Biology subjects

Kim, Y. S.

Publications and source records attributed to Kim, Y. S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

DNA Base Pair Polarities of Pyrimidine(s)-Purine(s) (Pyu) dsDNA induce stronger Hybridization and unique DNA Conformation compared to Purine(s)-Pyrimidine(s) (Puy) dsDNA

The present study introduces a concept of DNA base pair polarity in double-stranded DNA (dsDNA), which has led the serial investigation of hydrogen bonding magnetic resonance-based gene regulation. DNA base pair polarity is characterized by hydrogen bonding between electrostatically positive purine and negative pyrimidine, exerting the stabilization and organization of dsDNA. Comparison of DNA hybridization potential between Pyu (pyrimidine(s)-purine(s)) and Puy (purine(s)-pyrimidine(s)) oligo-dsDNAs in EtBr intercalated electrophoresis showed that Pyu ds(nTnA) (n=3, 4,5,6,7,8,10,12) and ds(nCnA) (n=7,8,10,12) exhibit stronger hybridization than Puy ds(nAnT) and ds(nAnC), respectively. In the HPLC for DNA conformation change in 0.1M NaCl solution, Pyu oligo-dsDNA tended to have a larger peak area than Puy oligo-dsDNA, indicating that the former had a more extended and organized DNA conformation than the latter. FT-IR results showed that Pyu ds3T3A, ds3C3G, dsCTCGAG, and dsTCTAGA showed higher IR absorbance at 3400-3200 cm-1 than Puy ds3A3T, ds3G3C, dsGAGCTC, and dsAGATCT, respectively, indicating Pyu oligo-dsDNAs have increased N-H stretching in hybridization than Puy oligo-dsDNAs. In vitro RNA transcription from template plasmid DNAs inserted with six segments of Pyu ds6(TCTGA) and positive ds14(GA) produced more RNA than those with seven segments of palindromic Pyu ds(CATG) and negative ds14(CT), respectively, indicating the former plays a stimulating role for RNA transcription while the latter plays an inhibitory role. When comparing PCR primers between Pyu ds12/n(nCnA) and Puy ds12/n(nAnC) (n=1,2,3,4,6,12), Pyu primers can easily hybridize on template DNA and subsequently produce abundant DNA compared to Puy primers. The data suggest that Pyu oligo-dsDNAs hybridize strongly and form distinctive conformation, thereby stimulating RNA transcription more than Puy oligo-dsDNAs. Since Pyu oligo-dsDNA appears to preserve DNA signal and conformation relevant to DNA function more efficiently than Puy dsDNA, it is proposed that the entire DNA code can be divided into Pyu dsDNAs with unique DNA base pair polarities.

molecular biology↗

bioRxiv2024

A transgene-free, human peri-gastrulation embryo model with trilaminar embryonic disc-, amnion- and yolk sac-like structures

The ultimate outcome of the gastrulation in mammalian development is a recognizable trilaminar disc structure containing organized cell lineages with spatially defined identities in an emerging coordinate system1-4. Despite its importance in human development, gastrulation remains difficult to study. Stem cell-based embryo models, including those that recapitulate different aspects of pre- and peri-gastrulation human development5-15, are emerging as promising tools for studying human embryogenesis16-18. However, it remains unclear whether existing human embryo models are capable of modeling the development of the trilaminar embryonic disc structure, a hallmark of human gastrulation. Here we report a transgene-free human embryo model derived solely from primed human pluripotent stem cells (hPSCs), which recapitulates various aspects of peri-gastrulation human development, including formation of trilaminar embryonic layers situated between dorsal amnion and ventral definitive yolk sac and primary hematopoiesis. We term this model the peri-gastrulation trilaminar embryonic disc (PTED) embryoid. The development of PTED embryoid does not follow natural developmental sequences of cell lineage diversification or spatial organization. Instead, it exploits both extrinsic control of tissue boundaries and intrinsic self-organizing properties and embryonic plasticity of the diverse peri-gastrulation-stage cell lineages, leading to the emergence of in vivo-like tissue organization and function at a global scale. Our lineage tracing study reveals that in PTED embryoids, embryonic and extraembryonic mesoderm cells, as well as embryonic and extraembryonic endoderm cells, share common progenitors emerging during peri-gastrulation development. Active hematopoiesis and blood cell generation are evident in the yolk sac-like structure of PTED embryoids. Together, PTED embryoids provide a promising and ethically less challenging model for studying self-organizing properties of peri-gastrulation human development.

