Biology subjects

Kim, T.

Publications and source records attributed to Kim, T..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Mitochondrial NADK2-dependent NADPH controls Tau oligomer uptake in human neurons

Alterations in NADH and NADPH metabolism are associated with aging, cancer, and Alzheimers Disease. Using 2P-FLIM imaging of the mitochondrial NAD(P)H in live human neurons and PS19 mouse brains, we show that tau oligomers (TauO) upregulate the mitochondrial de novo NADPH synthesis through NADK2. This process controls LRP1-mediated internalization of TauO, setting a vicious cycle for further TauO internalization. Thus, mitochondrial NADK2-dependent NADPH controls a key step in TauO toxicity.

neuroscience↗

bioRxiv2024

APOBEC3A, not APOBEC3B, drives deaminase mutagenesis in human gastric epithelium

Cancer genomes frequently carry APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like)-associated DNA mutations, suggesting APOBEC enzymes as innate mutagens during cancer initiation and evolution. However, the pure mutagenic impacts of the specific enzymes among this family that are responsible for APOBEC-associated mutagenesis remain unclear in human normal cell lineage, particularly the comparative mutagenic activities of APOBEC3A and APOBEC3B. Here, we investigated the mutagenic contributions of these enzymes through whole-genome sequencing of human normal gastric organoid lines carrying doxycycline-inducible APOBEC3A or APOBEC3B cassettes. Our findings demonstrated that transcriptional APOBEC3A upregulation led to the acquisition of a massive number of genomic mutations in a few cell cycles. By contrast, APOBEC3B upregulation did not generate a substantial number of mutations in gastric epithelium. APOBEC3B-associated mutagenesis remained insignificant even after a combined inactivation of TP53. Based on the spectrum of acquired mutations after APOBEC3A upregulation, we further analyzed APOBEC3A-associated mutational signatures, encompassing indels mainly composed of 1bp deletions, characteristics of clustered mutations, and selective pressures operative on cells carrying the mutations. Our observations provide a clear foundation for understanding the mutational impact of APOBEC enzymes in human cells.

genomics↗

bioRxiv2024

RBP4 functions as an antagonistic ligand of orphan DR6

Death receptor (DR) is a unique transmembrane receptor mediating extrinsic factor-induced programmed cell death pathways. For most DRs, the regulatory mechanism has been revealed to involve agonistic death ligand (e.g., TRAIL, TNF-)-driven receptor clustering and activation, offering an in-depth understanding of the mechanistic basis for cell death regulation and therapeutic strategies. However, only death receptor 6 (DR6) remains orphan, and its molecular mechanism is poorly understood. Here, we identified retinol binding protein 4 (RBP4), a major vitamin-A carrier protein, as an antagonistic ligand of DR6. Single-molecule photobleaching and single-receptor tracking analyses revealed that RBP4 drives DR6 dimerization on the plasma membrane to prevent DR6 prodegenerative heterointeractions and consequent neurodegenerative processes. DR6 homodimerization predominantly depends on an extracellular intrinsically disordered region, allowing the agonistic propensity of increasing Ca2+ levels. Collectively, our study establishes the distinct molecular mechanism of DR6 and highlights the non-canonical role of RBP4 as a cell death regulator.

molecular biology↗

bioRxiv2024

Growth-coupled microbial biosynthesis of the animal pigment xanthommatin

The mining of genomes across life has unearthed a bounty of biosynthetic potential to diverse molecules key to a biobased future. While the heterologous expression of metabolic pathways has achieved broad success, most approaches suffer a similar fate in low initial production levels that require extensive, resource-heavy iterative strain engineering refinement. Herein we introduce a growth-coupled biosynthetic (GrowBio) strategy that irrevocably connects microbial growth with specialized compound production. We demonstrate the plug-and-play versatility of GrowBio in the production of the structurally complex animal biopigment xanthommatin, a color-changing ommochrome with material and cosmetic potential. Xanthommatin biosynthesis directly fuels growth of a newly designed Pseudomonas putida 5,10-methylenetetrahydrofolate auxotroph (PUMA). Aided by genome-scale metabolic modeling, PUMA was designed and built to be controlled by endogenous formate co-produced as a coupled biosynthetic byproduct in the multistep conversion of tryptophan to xanthommatin. Adaptive laboratory evolution was utilized to streamline xanthommatins gram-scale bioproduction via growth rate selection, establishing GrowBio as a promising biotechnological approach for establishing and optimizing the microbial production of value-added molecules.

