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1.
Sports Biomech ; : 1-12, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38742268

ABSTRACT

This study aims to identify the relationship between jump height and the kinetic and kinematic parameters of the hip, knee, and ankle joints during countermovement jump (CMJ) in elite male athletes. Sixty-six elite male athletes from various sports (strength and power, winter downhill, combat, ball game, and aquatic) performed maximal effort CMJs with hands and arms crossed against their chests on force platforms. Jumping motion in the sagittal plane was recorded using video analysis and the peak torque, power, and angular velocity of the right hip, knee, and ankle joints were calculated during the propulsive phase. Correlations between the CMJ height and kinetic and kinematic parameters were investigated using Pearson's product-moment coefficient (r) and Spearman's rank correlation coefficient (ρ). CMJ height was highly correlated with peak hip power (ρ = 0.686, p < 0.001) and peak knee angular velocity (r = 0.517, p < 0.001), and moderately correlated with peak hip angular velocity (r = 0.438, p < 0.001) and peak hip torque (r = 0.398, p = 0.001). These results indicate that notable hip torque and power can contribute to increased angular velocity in both the knee and hip joints, ultimately increasing the CMJ height in elite male athletes.

2.
J Toxicol Sci ; 49(5): 241-248, 2024.
Article in English | MEDLINE | ID: mdl-38692911

ABSTRACT

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.


Subject(s)
ATP-Binding Cassette Transporters , Cell Survival , Ganglia, Spinal , Methylmercury Compounds , Schwann Cells , Animals , Ganglia, Spinal/metabolism , Ganglia, Spinal/drug effects , Methylmercury Compounds/toxicity , Schwann Cells/drug effects , Schwann Cells/metabolism , Cell Survival/drug effects , Cells, Cultured , Large Neutral Amino Acid-Transporter 1/metabolism , Large Neutral Amino Acid-Transporter 1/genetics , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/genetics , Peripheral Nerves/metabolism , Peripheral Nerves/drug effects , Male , Rats , Multidrug Resistance-Associated Protein 2
3.
J Perioper Pract ; : 17504589241232503, 2024 Apr 08.
Article in English | MEDLINE | ID: mdl-38590001

ABSTRACT

BACKGROUND: Postoperative temperature dysregulation affects the length of hospital stay and prognosis. This study evaluated the factors that influence the occurrence of fever in patients after aortic valve replacement surgery. METHODS: Eighty-seven consecutive patients who underwent aortic valve replacement surgery were included. Patients' age, sex and body mass index; presence of diabetes mellitus; operation time; blood loss; blood transfusion volume; preoperative and postoperative laboratory findings; presence or absence of oral function management; and fever >38°C were retrospectively analysed through univariate and multiple logistic regression analyses. RESULTS: Among the variables, only diabetes mellitus status was significantly associated with fever ⩾38°C. Postoperatively, patients with diabetes mellitus were significantly less likely to develop fever above 38°C and a fever rising to 38°C. CONCLUSIONS: This study shows that the presence of comorbid diabetes mellitus decreases the frequency of developing fever >38°C after aortic valve replacement surgery.

4.
Yeast ; 41(5): 349-363, 2024 May.
Article in English | MEDLINE | ID: mdl-38583078

ABSTRACT

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.


Subject(s)
Cyclic AMP-Dependent Protein Kinases , Optogenetics , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Signal Transduction , Schizosaccharomyces/genetics , Schizosaccharomyces/enzymology , Schizosaccharomyces/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Cyclic AMP/metabolism , Biosensing Techniques , Optical Imaging/methods , Cell Nucleus/metabolism , Cytoplasm/metabolism , Transcription Factors
5.
Materials (Basel) ; 17(4)2024 Feb 14.
Article in English | MEDLINE | ID: mdl-38399131