developmental biology↗

bioRxiv2024

Salience signaling and stimulus scaling of ventral tegmental area glutamate neuron subtypes

Ventral tegmental area (VTA) glutamatergic neurons participate in reward, aversion, drug-seeking, and stress. Subsets of VTA VGluT2+ neurons are capable of co-transmitting glutamate and GABA (VGluT2+VGaT+ neurons), transmitting glutamate without GABA (VGluT2+VGaT- neurons), or co-transmitting glutamate and dopamine (VGluT2+TH+ neurons), but whether these molecularly distinct subpopulations show behavior-related differences is not wholly understood. We identified that neuronal activity of each VGluT2+ subpopulation is sensitive to reward value but signaled this in different ways. The phasic maximum activity of VGluT2+VGaT+ neurons increased with sucrose concentration, whereas VGluT2+VGaT- neurons increased maximum and sustained activity with sucrose concentration, and VGluT2+TH+ neurons increased sustained but not maximum activity with sucrose concentration. Additionally, VGluT2+ subpopulations signaled consummatory preferences in different ways. VGluT2+VGaT- neurons and VGluT2+TH+ neurons showed a signaling preference for a behaviorally-preferred fat reward over sucrose, but in temporally-distinct ways. In contrast, VGluT2+VGaT+ neurons uniquely signaled a less behaviorally-preferred sucrose reward compared with fat. Further experiments suggested that VGluT2+VGaT+ consummatory reward-related activity was related to sweetness, partially modulated by hunger state, and not dependent on caloric content or behavioral preference. All VGluT2+ subtypes increased neuronal activity following aversive stimuli but VGluT2+VGaT+ neurons uniquely scaled their magnitude and sustained activity with footshock intensity. Optogenetic activation of VGluT2+VGaT+ neurons during low intensity footshock enhanced fear-related behavior without inducing place preference or aversion. We interpret these data such that VTA glutamatergic subpopulations signal different elements of rewarding and aversive experiences and highlight the unique role of VTA VGluT2+VGaT+ neurons in enhancing the salience of behavioral experiences.

neuroscience↗

bioRxiv2024

AIBP controls amyloid beta induced TLR4 inflammarafts and mitochondrial dysfunction in microglia

Microglia-driven neuroinflammation plays an important role in the development of Alzheimers disease (AD). Microglia activation is accompanied by the formation and chronic maintenance of TLR4 inflammarafts, defined as enlarged and cholesterol-rich lipid rafts serving as an assembly platform for TLR4 dimers and complexes of other inflammatory receptors. The secreted apoA-I binding protein (APOA1BP or AIBP) binds TLR4 and selectively targets cholesterol depletion machinery to TLR4 inflammaraft expressing inflammatory, but not homeostatic microglia. Here we demonstrated that amyloid-beta (A{beta}) induced formation of TLR4 inflammarafts in microglia in vitro and in the brain of APP/PS1 mice. Mitochondria in Apoa1bp-/- APP/PS1 microglia were hyperbranched and cupped, which was accompanied by increased ROS and the dilated ER. The size and number of A{beta} plaques and neuronal cell death were significantly increased, and the animal survival was decreased in Apoa1bp-/- APP/PS1 compared to APP/PS1 female mice. These results suggest that AIBP exerts control of TLR4 inflammarafts and mitochondrial dynamics in microglia and plays a protective role in AD associated oxidative stress and neurodegeneration.

neuroscience↗

bioRxiv2024

Forced mouth opening induces post-traumatic hyperalgesia and associated peripheral sensitization after temporomandibular joints injury in mice

