCultured human endothelial cells were treated with either vehicle or rhFABP3 (50 ng/ml, 6h) and then RNA-sequencing was performed. Gene expression analysis followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify differentially expressed genes and affected cellular functions and pathways. ResultsDifferential gene expression analysis revealed kinesin family member 26b (KIF26B) as the most upregulated and survival of motor neuron 2 (SMN2) as the most downregulated genes in rhFABP3-treated in comparison to vehicle-treated endothelial cells. Most of the differentially expressed genes were associated with endothelial cell motility, immune response, and angiogenesis. GO and KEGG analyses indicated that rhFABP3 exposure impacts several crucial pathways, prominently "Regulation of leukocyte mediated cytotoxicity" and "Natural killer cell mediated cytotoxicity", suggesting its involvement in endothelial cell physiology and response mechanisms to cardiovascular stress. ConclusionThis is the first study evaluating rhFABP3-induced transcriptomics in human endothelial cells. Our data reveal novel genes and pathways affected by FABP3-exposure to endothelial cells. Further research is necessary to validate these findings and fully understand FABP3s role in endothelial biology and in cardiovascular diseases like myocardial infarction and peripheral artery disease.

biochemistry↗

Chloroquine Up-regulates Expression of SARS-CoV-2 receptor Angiotensin Converting Enzyme-2 in Endothelial Cells

Background and PurposeThe novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) posed a serious threat to global public health. Hydroxychloroquine (HCQ), which is a derivative of Chloroquine (CQ), was a WHO-recommended drug to treat COVID-19 with mixed effects. The purpose of the present study is to evaluate the plausible mechanisms of HCQ actions behind its observed mixed effect. Key ResultsWe demonstrate that CQ-treatment significantly up-regulates mesenchymal markers and SARS-CoV-2 receptor ACE2 in cultured endothelial cells. Conclusions & ImplicationsThe detrimental effect of HCQ in seriously ill COVID-19 patients might be due to CQ-induced increased expression of endothelial ACE2 exacerbating the severity of SARS-CoV-2 infection. Our study warrants further investigation in animal models and humans and caution while prescribing HCQ to patients with an impaired renin-angiotensin-aldosterone system, such as in hypertension, cardiovascular diseases, or chronic kidney disease; particularly with ACE-inhibitors or statin therapy.

cell biology↗

Pigment Binding in The Light-Dependent Protochlorophyllide Oxidoreductase

The Light-Dependent Protochlorophyllide Oxidoreductase (LPOR) is a key enzyme in chlorophyll biosynthesis and its photocatalytic mechanism has long intrigued researchers. However, the lack of structural data for the active complex has impeded understanding of its reaction mechanism. A recent high-resolution structure of enzyme in the active conformation has established a robust foundation for validating hypotheses concerning pigment binding, residue involvement, and consequently, the reaction mechanism. Surprisingly, this new structure challenges previously proposed mechanisms, especially concerning the orientation of the bound protochlorophyllide (Pchlide) pigment. In this study, we employ molecular dynamics and hybrid quantum-mechanics/molecular-mechanics (QM/MM) simulations along with site-directed mutagenesis to compare two Pchlide binding modes: one aligned with previous proposals (mode A), and the other consistent with the recent experimental data (mode B). Binding energy calculations reveal thermodynamic instability of binding mode A due to nonspecific interactions, while mode B exhibits distinct stabilizing interactions yielding favorable binding. QM/MM-based local energy decomposition analysis unravels a complex interaction network that reinforces pigment stabilization in this conformation. Notably, interactions involving Tyr177, His319, and the carboxyl group at C131 influence the pigments excited state energy and potentially contributing to the substrate specificity of the enzyme. Our results uniformly favor binding mode B as represented in the new cryo-EM structure, over the previously assumed mode A. These findings challenge established interpretations and underscore the need for a comprehensive re-evaluation of the reaction mechanism of LPOR that correctly considers pigment interactions and substrate orientation in the binding pocket. Significance StatementA crucial step in the biosynthesis of the all-important photosynthetic pigment chlorophyll is the reduction of a double C=C bond in its precursor protochlorophyllide (PChlide). This is catalyzed by the Light-Dependent Protochlorophyllide Oxidoreductase (LPOR) via an extremely rare example of a biological photocatalytic reaction. Understanding of the LPOR mechanism has been hindered by limited insight into the structure of its active complex. A recent high-resolution LPOR cryo-EM structure substantiates pigment binding, residue interactions, and the reaction mechanism, but contrasts markedly with all previous assumptions regarding the binding mode of the substrate PChlide. Using molecular dynamics simulations, quantum-mechanics/molecular-mechanics calculations, and mutagenesis, we compare and evaluate the two possible Pchlide binding modes, the one assumed previously (mode A) and the one supported by recent data (mode B). Our findings conclusively favor mode B, challenging prior assumptions and pointing toward novel mechanistic possibilities for this unique photocatalytic reaction.