ABSTRACT

BACKGROUND: Bone morphogenetic protein-2 (bmp-2) has a high potential to induce bone tissue formation in skeletal muscles. We developed a bone induction system in skeletal muscles using the bmp-2 gene through in vivo electroporation. Natural bone tissues with skeletal muscles can be considered potential candidates for biomaterials. However, our previous system using plate-type electrodes did not achieve a 100% success rate in inducing bone tissues in skeletal muscles. In this study, we aimed to enhance the efficiency of bone tissue formation in skeletal muscles by using a non-viral bmp-2 gene expression plasmid vector (pCAGGS-bmp-2) and needle-type electrodes. METHODS: We injected the bmp-2 gene with pCAGGS-bmp-2 into the skeletal muscles of rats' legs and immediately placed needle-type electrodes there. Skeletal tissues were then observed on the 21st day after gene transfer using soft X-ray and histological analyses. RESULTS: The use of needle-type electrodes resulted in a 100% success rate in inducing bone tissues in skeletal muscles. In contrast, the plate-type electrodes only exhibited a 33% success rate. Thus, needle-type electrodes can be more efficient and reliable for transferring the bmp-2 gene to skeletal muscles, making them potential biomaterials for repairing bone defects.

6.
Glycobiology ; 34(4)2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38253038

ABSTRACT

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 µg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.


Subject(s)
Placenta , Proteins , Humans , Female , Pregnancy , Placenta/metabolism , Proteins/metabolism , Phosphorylation , Acetylglucosamine/metabolism , Protein Processing, Post-Translational
7.
Dev Cell ; 59(4): 545-557.e4, 2024 Feb 26.
Article in English | MEDLINE | ID: mdl-38228139

ABSTRACT

Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how CDK regulators coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by developing a CDK activity biosensor, Eevee-spCDK. We find that although CDK activity does not necessarily correlate with cyclin levels, it converges to the same level around mitotic onset in several mutant backgrounds, including pom1Δ cells and wee1 or cdc25 overexpressing cells. These data provide direct evidence that cells enter the M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.


Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Animals , Phosphorylation , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Mitosis , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Mammals/metabolism , Protein-Tyrosine Kinases/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism
8.
bioRxiv ; 2024 Jan 17.
Article in English | MEDLINE | ID: mdl-38293146

ABSTRACT

Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function-dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

9.
J Dent Educ ; 88(3): 314-321, 2024 Mar.
Article in English | MEDLINE | ID: mdl-37957835

ABSTRACT

PURPOSE/OBJECTIVES: Tooth carvings are evaluated subjectively. However, subjective evaluations are associated with low intra- and inter-evaluator reliability in providing feedback for the fabrication of better tooth carvings. Therefore, this study aimed to analyze the relationship between subjective evaluation and the morphological characteristics of tooth carvings and their morphological characteristics using the feedback method in the low-scoring group. METHODS: Between April 2013 and September 2021, 120 maxillary left first molar carvings fabricated by undergraduate students were subjectively evaluated by an experienced instructor using a five-point scale. The tooth carvings were scanned to obtain three-dimensional shape data and divided into two groups, the high- and low-scoring groups, for principal component analysis. Homologous models with dimensions matching those of the average model were created, and principal component analysis was performed to evaluate the morphological characteristics of the tooth. RESULTS: Principal component analysis indicated that an objective evaluation was possible using homologous model. On increasing the subjective evaluation, the occlusal inclination angle and the cervical region of the tooth became steeper, and the shape of the occlusal surface resembled a parallelogram. In addition, large morphological differences were observed in the position of the cervical region of the tooth, height of contour, and shape of the occlusal surface in the low-scoring group, whereas no such difference was observed in the high-scoring group. CONCLUSION: Objective evaluation of tooth carving was possible using homologous model. The evaluation of tooth morphological characteristics could be effective in providing feedback to undergraduate students.