synthetic biology↗

bioRxiv2024

SUMO2 Inhibition Reverses Aberrant Epigenetic Rewiring Driven by Synovial Sarcoma Fusion Oncoproteins and Impairs Sarcomagenesis

Synovial Sarcoma (SySa) is an aggressive soft tissue sarcoma that accounts for 5 - 10% of all soft tissue sarcomas. Current treatment involves radiation and radical surgery including limb amputation, highlighting the urgent need to develop targeted therapies. We reasoned that transcriptional rewiring by the fusion protein SS18-SSX, the sole oncogenic driver in SySa, creates specific vulnerabilities that can be exploited for treatment. To uncover genes that are selectively essential for SySa, we mined The Cancer Dependency Map (DepMap) data to identify genes that specifically impact the fitness of SySa compared to other tumor cell lines. Targeted CRISPR library screening of SySa-selective candidates revealed that the small ubiquitin-like modifier 2 (SUMO2) was one of the strongest dependencies both in vitro as well as in vivo. TAK-981, a clinical-stage small molecule SUMO2 inhibitor potently inhibited growth and colony-forming ability. Strikingly, transcriptomic studies showed that pharmacological SUMO2 inhibition with TAK-981 treatment elicited a profound reversal of a gene expression program orchestrated by SS18-SSX fusions. Of note, genetic or pharmacological SUMO2 inhibition reduced global and chromatin levels of the SS18-SSX fusion protein with a concomitant reduction in histone 2A lysine 119 ubiquitination (H2AK119ub), an epigenetic mark that plays an important role in SySa pathogenesis. Taken together, our studies identify SUMO2 as a novel, selective vulnerability in SySa. Since SUMO2 inhibitors are currently in Phase 1/2 clinical trials for other cancers, our findings present a novel avenue for targeted treatment of synovial sarcoma. SIGNIFICANCEOur study identifies SUMO2 as a selective dependency in synovial sarcoma. We demonstrate that the SUMO2/3 inhibitor TAK-981 impairs sarcomagenesis and reverses the SS18-SSX fusion-driven oncotranscriptome. Our study indicates that SUMO2 inhibition may be an attractive therapeutic option in synovial sarcoma.

cancer biology↗

bioRxiv2024

Characterization, enrichment, and computational modeling of cross-linked actin networks in trabecular meshwork cells

PurposeCross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM), yet their role in ocular hypertension remains unclear. We used a human TM cell line that spontaneously forms fluorescently-labeled CLANs (GTM3L) to explore the origin of CLANs, developed techniques to increase CLAN incidence in GMT3L cells, and computationally studied the biomechanical properties of CLAN-containing cells. MethodsGTM3L cells were fluorescently sorted for viral copy number analysis. CLAN incidence was increased by (i) differential sorting of cells by adhesion, (ii) cell deswelling, and (iii) cell selection based on cell stiffness. GTM3L cells were also cultured on glass or soft hydrogel to determine substrate stiffness effects on CLAN incidence. Computational models were constructed to mimic and study the biomechanical properties of CLANs. ResultsAll GTM3L cells had an average of 1 viral copy per cell. LifeAct-GFP expression level did not affect CLAN incidence rate, but CLAN rate was increased from [~]0.28% to [~]50% by a combination of adhesion selection, cell deswelling, and cell stiffness-based sorting. Further, GTM3L cells formed more CLANs on a stiff vs. a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus. ConclusionsIt is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the effect of CLANs on TM biomechanics and mechanobiology as well as the etiology of CLAN formation in the TM.