Temporomandibular disorder (TMD) is the most prevalent painful condition in the craniofacial area. The pathophysiology of TMD is not fully understood, and it is necessary to understand pathophysiology underlying painful TMD conditions to develop more effective treatment methods. Recent studies suggested that external or intrinsic trauma to TMJ is associated with chronic TMD in patients. Here, we investigated the effects of the TMJ trauma through forced-mouth opening (FMO) in mice to determine pain behaviors and peripheral sensitization of trigeminal nociceptors. FMO increased mechanical hyperalgesia assessed by von Frey test, spontaneous pain-like behaviors assessed by mouse grimace scale, and anxiety-like behaviors assessed by open-field test. In vivo GCaMP Ca2+ imaging of intact trigeminal ganglia (TG) showed increased spontaneous Ca2+ activity and mechanical hypersensitivity of TG neurons in the FMO compared to the sham group. Ca2+ responses evoked by cold, heat, and capsaicin stimuli were also increased. FMO-induced hyperalgesia and neuronal hyperactivities were not sex dependent. TG neurons sensitized following FMO were primarily small to medium-sized nociceptive afferents. Consistently, most TMJ afferents in the TG were small-sized peptidergic neurons expressing calcitonin gene-related peptides, whereas nonpeptidergic TMJ afferents were relatively low. FMO-induced intraneural inflammation in the surrounding tissues of the TMJ indicates potentially novel mechanisms of peripheral sensitization following TMJ injury. These results suggest that the TMJ injury leads to persistent post-traumatic hyperalgesia associated with peripheral sensitization of trigeminal nociceptors.

neuroscience↗

bioRxiv2024

Elucidation of neuronal activity in mouse models of TMJ injury by in vivo GCaMP Ca2+ imaging of intact trigeminal ganglion neurons

Patients with temporomandibular disorders (TMD) typically experience facial pain and discomfort or tenderness in the temporomandibular joint (TMJ), causing disability in daily life. Unfortunately, existing treatments for TMD are not always effective, creating a need for more advanced, mechanism-based therapies. In this study, we used in vivo GCaMP3 Ca2+ imaging of intact trigeminal ganglia (TG) to characterize functional activity of the TG neurons in vivo, specifically in TMJ animal models. This system allows us to observe neuronal activity in intact anatomical, physiological, and clinical conditions and to assess neuronal function and response to various stimuli. We observed a significant increase in spontaneously and transiently activated neurons responding to mechanical, thermal, and chemical stimuli in the TG of forced mouth open (FMO) mice. An inhibitor of the CGRP receptor significantly attenuated FMO-induced facial hypersensitivity. In addition, we confirmed the attenuating effect of CGRP antagonist on FMO-induced sensitization by in vivo GCaMP3 Ca2+ imaging of intact TG. Our results contribute to unraveling the role and activity of TG neurons in the TMJ pain animal models of TMD, bringing us closer understanding the pathophysiological processes underlying TMD. Our study also illustrates the utility of in vivo GCaMP3 Ca2+ imaging of intact TG for studies aimed at developing more targeted and effective treatments for TMD.

neuroscience↗

bioRxiv2023

A human pluripotent stem cell-based somitogenesis model using microfluidics

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation.

bioengineering↗

bioRxiv2023

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration

Parkinsons disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1{beta} as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

neuroscience↗

bioRxiv2023

Alternate splice variants of the mitochondrial fission protein DNM1L/Drp1 regulate mitochondrial dynamics and cell fate in ovarian cancer.

Aberrant mitochondrial fission/fusion dynamics have been reported in cancer cells. While post translational modifications are known regulators of the mitochondrial fission/fusion machinery, we show that alternative splice variants of the fission protein Drp1 (DNM1L) have specific and unique roles in cancer, adding to the complexity of mitochondrial fission/fusion regulation in tumor cells. Ovarian cancer specimens express an alternative splice transcript variant of Drp1 lacking exon 16 of the variable domain, and high expression of this splice variant relative to other transcripts is associated with poor patient outcome. Unlike the full-length variant, expression of Drp1 lacking exon 16 leads to decreased association of Drp1 to mitochondrial fission sites, more fused mitochondrial networks, enhanced respiration, and TCA cycle metabolites, and is associated with a more metastatic phenotype in vitro and in vivo. These pro-tumorigenic effects can also be inhibited by specific siRNA-mediated inhibition of the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the significance of the pathophysiological consequences of Drp1 alternative splicing and divergent functions of Drp1 splice variants, and strongly warrant consideration of Drp1 splicing in future studies.

cancer biology↗

bioRxiv2023

Virtual biomarkers: predicting immune status using label-free holotomography of individual human monocytes and machine learning analysis

