Biology subjects

Kim, K.

Publications and source records attributed to Kim, K..

At least 163 records · Page 9Linked to original sources

bioRxiv2024

PIEZO acts in an intestinal valve to regulate swallowing in C. elegans

Sensations of the internal state of the body play crucial roles in regulating the physiological processes and maintaining homeostasis of an organism. However, our understanding of how internal signals are sensed, processed, and integrated to generate appropriate biological responses remains limited. Here, we show that the C. elegans PIEZO channel, encoded by pezo-1, regulates food movement in the intestine by detecting food accumulation in the anterior part of the intestinal lumen, thereby triggering rhythmical movement of the pharynx, referred to as the pharyngeal plunge. pezo-1 deletion mutants exhibit defects in the pharyngeal plunge, which is rescued by PEZO-1 or mouse PIEZO1 expression, but not by PIEZO2, in a single isolated non-neuronal tissue of the digestive tract, the pharyngeal-intestinal valve. Genetic ablation or optogenetic activation of this valve inhibits or induces the pharyngeal plunge, respectively. Moreover, pressure built in the anterior lumen of the intestine results in a pezo-1-dependent pharyngeal plunge, which is driven by head muscle contraction. These findings illustrate how interoceptive processes in a digestive organ regulate swallowing through the PIEZO channel, providing insights into how interoception coordinates ingestive processes in higher animals, including humans.

neuroscience↗

bioRxiv2024

Stress hyperglycemia exacerbates inflammatory brain injury after stroke

Post-stroke hyperglycemia occurs in 30% - 60% of ischemic stroke patients as part of the systemic stress response, but neither clinical evidence nor pre-clinical studies indicate whether post-stroke hyperglycemia affects stroke outcome. Here we investigated this issue using a mouse model of permanent ischemia. Mice were maintained either normoglycemic or hyperglycemic during the interval of 17 - 48 hours after ischemia onset. Post-stroke hyperglycemia was found to increase infarct volume, blood-brain barrier disruption, and hemorrhage formation, and to impair motor recovery. Post-stroke hyperglycemia also increased superoxide formation by peri-infarct microglia/macrophages. In contrast, post-stroke hyperglycemia did not increase superoxide formation or exacerbate motor impairment in p47phox-/- mice, which cannot form an active superoxide-producing NADPH oxidase-2 complex. These results suggest that hyperglycemia occurring hours-to-days after ischemia can increase oxidative stress in peri-infarct tissues by fueling NADPH oxidase activity in reactive microglia/macrophages, and by this mechanism contribute to worsened functional outcome.

neuroscience↗

bioRxiv2024

High-throughput protein characterization by complementation using DNA barcoded fragment libraries

Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we use complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. Although assay effectiveness ranged with respect to source genome, with 41% of expected enzymes recovered, even distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron showed success. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein on an amino acid basis. Among unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seqs utility is demonstrated, with future applications in ecology, health, and engineering.

genomics↗

bioRxiv2024

Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.

cell biology↗

bioRxiv2024

A mismatch between striatal cholinergic pauses and dopaminergic reward prediction errors

Movement, motivation and reward-related learning depend strongly on striatal dopamine and acetylcholine. These neuromodulators each regulate the other, and disturbances to their coordinated signals contribute to human disorders ranging from Parkinsons Disease to depression and addiction. Pauses in the firing of cholinergic interneurons (CINs) are thought to coincide with pulses in dopamine release that encode reward prediction errors (RPEs), together shaping synaptic plasticity and thereby learning. However, such models are based upon recordings from unidentified neurons, and do not incorporate the distinct characteristics of striatal subregions. Here we compare the firing of identified, individual CINs to dopamine release as unrestrained rats performed a probabilistic decision-making task. The relationships between CIN spiking, dopamine release, and behavior varied strongly by subregion. In dorsal-lateral striatum a Go! cue evoked burst-pause CIN spiking, quickly followed by a very brief (~150ms) dopamine pulse that was unrelated to RPE. In dorsal-medial striatum the same cue evoked only a CIN pause; this pause was curtailed by a movement-selective rebound in firing. Finally in ventral striatum a reward cue evoked slower, RPE-coding increases in both dopamine and CIN firing, without any distinct pause. Our results demonstrate a spatial and temporal dissociation between CIN pauses and dopamine RPE signals, and will inform new models of striatal microcircuits and their contributions to behavior.