cell biology↗

bioRxiv2024

Characterization and analysis of neuronal signaling using microelectrode array combined with rapid and localized cooling device for cryo-neuromodulation

Cryoanesthesia--a purely physical anesthesia treatment that freezes tissue and attenuate nerve activity--can provide fast treatment through freezing and thawing of cryo-machine and is inexpensive compared to other anesthetics. However, cryoanesthesia has not been widely adopted because securing safe and effective conditions requires quantitative measurement and analysis of neuronal signaling during freezing and recovery, for which research tools are limited. A lack of rapid and localized cooling technologies for quantitative cellular level analysis, in particular, hinders research on not only the optimal cryo-modulation of neuronal activities but also its influence to neighboring cells via cellular networks. Here, we introduce a novel cryo-neuromodulation platform, a high-speed precision probe-type cooling device ([~]20{degrees}C/s at cooling) that provides localized cooling combined with a microelectrode array (MEA) system. We explored the temperature conditions for efficient silencing and recovery of neuronal activities without cell damage. We found that electrical activities of neurons were fully recovered within 1 minute of cooling duration with the maximum cooling speed, which was also confirmed with calcium imaging. The impact of silenced neurons on the neighboring neural network was explored using the localized cooling and we perceived that its influence can be transmitted if the neuronal network is well organized. Our new cryo-device provides rapid and reversible control of neural activities, which allows not just quantitative analysis of the network dynamics, but also new applications in clinical settings.

neuroscience↗

bioRxiv2024

Inhibiting monocyte migration reduces arterial thrombosis and facilitates thrombolysis