Precise evaluation of immune status is critical for managing diseases such as sepsis, in which the immune system transitions between hyper-inflammatory and immune-suppressed states. However, current biomarkers are limited by low specificity and time-consuming protocols. Here, we present a label-free, imaging-based framework for single-cell immune profiling of human monocytes using three-dimensional holotomography (HT) and deep learning. HT captures subcellular refractive index (RI) distributions of live, unlabeled cells, enabling quantitative extraction of morphological and biophysical features. Using an in vitro LPS-based model, we classified three functional immune states--control, hyper-inflammation, and immune suppression--based on 4,059 holotomograms from 11 donors. Immune state transitions were associated with significant changes in cell volume, surface area, RI variability, and the abundance of lipid droplets. A 3D convolutional neural network trained on HT images achieved 83.7% accuracy for single-cell predictions, increasing to 99.9% with ensemble averaging. This study establishes HT as a scalable, label-free platform for real-time immune monitoring and introduces subcellular RI features as robust correlates of immune dysregulation.

immunology↗

bioRxiv2023

Tissue-intrinsic Wnt signals antagonize Nodal-driven AVE differentiation

The anterior-posterior axis of the mammalian embryo is laid down by the anterior visceral endoderm (AVE), an extraembryonic signaling center that is specified within the visceral endoderm. Current models posit that AVE differentiation is promoted globally by epiblast-derived Nodal signals, and spatially restricted by a BMP gradient established by the extraembryonic ectoderm. Here, we report spatially restricted AVE differentiation in bilayered embryo-like aggregates made from mouse embryonic stem cells that lack an extraembryonic ectoderm. Notably, clusters of AVE cells also form in pure visceral endoderm cultures upon activation of Nodal signaling, indicating that tissue-intrinsic factors restrict AVE differentiation. We identify Wnt signaling as a tissue-intrinsic factor that antagonizes AVE-inducing Nodal signals. Together, our results suggest that interactions between epiblast and visceral endoderm alone enable local AVE differentiation in the absence of graded BMP signals. This may be a flexible solution for axis patterning in a wide range of embryo geometries.

developmental biology↗

bioRxiv2023

Monosynaptic inputs to ventral tegmental area glutamate and GABA co-transmitting neurons

A unique population of ventral tegmental area (VTA) neurons co-transmits glutamate and GABA as well as functionally signals rewarding and aversive outcomes. However, the circuit inputs to VTA VGluT2+VGaT+ neurons are unknown, limiting our understanding of the functional capabilities of these neurons. To identify the inputs to VTA VGluT2+VGaT+ neurons, we coupled monosynaptic rabies tracing with intersectional genetic targeting of VTA VGluT2+VGaT+ neurons in mice. We found that VTA VGluT2+VGaT+ neurons received diverse brain-wide inputs. The largest numbers of monosynaptic inputs to VTA VGluT2+VGaT+ neurons were from superior colliculus, lateral hypothalamus, midbrain reticular nucleus, and periaqueductal gray, whereas the densest inputs relative to brain region volume were from dorsal raphe nucleus, lateral habenula, and ventral tegmental area. Based on these and prior data, we hypothesized that lateral hypothalamus and superior colliculus inputs were glutamatergic neurons. Optical activation of glutamatergic lateral hypothalamus neurons robustly activated VTA VGluT2+VGaT+ neurons regardless of stimulation frequency and resulted in flee-like ambulatory behavior. In contrast, optical activation of glutamatergic superior colliculus neurons activated VTA VGluT2+VGaT+ neurons for a brief period of time at high stimulation frequency and resulted in head rotation and arrested ambulatory behavior (freezing). For both pathways, behaviors induced by stimulation were uncorrelated with VTA VGluT2+VGaT+ neuron activity. However, stimulation of glutamatergic lateral hypothalamus neurons, but not glutamatergic superior colliculus neurons, was associated with VTA VGluT2+VGaT+ footshock-induced activity. We interpret these results such that inputs to VTA VGluT2+VGaT+ neurons may integrate diverse signals related to the detection and processing of motivationally-salient outcomes. Further, VTA VGluT2+VGaT+ neurons may signal threat-related outcomes, possibly via input from lateral hypothalamus glutamate neurons, but not threat-induced behavioral kinematics.