biochemistry↗

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

biophysics↗

Photocatalytic plant LPOR forms helical lattices that shape membranes for chlorophyll synthesis

Chlorophyll (Chl) biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic1. In flowering plants, the enzyme Light-dependent Protochlorophyllide OxidoReductase (LPOR) captures photons to catalyze the penultimate reaction: the reduction of a double-bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide)2,3. In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis4,5. However, the complete 3D structure of LPOR, the higher-order architecture of LPOR oligomers, and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the cofactor NADPH, remain unknown. Here we report the atomic structure of LPOR assemblies by electron cryo-microscopy (cryoEM). LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolammelar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Our structure of the catalytic site, moreover, challenges previously proposed reaction mechanisms6. Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants orchestrated by LPOR.

biophysics↗

Membrane constriction and thinning by sequential ESCRT-III polymerization

The Endosomal Sorting Complexes Required for Transport (ESCRTs) mediate diverse membrane remodeling events. These activities typically require ESCRT-III proteins to stabilize negatively-curved membranes, although recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane curvature shaping by these proteins remains poorly understood. To learn how ESCRT-IIIs shape membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B+IST1, and IST1-only filaments by electron cryomicroscopy. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand upon the CHMP1B filament, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction, moreover, thins the underlying bilayer and lowers the barrier to membrane fission. Together, our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, tube constriction, and bilayer thinning.

biophysics↗

Whole genome sequencing of 358 brown planthoppers uncovers the landscape of their migration and dispersal worldwide

The brown planthopper (BPH), Nilaparvata lugens, is a serious migratory rice pest, which is distributed in the broad area of the tropical and temperate Asian-Pacific region. However, we know little about key aspects regarding its evolution such as how they diverged and dispersed worldwide. By resequencing and analyzing 358 BPH genomes from 92 populations across the world, we uncover the genetic relationships among their worldwide populations and the history of their global dispersal. We recovered five genetic groups representing the major population structures. Of these, Australian BPHs were shown large genetic divergence with Asian BPHs; two distinct groups have formed in South and Southeast/East Asia that show strong genetic admixture in the southwest border regions of China and west Thailand with Myanmar; two local populations in Bangladesh and Fujian province of China, respectively, unexpectedly separated with surrounding populations. We also find the genetic similarity and closely phylogenetic relationships between majority of East Asian BPHs and Indo-china peninsula BPHs, indicating that Southeast Asia mainland is the major insect sources and overwintering sites for East Asia. Our study provides important molecular evidence to address BPH evolution and other key aspects of its biology such as insecticides resistance and rice varieties virulence.

evolutionary biology↗

Structural basis of eIF2B-catalyzed GDP exchange and phosphoregulation by the integrated stress response

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a GTPase, that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2s dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryoEM structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2Bs bipartite catalytic centers to catalyze guanine nucleotide exchange. Phosphorylation refolds eIF2, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequesters it into a non-productive complex.\n\nOne Sentence SummaryStructures of translation factors eIF2 and eIF2B reveal the mechanism of nucleotide exchange and its phosphoregulation during stress.

biochemistry↗

The ESCRT-III proteins IST1 and CHMP1B assemble around nucleic acids

ESCRT-III proteins can promote inside-out or outside-in membrane tubulation and fission. In addition, several observations suggest that ESCRT factors may also associate with nucleic acids during development, different stages of the cell cycle, and during retro-transposition of parasitic nucleic acids like LINE1 elements. Two ESCRT-III subunits, IST1 (aka CHMP8) and CHMP1B, can coassemble as an external protein coat around liposomes in vitro and around recycling endosomal tubules in living cells. Here we show that recombinant IST1 and CHMP1B can also copolymerize into double stranded filaments that surround nucleic acids. Electron cryo-microscopy reconstructions of nucleic acid-bound IST1-CHMP1B copolymers revealed that the polynucleotides track along a binding groove formed between filaments of the inner CHMP1B strand. The well-ordered structures also reveal that the C-terminal tails of CHMP1B subunits extrude through the outer IST1 layer to the tube exterior. As a result, the MIT domain binding motifs of both CHMP1B and IST1 are arrayed on the outer surface of the copolymer, where they could bind and recruit MIT domain-containing co-factors, such as the SPASTIN ATPase or the USP8 ubiquitin protease. Our structure raises the possibility that ESCRT-III proteins may form nucleic acid complexes in mammalian cells.

biochemistry↗

Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from substrate to competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation and, remarkably, in rodents enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by electron cryo-microscopy. Structural and biochemical analyses revealed that formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and that ISRIB promoted this step by cross-bridging a central symmetry interface. Regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.

biochemistry↗