BackgroundMonocytes contribute to the initiation and propagation of venous thrombosis. Little is known about the roles monocytes play in arterial thrombosis, the cause of stroke and MI. MethodsWe investigated how chemokine receptor 2 (CCR2) knockout (-/-) affects platelet function, blood coagulation, thrombus volume, and thrombolytic susceptibility in 627 mice with FeCl3-mediated carotid arterial thrombosis: three CX3CR1-GFP mice, 326 C57BL/6 mice, and 288 CCR2-/- mice. We performed i) intravital microscopy imaging of leukocyte recruitment to carotid thrombus, ii) flow cytometry-based quantification of leukocytes in blood vs. thrombus and leukocyte-platelet aggregates in blood, iii) platelet aggregometry, iv) coagulation assays, v) micro-computed tomography (microCT)-based thrombus imaging using gold nanoparticles after tissue plasminogen activator (tPA) therapy with or without either a) CCR2 siRNA pretreatment (for 3 days) or post-treatment or b) clopidogrel pretreatment (for 7 days), and vi) histology including scanning electron microscopy. ResultsIntravital microscopy and flow cytometry showed that both neutrophils and monocytes were recruited to the acute arterial thrombus, as observed 30 minutes post-thrombosis. Platelet function tests demonstrated platelet aggregation to be lower in the whole blood of CCR2-/- mice (vs. C57BL/6 mice) but not in their leukocyte-free platelet rich plasma, suggesting this platelet dysfunction is cell-mediated. Flow cytometry experiments revealed lower numbers of monocyte - platelet aggregates (MPAs), a marker of platelet activity, in the blood of CCR2-/- mice, compared to C57BL/6 mice. Blood levels of FXIII and monocyte levels of FXIII-A were increased after carotid thrombosis in C57BL/6 mice but not CCR2-/- mice. Further, in vivo microCT and histology, respectively, showed that CCR2-/- mice had smaller and more porous thrombi with less fibrin cross-linking, compared with C57BL/6 mice. MicroCT also demonstrated that tPA-mediated thrombolysis was faster in CCR2-/- mice and CCR2 siRNA-treated mice, compared to C57BL/6 mice. In addition, clopidogrel had a greater effect on inducing thrombus formation with smaller sizes after FeCl3 application, while CCR2-/- had a greater effect on dissolving thrombus faster after tPA administration. ConclusionsCCR2 antagonism decreases platelet aggregation and reduces FXIII levels in blood and monocytes, thus driving arterial thrombosis towards the generation of a relatively small, porous, more lysable clot. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIn venous thrombosis, monocytes cooperate with platelets and neutrophils to promote thrombus formation; however, few studies have investigated the roles of monocytes in arterial thrombosis. C_LIO_LIAn in vitro study using arterial blood samples from patients with acute coronary syndrome showed that P-selectin on activated platelets upregulated monocyte chemokine receptor 2 (CCR2) expression, which was higher in monocyte-platelet aggregates (MPAs) than in monocytes without platelets. C_LIO_LIIn a mouse study using an in vivo model of FeCl3-induced arterial thrombosis, the time to thrombotic occlusion was prolonged in CC chemokine ligand 2 (CCL2) knockout (-/-) mice compared with wild-type mice. C_LI What New Information Does This Article Contribute?O_LIThis study is the first to demonstrate that CCR2-/--mediated monocyte deficiency exerts antithrombotic and tissue plasminogen activator (tPA)-facilitating effects in acute arterial thrombosis. C_LIO_LICCR2-/- mice had a) decreased platelet aggregability, probably due in part to reduced post-thrombosis MPA numbers, and b) lower blood and monocyte levels of Factor XIII (FXIII), compared to those of C57BL/6 mice. C_LIO_LIMicro-computed tomography (microCT) thrombus imaging showed that i) CCR2-/- mice, compared to C57BL/6 mice, had smaller thrombi, with more porosity and less fibrin cross-linking on histology and ii) CCR2-/- or CCR2 siRNA facilitates tPA-mediated thrombolysis. C_LI The majority of ischemic strokes occur due to thromboembolic occlusion of cerebral arteries. Tissue plasminogen activator (tPA), which is the only FDA-approved drug for acute ischemic stroke, has only a moderate efficacy rate regarding early recanalization, which is closely associated with thrombus volume and thrombolytic resistance. Monocytes, in cooperation with platelets and neutrophils, contribute to thrombus formation in venous thrombosis. To date, only a few studies have detailed potential prothrombotic roles for monocytes in arterial thrombosis. In the present study, using a mouse model of FeCl3-mediated carotid artery thrombosis and high-resolution microCT thrombus imaging, we demonstrate that CCR2-/--mediated monocyte deficiency exerts antithrombotic and tPA-facilitating effects. Compared to C57BL/6 mice, CCR2-/- mice exhibited decreased platelet aggregability along with lower numbers of circulating MPAs (a marker of platelet activity) and reduced monocyte recruitment to arterial thrombi. Moreover, CCR2-/- mice had lower levels of both circulating FXIII and monocyte FXIII-A, which are known to strengthen and stabilize thrombi. In line with these findings, i) CCR2-/- mice had smaller, more porous thrombi with less fibrin cross-linking and ii) tPA-mediated thrombolysis was faster in CCR2-/- mice or with CCR2 siRNA.

immunology↗

bioRxiv2024

Inhibition of nitric oxide synthase transforms carotid occlusion-mediated benign oligemia into de novo large cerebral infarction

It remains unclear why unilateral proximal carotid artery occlusion (UCAO) causes benign oligemia, without progressing to cerebral infarction, in mice, yet leads to a wide variety of outcomes (ranging from asymptomatic to death) in humans. We hypothesized that inhibition of NOS both transforms UCAO-mediated oligemia into full infarction and expands pre-existing infarction. In support, intraperitoneal administration of N{omega}-nitro-L-arginine methyl ester (L-NAME) followed by UCAO induced large-arterial infarction in mice, unlike UCAO alone. Six-hour laser-speckle-contrast imaging detected spreading ischemia in mice with infarction as assessed at 24h. In agreement with vasoconstriction/microthrombus formation shown by intravital microscopy, the NO-donor, molsidomine and the endothelial-NOS- activating antiplatelet, cilostazol, attenuated or prevented progression to infarction. Moreover, UCAO without L-NAME caused infarction in mice with hyperglycemia and hyperlipidemia, which, in turn, were associated with greater symmetric dimethylarginine (SDMA) levels. Further, increased levels of glucose and cholesterol associated with significantly larger infarct volumes in 438 consecutive patients with UCAO-mediated infarction. Lastly, Mendelian randomization identified a causative role of NOS inhibition, particularly in elevated SDMA concentration, in ischemic stroke risk. Therefore, NOS activity is a key factor determining the fate of hypoperfused brain following acute carotid occlusion, where SDMA could be a potential risk predictor.