neuroscience↗

bioRxiv2023

Volumetric refractive index measurement and quantitative density analysis of mouse brain tissue with sub-micrometer spatial resolution

High-resolution structural imaging of brain tissue is important for neuroscience research. However, conventional approaches have several limitations, such as the need for exogenous staining, limited accessibility to volumetric information, and qualitative analysis. Herein, we present high-resolution label-free volumetric imaging and analysis of mouse brain tissue using three-dimensional quantitative phase imaging. Measurement of the refractive index distribution of tissue enables direct imaging of the cellular and subcellular structures. Quantification of subcellular organelles is performed in the anatomical regions of the somatosensory cortex, corpus callosum, caudoputamen, and thalamus regions.

biophysics↗

bioRxiv2022

Unique functional responses differentially map onto genetic subtypes of dopamine neurons

Dopamine neurons are characterized by their response to unexpected rewards, but they also fire during movement and aversive stimuli. Dopamine neuron diversity has been observed based on molecular expression profiles; however, whether different functions map onto such genetic subtypes remains unclear. Here, we establish that three genetic dopamine subtypes within the substantia nigra pars compacta each have a unique set of responses to rewards, aversive stimuli, accelerations and decelerations, and these signaling patterns are highly-correlated between somas and axons within subtypes. Remarkably, reward responses were not detected in one subtype, which instead displayed acceleration-correlated signaling. Our findings establish a connection between functional and genetic dopamine subtypes and demonstrate that molecular expression patterns can serve as a common framework to dissect dopaminergic functions.

neuroscience↗

bioRxiv2022

Structural basis for ion selectivity in potassium-selective channelrhodopsins

The KCR channelrhodopsins are recently-discovered light-gated ion channels with high K+ selectivity, a property that has attracted broad attention among biologists- due to intense interest in creating novel inhibitory tools for optogenetics leveraging this K+ selectivity, and due to the mystery of how this selectivity is achieved in the first place. Indeed, the molecular and structural mechanism for K+ selectivity in KCRs has remained especially puzzling since these 7-transmembrane retinal-binding proteins completely lack structural similarity with known K+ channels, which generally coordinate K+ in a precisely symmetric conduction pathway formed by a tight interface among multiple small monomeric channel subunits (presumably not an accessible mechanism for the large KCR rhodopsin proteins). Here we present the cryo-electron microscopy structures of two KCRs from Hyphochytrium catenoides with distinct spectral properties for light absorption and channel actuation, HcKCR1, and HcKCR2, at resolutions of 2.6 and 2.5 [A], respectively. Structural comparison revealed first an unusually-shaped retinal binding pocket which induces rotation of the retinal in HcKCR2, explaining the large spectral difference between HcKCR1 and 2. Next, our combined structural, electrophysiological, computational, and spectroscopic analyses revealed a new solution to the challenging problem of K+-selective transport. KCRs indeed do not exhibit the canonical tetrameric K+ selectivity filter that specifically coordinates dehydrated K+; instead, single KCR monomers form a size exclusion filter using aromatic residues at the extracellular side of the pore which inhibits passage of bulky hydrated ions. This unique feature allows KCRs to function as K+ channels under relevant physiological conditions, providing not only a novel mechanism for achieving high K+ permeability ratios in biological ion channels, but also a framework for designing the next generation of inhibitory optogenetic tools. In BriefThe first structures of K+-selective channelrhodopsins (HcKCR1 and 2) are determined, revealing a K+ selectivity mechanism distinctly different from canonical K+ channels. HighlightsO_LIThe cryo-EM structures of K+-selective channelrhodopsins, HcKCR1 and 2, in nanodisc C_LIO_LIConditions under which naturally-occurring microbial rhodopsins have a 6-s-cis retinal C_LIO_LIIdentification of key residues for high K+ permeability ratios C_LIO_LIThe unique K+ selectivity mechanism of KCRs C_LI

biophysics↗

bioRxiv2022

Dopaminergic axons track somatic signaling in behaving mice

Striatal dopamine released from the axons of midbrain dopamine neurons has been linked to a wide range of functions, including movement control and reward-based learning. Recent studies have reported functional signaling differences between axons and somas of dopamine neurons, suggesting that local modulation controls dopamine release and calling into question the classical view of somatic control. However, these experiments are technically challenging, making it difficult to ensure that axonal and somatic recordings come from the same neurons, particularly given the heterogeneity of dopaminergic cell types. Here we used genetic strategies to isolate key dopaminergic neuron subtypes and monitor their axonal and somatic signaling patterns in behaving mice. Contrary to the inferences drawn from previous studies, these experiments revealed a robust correlation between somatic and axonal signaling. Thus, by exploiting a previously unknown connection between genetic and functional diversity in dopamine neurons, we establish that subtypes must be considered to understand the mechanisms of dopamine release in striatum during behavior.