neuroscience↗

bioRxiv2024

3D bioprinting of Liver Microenvironment Model Using Photocrosslinkable Decellularized Extracellular Matrix based Hydrogel

The liver, as one of the vital organs in the body, plays a crucial role in various bodily functions. Numerous factors can cause liver damage, that the sole remedy for severe liver conditions is the transplantation of healthy liver tissue. In response to the transplantation challenges, innovative approaches involving hydrogel-based technologies have emerged, leading to the creation of highly functionalized tissues. The development of three-dimensional printing and patterning of cell-laden biomaterial matrices offers promising advances for creating tissue-specific structures in tissue engineering and bioprinting. However, the matrix materials currently employed in bioprinting liver microtissue often fail to capture the complexity of the natural extracellular matrix (ECM), hindering their ability to restore innate cellular shapes and functions. Liver ECM-based hydrogels are increasingly recognized for their potential as biomimetic 3D cell culture systems that facilitate the exploration of liver disease, metabolism, and toxicity mechanisms. Yet, the conventional production of these hydrogels relies on slow thermal gelation processes, which restrict the manipulation of their mechanical characteristics. In this research, we introduce a novel approach with a functionalized photocrosslinkable liver decellularized extracellular matrix (dECM). By combining liver dECM methacrylate (LdECMMA) with gelatin methacrylate (GelMA), we achieved accelerated crosslinking under visible light irradiation and the ability to tune the mechanical, rheological, and physiological properties of the material. We encapsulated human hepatocellular carcinoma cells within an optimal concentration of the GelMA-LdECMMA hybrid hydrogel and examined cell proliferation and function over an extended period. The findings revealed that the GelMA-LdECMMA hybrid hydrogel enhances liver cell proliferation and function, holding significant promise for applications in drug screening and liver cancer metastasis research.

bioengineering↗

bioRxiv2024

Gpr37 modulates the severity of inflammation-induced GI dysmotility by regulating enteric reactive gliosis.

The enteric nervous system (ENS) is contained within two layers of the gut wall and is made up of neurons, immune cells, and enteric glia cells (EGCs) that regulate gastrointestinal (GI) function. EGCs in both inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) change in response to inflammation, referred to as reactive gliosis. Whether EGCs restricted to a specific layer or region within the GI tract alone can influence intestinal immune response is unknown. Using bulk RNA-sequencing and in situ hybridization, we identify G-protein coupled receptor Gpr37, as a gene expressed only in EGCs of the myenteric plexus, one of the two layers of the ENS. We show that Gpr37 contributes to key components of LPS-induced reactive gliosis including activation of NF-kB and IFN-y signaling and response genes, lymphocyte recruitment, and inflammation-induced GI dysmotility. Targeting Gpr37 in EGCs presents a potential avenue for modifying inflammatory processes in the ENS.

neuroscience↗

bioRxiv2024

Robust single nucleus RNA sequencing reveals depot-specific cell population dynamics in adipose tissue remodeling during obesity

Single nucleus RNA sequencing (snRNA-seq), an alternative to single cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying adipose tissue remodeling during obesity. By integrating bulk nuclear RNA-seq from adipocyte nuclei of different sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. These subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot. Specifically, we identify a key molecular feature of dysfunctional hypertrophic adipocytes, a global shutdown in gene expression, along with elevated stress and inflammatory responses. Furthermore, our differential gene expression analysis reveals distinct contributions of adipocyte subpopulations to the overall pathophysiology of adipose tissue. Our study establishes a robust snRNA-seq method, providing novel insights into the biological processes involved in adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.