neuroscience↗

bioRxiv2024

Developmental changes in mouse motor skill learning and cortical circuitry

Learning motor skills requires plasticity in the primary motor cortex (M1). But the capacity for cortical circuit plasticity varies over developmental age in sensory cortex. This study assesses the normal developmental trajectory of motor learning to assess how aptitude might vary with age. We trained mice of both sexes to run on a custom accelerating rotarod at ages from postnatal day (P) 20 to P120, tracking paw position and quantifying time to fall and changes in gait pattern. While animals of all ages were able to perform better after five training sessions, performance improved most rapidly on the first training day for mice between ages P30-60, suggesting an age with heightened plasticity. Learning this task required M1, because pharmacological inactivation of M1 prevented improvement in task performance. Paw position and gait patterns changed with learning, though differently between age groups. Successful mice learned to shift their gait from hopping to walking. Notably, this shift in gait happened earlier in the trial for forelimbs in comparison to hindlimbs. Thus, motor plasticity might more readily occur in forelimbs. Changes in gait and other kinematic parameters are an additional learning metric beyond time to fall, offering insight into how mice improve performance. Overall, these results suggest mouse motor learning has a developmental trajectory. SignificancePlasticity in sensory cortex is restricted to a limited developmental window. Learning motor skills requires motor cortex plasticity, but it is unknown whether learning aptitude changes over development. Here, we define the developmental trajectory of motor learning aptitude for the accelerating rotarod task in mice, demonstrating a sensitive period for motor learning. Learning peaks at P30-P60, with mice learning to shift from hopping to walking gait to stay on the rotarod longer. Learning this task depends on M1. Further, the gait shift in forelimbs precedes hindlimbs. Knowing the peak in motor plasticity identifies the time window at which we should seek to understand the circuit basis of motor learning plasticity in cortex.

neuroscience↗

bioRxiv2024

AIVE: accurate predictions of SARS-CoV-2 infectivity from comprehensive analysis

An unprecedented amount of SARS-CoV-2 data has been accumulated compared with previous infectious diseases, enabling insights into its evolutionary process and more thorough analyses. This study investigates SARS-CoV-2 features as it evolved to evaluate its infectivity. We examined viral sequences and identified the polarity of amino acids in the Receptor Binding Motif (RBM) region. We detected an increased frequency of amino acid substitutions to lysine (K) and arginine (R) in Variants of Concern (VOCs). As the virus evolved to Omicron, commonly occurring mutations became fixed components of the new viral sequence. Furthermore, at specific positions of VOCs, only one type of amino acid substitution and a notable absence of mutations at D467 was detected. We found that the binding affinity of SARS-CoV-2 lineages to the ACE2 receptor was impacted by amino acid substitutions. Based on our discoveries, we developed APESS, an evaluation model evaluating infectivity from biochemical and mutational properties. In silico evaluation using real-world sequences and in vitro viral entry assays validated the accuracy of APESS and our discoveries. Using Machine Learning, we predicted mutations that had the potential to become more prominent. We created AIVE, a web-based system, accessible at https://ai-ve.org to provide infectivity measurements of mutations entered by users. Ultimately, we established a clear link between specific viral properties and increased infectivity, enhancing our understanding of SARS-CoV-2 and enabling more accurate predictions of the virus.

genomics↗

bioRxiv2024

Genome-Wide Transcriptome Dynamics in Auxin Homeostasis During Fruit Development in Strawberry (F. x ananassa)

The plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry (Fragaria vesca), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle. Consistently, genes functionating in auxin biosynthesis and conjugation, such as TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAAs), YUCCA (YUCs), and GRETCHEN HAGEN 3 (GH3s) were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like PIN-FORMED (PINs), AUXIN/INDOLE-3-ACETIC ACID proteins (Aux/IAAs), TRANSPORT INHIBITOR RESPONSE 1 / AUXIN-SIGNALING F-BOX (TIR/AFBs) and AUXIN RESPONSE FACTOR (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), BASIC REGION/ LEUCINE ZIPPER motif (bZIP), and APETALA2/Ethylene Responsive Factor (AP2/ERF) groups. These results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries.

developmental biology↗

bioRxiv2024

A generalized framework to identify SARS-CoV-2 broadly neutralizing antibodies

Monoclonal antibodies (mAbs) targeting the SARS-CoV-2 receptor-binding domain (RBD) are used to treat and prevent COVID-19. However, the rapid evolution of SARS-CoV-2 drives continuous escape from therapeutic mAbs. Therefore, the ability to identify broadly neutralizing antibodies (bnAbs) against future variants is needed. Here, we use deep mutational scanning (DMS) to predict viral RBD evolution and to select for mAbs neutralizing both existing and prospective variants. A retrospective analysis of 1,103 SARS-CoV-2 wildtype-elicited mAbs shows that this method can increase the probability of identifying effective bnAbs against the XBB.1.5 strain from 1% to 40% in an early pandemic setup. Among these bnAbs, BD55-1205 exhibited potent activity against all tested variants. Cryo-EM structural analyses revealed the receptor mimicry of BD55-1205, explaining its broad reactivity. Delivery of mRNA-LNPs encoding BD55-1205-IgG in mice resulted in ~5,000 serum NT50 against XBB.1.5, HK.3.1, and JN.1 variants. Combining bnAb identification using viral evolution prediction with the versatility of mRNA delivery technology can enable rapid development of next-generation antibody-based countermeasures against SARS-CoV-2 and potentially other pathogens with pandemic potential.

immunology↗

bioRxiv2024

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1/BUB-3 complex to C. elegans kinetochores

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

cell biology↗

bioRxiv2024

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APPSAA knock-in mouse model of Alzheimer disease

IntroductionReduced brain energy metabolism, mTOR dysregulation, and extracellular amyloid-{beta} oligomer (xcA{beta}O) buildup characterize AD; how they collectively promote neurodegeneration is poorly understood. We previously reported that xcA{beta}Os inhibit Nutrient-induced Mitochondrial Activity (NiMA) in cultured neurons. We now report NiMA disruption in vivo. MethodsBrain energy metabolism and oxygen consumption were recorded in APPSAA/+ mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. ResultsNiMA is inhibited in APPSAA/+ mice before other defects are detected in these amyloid-{beta}-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. GSK3{beta} signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3{beta} with lithium or TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APPSAA mice. ConclusionNiMA disruption in vivo occurs before histopathological changes and cognitive decline in APPSAA mice, and may represent an early stage in human AD.

neuroscience↗

bioRxiv2024

Ensemble learning and ground-truth validation of synaptic connectivity inferred from spike trains