neuroscience↗

bioRxiv2022

Targeted deletion of uterine glandular Foxa2 induces embryonic diapause in mice

Embryonic diapause is a reproductive strategy in which embryo development and growth is temporarily arrested within the uterus to ensure the survival of neonates and mothers during unfavorable conditions. Pregnancy is reinitiated when conditions become favorable for neonatal survival. The mechanism of how the uterus enters diapause remains unclear. Mice with uterine deletion of Foxa2, a transcription factor, are infertile. In this study, we show that dormant blastocysts are recovered from these mice on day 8 of pregnancy with persistent uterine Msx1 expression, a gene critical to maintaining the uterine quiescent state, suggesting that these mice enter embryonic diapause. Leukemia inhibitory factor (LIF) can resume implantation in these mice. Although estrogen is critical for implantation in progesterone (P4)-primed uterus, our current model reveals that FOXA2-independent estrogenic effects are detrimental to sustaining uterine quiescence. Interestingly, P4 and anti-estrogen can prolong uterine quiescence in the absence of FOXA2. While we find that Msx1 expression persists in the uterus deficient in Foxa2, the complex relationship of FOXA2 with Msx genes and estrogen receptors remains to be explored.

developmental biology↗

bioRxiv2021

Pentoxifylline-induced Protein Expression Change in RAW 264.7 Cells as Determined by Immunoprecipitation-based High Performance Liquid Chromatography

Although pentoxifylline (PTX) was identified as a competitive non-selective phosphodiesterase inhibitor, its pharmacological effect has not been clearly elucidated. The present study explored the effect of low dose 10 g/mL PTX (therapeutic dose) compared to high dose 300 g/mL PTX (experimental dose) in RAW 264.7 cells through immunoprecipitation-based high performance liquid chromatography (IP-HPLC), immunohistochemistry, and western blot. 10 g/mL PTX increased the expression of proliferation (Ki-67, PCNA, cyclin D2, cdc25A), epigenetic modification (KDM4D, PCAF), protein translation (DOHH, DHPS, eIF5A1), RAS signaling (KRAS, pAKT1/2/3, PI3K), NFkB signaling (NFkB, GADD45, p38), protection (HSP70, SOD1, GSTO1/2), neuromuscular differentiation (NSE{gamma}, myosin-1a, desmin), osteoblastic differentiation (BMP2, RUNX2, osterix), acute inflammation (TNF, IL-1, CXCR4), innate immunity ({beta}-defensin 1, lactoferrin, TLR-3, -4), cell-mediated immunity (CD4, CD8, CD80), while decreased the expression of ER stress (eIF2, eIF2AK3, ATF6), fibrosis (FGF2, CTGF, collagen 3A1), and chronic inflammation (CD68, MMP-2, -3, COX2) versus the untreated controls. The activation of proliferation by 10 g/mL PTX was also supported by the increase of cMyc-MAX heterodimer and {beta}-catenin-TCF1 complex in double IP-HPLC. 10 g/mL PTX enhanced FAS-mediated apoptosis but diminished p53-mediated apoptosis, and downregulated many angiogenesis proteins (angiogenin, VEGF-A, and FLT4), but upregulated HIF1, VEGFR2, and CMG2 reactively. Whereas, 300 g/mL PTX consistently decreased proliferation, epigenetic modification, RAS and NFkB signaling, neuromuscular and osteoblastic differentiation, but increased apoptosis, ER stress, and fibrosis compared to 10 g/mL PTX. These data suggest PTX has different biological effect on RWA 264.7 cells depending on the concentration of 10 g/mL and 300 g/mL PTX. The low dose 10 g/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, while the high dose 300 g/mL PTX was found to alleviate the 10 g/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation.

pharmacology and toxicology↗