molecular biology↗

bioRxiv2024

Enhanced extracellular matrix remodeling due to embedded spheroid fluidization

Tumor spheroids are in vitro three-dimensional, cellular collectives consisting of cancerous cells. Embedding these spheroids in an in vitro fibrous environment, such as a collagen network, to mimic the extracellular matrix (ECM) provides an essential platform to quantitatively investigate the biophysical mechanisms leading to tumor invasion of the ECM. To understand the mechanical interplay between tumor spheroids and the ECM, we computationally construct and study a three-dimensional vertex model for a tumor spheroid that is mechanically coupled to a cross-linked network of fibers. In such a vertex model, cells are represented as deformable polyhedrons that share faces. Some fraction of the boundary faces of the tumor spheroid contain linker springs connecting the center of the boundary face to the nearest node in the fiber network. As these linker springs actively contract, the fiber network remodels. By toggling between fluid-like and solid-like spheroids via changing the dimensionless cell shape index, we find that the spheroid rheology affects the remodeling of the fiber network. More precisely, fluid-like spheroids displace the fiber network more on average near the vicinity of the spheroid than solid-like spheroids. We also find more densification of the fiber network near the spheroid for the fluid-like spheroids. These spheroid rheology-dependent effects are the result of cellular motility due to active cellular rearrangements that emerge over time in the fluid-like spheroids to generate spheroid shape fluctuations. These shape fluctuations lead to emergent feedback between the spheroid and the fiber network to further remodel the fiber network with, for example, lower radial alignment of the higher-tensioned fibers given the breaking of spheroidal radial symmetry, which can then further remodel the spheroid. Our results uncover intricate morphological-mechanical interplay between an embedded spheroid and its surrounding fiber network with both spheroid contractile strength and spheroid shape fluctuations playing important roles in the pre-invasion stages of tumor invasion.

biophysics↗

bioRxiv2024

Optical quantification of molecular interaction strength in protein condensates

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques - Brillouin microscopy and quantitative phase imaging (QPI) - to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

biophysics↗

bioRxiv2024

Assessing Long-Term Stored Tissues for Multi-Omics Data Quality and Proteogenomics Suitability

As research into the complexities of cancer biology deepens, the integration of multi-omics analyses has emerged as a powerful approach to unravel the complex molecular basis of cancers. However, challenges related to sample availability, including size, collection procedures, and storage duration, hinder the broad application of this methodology. Despite these limitations, there is a growing interest in exploring the potential of archived samples to expand the scope of multi-omics research. Our study aims to investigate the impact of storage duration on the measurment in genomic, transcriptomic, and proteomic profiles of archived samples, demonstrating their viability for advancing our understanding of cancer biology. To comprehensively address these trends and limitations, we systematically examined archived samples collected over a decade, focusing on their transcriptomic, proteomic, and phosphoproteomic attributes. Analysis revealed intricate patterns and dynamic shifts, especially in long-term transcriptomic data, with observed declines in read counts related to protein coding and gene coverage. However, these changes did not compromise the fundamental gene expression landscape. Proteomic result also demonstrated that storage period did not significantly influence proteomic measurement. Comparisons of housekeeping gene (HKG) and housekeeping protein (HKP) expressions unveiled consistent transcriptomic levels across samples, while distinctive proteomic disparities between tumor and normal tissues. In conclusion, the challenges posed by limited sample availability in multi-omics studies can be partially overcome through the strategic integration of archived samples. While technical shifts were evident in certain aspects of transcriptomic data, core gene expression patterns remained robust, and the functionality of essential transcription factors (TFs) and kinases remained unaffected. These findings underscore the potential of archived samples as valuable resources for multi-omics research, providing a broader landscape for investigating cancer biology and paving the way for more comprehensive insights into this intricate field.

cancer biology↗

bioRxiv2024

Direct Ink Writing of Graphene Oxide Reinforced 13-93B1 Bioactive Glass Scaffolds for Bone Tissue Engineering Applications

Graphene-reinforced bioactive glass scaffolds have gained significant attention in the field of bone tissue engineering due to their unique combination of mechanical strength, bioactivity, and electrical conductivity. Additive manufacturing techniques, such as 3D printing, provide a versatile platform for fabricating these scaffolds with precise control over their architecture and composition. Consequently, in this work, we have fabricated graphene oxide (GO)-reinforced 13-93B1 bioactive glass scaffold using the direct ink writing technique. A Pluronic F-127- based ink was prepared for scaffold fabrication, and its rheological properties were assessed for shear thinning behaviour, structural support, and recovery. Further, the fabricated scaffolds were characterized using micro-computed tomography, scanning electron microscopy, and energy dispersive x-ray spectroscopy. Additionally, computational fluid dynamics simulations with Dulbeccos modified eagle medium and blood were performed to evaluate the perfusion kinetics of the scaffolds. The inclusion of GO enhanced the compressive strength of the fabricated scaffolds by [~]225%. The morphological characterization based on micro-computed tomography showed that additively manufactured scaffolds have appropriate porosity, pore size, pore throat size, and interconnectivity. The live-dead assay results showed no cytotoxicity towards C2C12 mouse myoblast cells. Also, cell adhesion and cell viability results show better cell growth on the nanocomposite scaffolds. Overall, the fabricated scaffold is found suitable for bone tissue engineering applications.