Subject(s)
Tooth , Humans , Reproducibility of Results , Tooth/anatomy & histology , Molar/anatomy & histology , Feedback
10.
Proc Natl Acad Sci U S A ; 120(52): e2313514120, 2023 Dec 26.
Article in English | MEDLINE | ID: mdl-38109538

ABSTRACT

To cope with seasonal environmental changes, organisms have evolved approximately 1-y endogenous circannual clocks. These circannual clocks regulate various physiological properties and behaviors such as reproduction, hibernation, migration, and molting, thus providing organisms with adaptive advantages. Although several hypotheses have been proposed, the genes that regulate circannual rhythms and the underlying mechanisms controlling long-term circannual clocks remain unknown in any organism. Here, we show a transcriptional program underlying the circannual clock in medaka fish (Oryzias latipes). We monitored the seasonal reproductive rhythms of medaka kept under natural outdoor conditions for 2 y. Linear regression analysis suggested that seasonal changes in reproductive activity were predominantly determined by an endogenous program. Medaka hypothalamic and pituitary transcriptomes were obtained monthly over 2 y and daily on all equinoxes and solstices. Analysis identified 3,341 seasonally oscillating genes and 1,381 daily oscillating genes. We then examined the existence of circannual rhythms in medaka via maintaining them under constant photoperiodic conditions. Medaka exhibited approximately 6-mo free-running circannual rhythms under constant conditions, and monthly transcriptomes under constant conditions identified 518 circannual genes. Gene ontology analysis of circannual genes highlighted the enrichment of genes related to cell proliferation and differentiation. Altogether, our findings support the "histogenesis hypothesis" that postulates the involvement of tissue remodeling in circannual time-keeping.


Subject(s)
Oryzias , Animals , Oryzias/genetics , Seasons , Circadian Rhythm/physiology , Gonads , Photoperiod
11.
ACS Chem Biol ; 18(12): 2506-2515, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-37990966

ABSTRACT

Cells sense extracellular stimuli through membrane receptors and process information through an intracellular signaling network. Protein translocation triggers intracellular signaling, and techniques such as chemically induced dimerization (CID) have been used to manipulate signaling pathways by altering the subcellular localization of signaling molecules. However, in the fission yeast Schizosaccharomyces pombe, the commonly used FKBP-FRB system has technical limitations, and therefore, perturbation tools with low cytotoxicity and high temporal resolution are needed. We here applied our recently developed self-localizing ligand-induced protein translocation (SLIPT) system to S. pombe and successfully perturbed several cell cycle-related proteins. The SLIPT system utilizes self-localizing ligands to recruit binding partners to specific subcellular compartments such as the plasma membrane or nucleus. We optimized the self-localizing ligands to maintain the long-term recruitment of target molecules to the plasma membrane. By knocking in genes encoding the binding partners for self-localizing ligands, we observed changes in the localization of several endogenous molecules and found perturbations in the cell cycle and associated phenotypes. This study demonstrates the effectiveness of the SLIPT system as a chemogenetic tool for rapid perturbation of endogenous molecules in S. pombe, providing a valuable approach for studying intracellular signaling and cell cycle regulation with an improved temporal resolution.


Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Ligands , Protein Transport , Cell Cycle Proteins/metabolism , Protein Translocation Systems/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism
12.
Hum Mol Genet ; 32(24): 3323-3341, 2023 Dec 01.
Article in English | MEDLINE | ID: mdl-37676252

ABSTRACT

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms underlying the neural phenotypes associated with GM3SD are unknown. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in cells carrying either variant, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated GSLs compared to WT. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.


Subject(s)
Gangliosides , Glycosphingolipids , Humans , Erlotinib Hydrochloride , Glycosphingolipids/metabolism , G(M3) Ganglioside/genetics , G(M3) Ganglioside/metabolism , Sialyltransferases/genetics , Sialyltransferases/metabolism , Signal Transduction
13.
Mar Pollut Bull ; 195: 115472, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37657157

ABSTRACT

Bloom dynamics of K. mikimotoi during summer 2015 in the Yatsushiro Sea, Japan, which caused fish mortality was studied using field survey data and satellite data. The bloom initially formed in the western area, subsequently appeared in the southern area, and finally expanded to the central area. The field-survey detected the horizontal displacement of the bloom which was also assessed by satellite data. Acoustic backscattering intensity of the current meter captured the modulation of the diurnal vertical migration of K. mikimotoi. After the modulation, K. mikimotoi distributed at a shallower depth in the nighttime than the period prior to the modulation. Factors affecting the modulation are suggested to be the continuous low nutrient conditions. Synchronization between the shallowed distribution during the nighttime and the wind driven surface northeastward current enabled a sudden horizontal transport toward the central area. Satellite and acoustic backscattering data are beneficial subsidiary tools for detecting blooms.