Probing the architecture of neuronal circuits and the principles that underlie their functional organization remains an important challenge of modern neurosciences. This holds true, in particular, for the inference of neuronal connectivity from large-scale extracellular recordings. Despite the popularity of this approach and a number of elaborate methods to reconstruct networks, the degree to which synaptic connections can be reconstructed from spike-train recordings alone remains controversial. Here, we provide a framework to probe and compare connectivity inference algorithms, using a combination of synthetic and empirical ground-truth data sets, obtained from simulations and parallel single-cell patch-clamp and high-density microelectrode array (HD-MEA) recordings in vitro. We find that reconstruction performance critically depends on the regularity of the recorded spontaneous activity, i.e., their dynamical regime, the type of connectivity, and the amount of available spike train data. We find gross differences between different algorithms, and many algorithms have difficulties in detecting inhibitory connections. We therefore introduce an ensemble artificial neural network (eANN) to improve connectivity inference. We train the eANN on the validated outputs of six established inference algorithms, and show how it improves network reconstruction accuracy and robustness. Overall, the eANN was robust across different dynamical regimes, with shorter recording time, and ameliorated the identification of synaptic connections, in particular inhibitory ones. Results indicated that the eANN also improved the topological characterization of neuronal networks. The presented methodology contributes to advancing the performance of inference algorithms and facilitates our understanding of how neuronal activity relates to synaptic connectivity. Author summaryThis study introduces an ensemble artificial neural network (eANN) to infer neuronal connectivity from multi-unit spike time recordings. We compare the eANN to previous algorithms and validate it using simulations and HD-MEA/patch-clamp datasets. The latter is obtained from three single-cell patch-clamp recordings and high-density microelectrode array (HD-MEA) measurements, in parallel. Our results demonstrate that the eANN outperforms all other algorithms across different dynamical regimes and provides a more accurate description of the underlying topological organization of the studied networks. We also provide a SHAP analysis of the trained eANN to understand which input features of the eANN contribute most to this superior performance. The eANN is a promising approach to improve connectivity inference from spike-train data.

neuroscience↗

bioRxiv2023

Spatial dissection of cellular heterogeneity and functional crosstalk in the tumor microenvironments of gastric cancers

Spatial transcriptomics offers unprecedented challenges in characterizing tumor ecosystems, encompassing tumor cells and their surrounding tumor microenvironments (TME) along with cellular interactions therein. Here, we conducted Visium-based spatial transcriptomics on nine primary gastric cancers (GC) to translate the spatial organization of GC ecosystems into the functional landscape of cellular crosstalk across malignant, stromal and immune cells. We identified three distinct GC subtypes of 3 immunogenic, 3 epithelial and 3 stromal GCs with elevated infiltration of immune, malignant cells and fibroblasts reflecting the heterogeneity of the cell types and their interactions in GC TME. GC spatial architecture was further delineated into 6 regional compartments with varying degree of TME infiltration, among which the fibroblast-enriched TME was associated with the upregulation of epithelial-to-mesenchymal transformation (EMT) and immune response in malignant and immune cells, respectively. Cell type-specific transcriptional dynamics representing cellular crosstalk between TME cells were further identified, e.g., the infiltration of malignant and endothelial cells promote the cellular proliferations of TME cells whereas the fibroblasts and immune cells are associated with pro-cancer and anti-cancer immunity, respectively. Ligand-receptor analysis further revealed that CCL2-expressing fibroblasts promote the pro-cancer signaling of immune cells including JAK-STAT3 signaling and inflammatory response in tumor-infiltration macrophages. The CCL2+fibroblasts and macrophages with activated JAK-STAT3 signals are co-localized in spatial GC architecture and their co-abundance was associated with unfavorable clinical outcomes. Taken together, GC spatial transcriptomes can be regionally and functionally delineated into functional cellular crosstalk involving multiple cell types with potential clinical relevance, particularly the interaction between CCL2+ fibroblasts and JAK-STAT3+macrophages contributing to cancer-favoring immune contexture in GC TME.

cancer biology↗

bioRxiv2023

Whole-genome sequences reveal zygotic composition in chimeric twins

The monochorionic placenta in dizygotic twins allows in utero exchange of embryonic cells, resulting in chimerism in the twins. In practice, this chimerism is incidentally identified on mixed ABO blood types or in the presence of cells with a discordant sex chromosome. Here, we applied whole-genome sequencing to one triplet and one twin families to precisely understand their zygotic compositions, using millions of genomic variants as barcodes of zygotic origins. Peripheral blood showed asymmetrical contributions from two sister zygotes, where one of the zygotes was the major clone in both twins. Single-cell RNA sequencing of peripheral blood tissues further showed differential contributions from the two sister zygotes across blood cell types. In contrast, buccal tissues were pure in genetic composition, suggesting that in utero cellular exchanges were confined to the blood tissues. Our study illustrates the cellular history of twinning during human development, which is critical for managing the health of chimeric individuals in the era of genomic medicine.

genomics↗