bioengineering↗

bioRxiv2024

Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain

APOBEC3G (A3G) belongs to the AID/APOBEC cytidine deaminase family and is essential for antiviral immunity. It contains two zinc-coordinated cytidine-deaminase (CD) domains. The N-terminal CD1 domain is non-catalytic but has a strong affinity for nucleic acids, whereas the C-terminal CD2 domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains in DNA binding and editing is not fully understood. Here, our studies on rhesus macaque A3G (rA3G) show that the DNA editing function in linear and hairpin loop DNA is greatly enhanced by AA or GA dinucleotide motifs present downstream (in the 3-direction) but not upstream (in the 5-direction) of the target-C editing sites. The effective distance between AA/GA and the target-C sites depends on the local DNA secondary structure. We present two co-crystal structures of rA3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic CD1 domain in capturing AA/GA DNA and explaining our biochemical observations. Our structural and biochemical findings elucidate the molecular mechanism underlying the cooperative function between the non-catalytic and the catalytic domains of A3G, which is critical for its antiviral role and its contribution to genome mutations in cancer.

biochemistry↗

bioRxiv2024

Environmental and Historical Determinants of African Horse Sickness: Insights from Predictive Modeling

This study marks a pioneering effort in analyzing the global epidemiological patterns of African Horse Sickness (AHS) across different regions. By employing predictive modeling with a comprehensive set of environmental variables, we uncovered overarching global patterns in AHS dynamics, a first in this field. Our analysis revealed significant regional differences influenced by specific climatic conditions, highlighting the diseases complexity. The study also identifies new high-risk areas for AHS, underscoring the necessity for regionally tailored disease management strategies. Despite some limitations, such as the exclusion of wild equine data, this research offers critical insights for global AHS intervention and prevention, setting a path for future research incorporating broader datasets and socio-economic factors. Author SummaryAHS presents a significant challenge to the global equine industry, impacting both health and economic aspects. Our study highlights the profound effect of climate change, particularly the frequency of extreme climatic events including temperature and humidity variations, on the transmission dynamics of diseases like AHS. In our research, we focused on overcoming the challenges associated with identifying key environmental factors and determining the appropriate geographic scale for a comprehensive global understanding of AHS. Our aim was to bridge existing knowledge gaps and elucidate the fundamental principles governing AHS transmission. This study establishes a solid foundation for understanding the intricate dynamics of AHS and offers practical pathways for conservation efforts. It emphasizes the urgent need for environmentally conscious strategies to protect horse populations and the industries dependent on them. By highlighting the relationship between environmental factors, vector presence, and AHS transmission, our research underscores the importance of a holistic approach to disease mitigation. In conclusion, the findings of our study not only contribute to the scientific understanding of AHS but also serve as a guide for policymakers and practitioners in developing effective strategies for disease management and prevention, tailored to the specific environmental conditions and challenges faced in different regions around the world.

ecology↗

bioRxiv2024

Fetal brain response to maternal inflammation requires microglia

In utero infection and maternal inflammation can adversely impact fetal brain development. Maternal systemic illness, even in the absence of direct fetal central nervous system infection, is associated with an increased risk of autism and schizophrenia in affected offspring. The cell types mediating the response of the fetal brain to maternal inflammation are largely unknown, hindering the development of therapies to prevent and treat adverse neuropsychiatric outcomes. Here, we show that microglia, the resident phagocytes of the brain, are enriched for expression of receptors for relevant pathogens and cytokines, throughout embryonic development. Using a rodent maternal immune activation (MIA) model in which polyinosinic:polycytidylic acid is injected into pregnant dams, we demonstrate long-lasting transcriptional changes in fetal microglia that persist into postnatal life. We find that MIA induces widespread gene expression changes in neuronal and non-neuronal cells; importantly, these responses are abolished by selective genetic deletion of microglia, indicating that microglia are required for the transcriptional response of other cortical cell types to MIA. These findings demonstrate that microglia play a critical, durable role in fetal response to maternal inflammation, pointing at microglia as a potential therapeutic cell target.

neuroscience↗

bioRxiv2024

Neuronal-class specific molecular cues drive differential myelination in the neocortex