14.
Int J Mol Sci ; 24(16)2023 Aug 16.
Article in English | MEDLINE | ID: mdl-37629048

ABSTRACT

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.


Subject(s)
Fluorescence Resonance Energy Transfer , Protein Disulfide-Isomerases , Protein Disulfide-Isomerases/genetics , Allosteric Regulation , Binding Sites , Oxidation-Reduction
15.
Curr Opin Cell Biol ; 84: 102217, 2023 10.
Article in English | MEDLINE | ID: mdl-37574635

ABSTRACT

Extracellular signal-regulated kinase (ERK) has been recognized as a critical regulator in various physiological and pathological processes. Extensive research has elucidated the signaling mechanisms governing ERK activation via biochemical regulations with upstream molecules, particularly receptor tyrosine kinases (RTKs). However, recent advances have highlighted the role of mechanical forces in activating the RTK-ERK signaling pathways, thereby opening new avenues of research into mechanochemical interplay in multicellular tissues. Here, we review the force-induced ERK activation in cells and propose possible mechanosensing mechanisms underlying the mechanoresponsive ERK activation. We conclude that mechanical forces are not merely passive factors shaping cells and tissues but also active regulators of cellular signaling pathways controlling collective cell behaviors.


Subject(s)
Extracellular Signal-Regulated MAP Kinases , MAP Kinase Signaling System , MAP Kinase Signaling System/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Signal Transduction/physiology , Receptor Protein-Tyrosine Kinases/metabolism
16.
Int J Mol Sci ; 24(11)2023 Jun 05.
Article in English | MEDLINE | ID: mdl-37298702

ABSTRACT

The receptor activator of NF-κB ligand (RANKL)-binding peptide is known to accelerate bone morphogenetic protein (BMP)-2-induced bone formation. Cholesterol-bearing pullulan (CHP)-OA nanogel-crosslinked PEG gel (CHP-OA nanogel-hydrogel) was shown to release the RANKL-binding peptide sustainably; however, an appropriate scaffold for peptide-accelerated bone formation is not determined yet. This study compares the osteoconductivity of CHP-OA hydrogel and another CHP nanogel, CHP-A nanogel-crosslinked PEG gel (CHP-A nanogel-hydrogel), in the bone formation induced by BMP-2 and the peptide. A calvarial defect model was performed in 5-week-old male mice, and scaffolds were placed in the defect. In vivo µCT was performed every week. Radiological and histological analyses after 4 weeks of scaffold placement revealed that the calcified bone area and the bone formation activity at the defect site in the CHP-OA hydrogel were significantly lower than those in the CHP-A hydrogel when the scaffolds were impregnated with both BMP-2 and the RANKL-binding peptide. The amount of induced bone was similar in both CHP-A and CHP-OA hydrogels when impregnated with BMP-2 alone. In conclusion, CHP-A hydrogel could be an appropriate scaffold compared to the CHP-OA hydrogel when the local bone formation was induced by the combination of RANKL-binding peptide and BMP-2, but not by BMP-2 alone.


Subject(s)
Hydrogels , Peptides , Animals , Male , Mice , Bone Morphogenetic Protein 2/pharmacology , Cholesterol , Hydrogels/pharmacology , Nanogels , Peptides/pharmacology , RANK Ligand/chemistry , RANK Ligand/metabolism
17.
Sci Rep ; 13(1): 9465, 2023 06 10.
Article in English | MEDLINE | ID: mdl-37301878