In the neocortex, oligodendrocytes produce distinct amounts of myelin in each cortical layer and along the axons of individual neuron types. Here we present a comprehensive single-cell molecular map of mouse cortical oligodendrocytes across different cortical layers and stages of myelination, spanning the initiation of cortical myelination into adulthood. We apply this dataset to show that neuron-class specific signals drive oligodendrocyte maturation and differential myelination across cortical layers. We find that each layer contains a similar compendium of oligodendrocyte classes, indicating that oligodendrocyte heterogeneity cannot explain layer-specific myelination. To evaluate whether neuronal diversity drives differential myelination across cortical layers, we generated a predicted ligand-receptor interactome between projection neuron types and oligodendrocyte states, across cortical layers and time. In vivo functional testing identified Fgf18, Ncam1, and Rspo3 as novel, neuron-derived pro-myelinating signals. Our results highlight neuron-class-dependent control of myelin distribution in the neocortex.

neuroscience↗

bioRxiv2024

Binding Position Dependent Modulation of Smoothened Activity by Cyclopamine

Cyclopamine is a natural alkaloid that is known to act as an agonist when it binds to the Cysteine Rich Domain (CRD) of the Smoothened receptor and as an antagonist when it binds to the Transmembrane Domain (TMD). To study the effect of cyclopamine binding to each binding site experimentally, mutations in the other site are required. Hence, simulations are critical for understanding the WT activity due to binding at different sites. Additionally, there is a possibility that cyclopamine could bind to both sites simultaneously especially at high concentration, the implications of which remain unknown. We performed three independent sets of simulations to observe the receptor activation with cyclopamine bound to each site independently (CRD, TMD) and bound to both sites simultaneously. Using multi-milliseconds long aggregate MD simulations combined with Markov state models and machine learning, we explored the dynamic behavior of cyclopamines interactions with different domains of WT SMO. A higher population of the active state at equilibrium, a lower activation free energy barrier of[~] 2 kcal/mol, and expansion of the hydrophobic tunnel to facilitate cholesterol transport agrees with the cyclopamines agonistic behavior when bound to the CRD of SMO. A higher population of the inactive state at equilibrium, a higher free energy barrier of[~] 4 kcal/mol and restricted the hydrophobic tunnel to impede cholesterol transport showed cyclopamines antagonistic behavior when bound to TMD. With cyclopamine bound to both sites, there was a slightly larger inactive population at equilibrium and an increased free energy barrier ([~] 3.5 kcal/mol). The tunnel was slightly larger than when solely bound to TMD, and showed a balance between agonism and antagonism with respect to residue movements exhibiting an overall weak antagonistic effect.

biophysics↗

bioRxiv2024

Stratification of viral shedding patterns in saliva of COVID-19 patients

Living with COVID-19 requires continued vigilance against the spread and emergence of variants of concern (VOCs). Rapid and accurate saliva diagnostic testing, alongside basic public health responses, is a viable option contributing to effective transmission control. Nevertheless, our knowledge regarding the dynamics of SARS-CoV-2 infection in saliva is not as advanced as our understanding of the respiratory tract. Here we analyzed longitudinal viral load data of SARS-CoV-2 in saliva samples from 144 patients with mild COVID-19 (a combination of our collected data and published data). Using a mathematical model, we quantified individual-level viral dynamics and stratified them into three groups using a clustering approach. Notably, the three groups exhibited distinct differences viral RNA detection durations: 11.5 days (95% CI: 10.6 to 12.4), 17.4 days (16.6 to 18.2), and 30.0 days (28.1 to 31.8), respectively. Surprisingly, this stratified grouping remained unexplained despite our analysis of 47 types of clinical data, including basic demographic information, clinical symptoms, results of blood tests, and vital signs. Additionally, we quantified the expression levels of 92 micro-RNAs in a subset of saliva samples, but these also failed to explain the observed stratification, although the mir-1846 level may have been weakly correlated with peak viral load. Our study provides insights into SARS-CoV-2 infection dynamics in saliva, highlighting the challenges in predicting the duration of viral RNA detection without indicators that directly reflect an individuals immune response, such as antibody induction. Given the significant individual heterogeneity in the kinetics of saliva viral shedding, identifying biomarker(s) for viral shedding patterns will be crucial for improving public health interventions in the era of living with COVID-19.

microbiology↗