ABSTRACT

The extracellular signal-regulated kinase (ERK) is a serine/threonine kinase that is known to regulate cellular events such as cell proliferation and differentiation. The ERK signaling pathway is activated by fibroblast growth factors, and is considered to be indispensable for the differentiation of primitive endoderm cells, not only in mouse preimplantation embryos, but also in embryonic stem cell (ESC) culture. To monitor ERK activity in living undifferentiated and differentiating ESCs, we established EKAREV-NLS-EB5 ESC lines that stably express EKAREV-NLS, a biosensor based on the principle of fluorescence resonance energy transfer. Using EKAREV-NLS-EB5, we found that ERK activity exhibited pulsatile dynamics. ESCs were classified into two groups: active cells showing high-frequency ERK pulses, and inactive cells demonstrating no detectable ERK pulses during live imaging. Pharmacological inhibition of major components in the ERK signaling pathway revealed that Raf plays an important role in determining the pattern of ERK pulses.


Subject(s)
Extracellular Signal-Regulated MAP Kinases , Mouse Embryonic Stem Cells , Animals , Mice , Mouse Embryonic Stem Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System , Cell Differentiation , Signal Transduction
18.
Proc Natl Acad Sci U S A ; 120(15): e2211807120, 2023 04 11.
Article in English | MEDLINE | ID: mdl-37014867

ABSTRACT

Intensity-based time-lapse fluorescence resonance energy transfer (FRET) microscopy has been a major tool for investigating cellular processes, converting otherwise unobservable molecular interactions into fluorescence time series. However, inferring the molecular interaction dynamics from the observables remains a challenging inverse problem, particularly when measurement noise and photobleaching are nonnegligible-a common situation in single-cell analysis. The conventional approach is to process the time-series data algebraically, but such methods inevitably accumulate the measurement noise and reduce the signal-to-noise ratio (SNR), limiting the scope of FRET microscopy. Here, we introduce an alternative probabilistic approach, B-FRET, generally applicable to standard 3-cube FRET-imaging data. Based on Bayesian filtering theory, B-FRET implements a statistically optimal way to infer molecular interactions and thus drastically improves the SNR. We validate B-FRET using simulated data and then apply it to real data, including the notoriously noisy in vivo FRET time series from individual bacterial cells to reveal signaling dynamics otherwise hidden in the noise.


Subject(s)
Fluorescence Resonance Energy Transfer , Microscopy , Fluorescence Resonance Energy Transfer/methods , Bayes Theorem
19.
Nihon Yakurigaku Zasshi ; 158(3): 246, 2023.
Article in Japanese | MEDLINE | ID: mdl-37121706
20.
JIMD Rep ; 64(2): 138-145, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36873089

ABSTRACT

GM3 synthase deficiency (GM3SD) is caused by biallelic variants in ST3GAL5. The ganglioside GM3, enriched in neuronal tissues, is a component of lipid rafts and regulates numerous signaling pathways. Affected individuals with GM3SD exhibit global developmental delay, progressive microcephaly, and dyskinetic movements. Hearing loss and altered skin pigmentation are also common. Most of the reported variants in ST3GAL5 are found in motifs conserved across all sialyltransferases within the GT29 family of enzymes. These motifs include motif L and motif S which contain amino acids responsible for substrate binding. These loss-of-function variants cause greatly reduced biosynthesis of GM3 and gangliosides derived from GM3. Here we describe an affected female with typical GM3SD features bearing two novel variants that reside in the other two conserved sialyltransferase motifs (motif 3 and motif VS). These missense alterations occur in amino acid residues that are strictly invariant across the entire GT29 family of sialyltransferases. The functional significance of these variants was confirmed by mass spectrometric analysis of plasma glycolipids, demonstrating a striking loss of GM3 and accumulation of lactosylceramide and Gb3 in the patient. The glycolipid profile changes were accompanied by an increase in ceramide chain length on LacCer. No changes in receptor tyrosine phosphorylation were observed in patient-derived lymphoblasts, indicating that GM3 synthase loss-of-function in this cell type does not impact receptor tyrosine kinase activity. These findings demonstrate the high prevalence of loss-of-function ST3GAL5 variants within highly conserved sialyltransferase motifs in affected individuals with GM3SD.

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