Abstract
One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human ...induced pluripotent stem cells (hiPSCs) carrying a TNNI1
EmGFP
and TNNI3
mCherry
double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.
Iron is involved in numerous physiologically essential processes in our body. However, excessive iron is a pathogenic factor in neurodegenerative diseases, causing aberrant oxidative stress. Divalent ...metal transporter 1 (DMT1) acts as a primary transporter of Fe(ii) ions. The intracellular delivery of DMT1 toward the cellular membrane
the trans-Golgi network during the endocytotic process is partially regulated by a retromer-mediated protein-sorting system comprising vacuolar protein-sorting proteins (VPSs). Thus, together with DMT1, the Golgi-apparatus acts as a hub organelle in the delivery system for intracellular Fe(ii) ions. Dysfunction of the VPS-relevant protein sorting system can induce the abnormal delivery of DMT1 toward lysosomes concomitantly with Fe(ii) ions. To explore this issue, we developed a fluorescent probe, Gol-SiRhoNox, for the Golgi-specific detection of Fe(ii) ions by integrating our original
-oxide-based Fe(ii)-specific chemical switch, a new Golgi-localizable chemical motif, and polarity-sensitive fluorogenic scaffold. Our synchronous imaging study using Gol-SiRhoNox and LysoRhoNox, a previously developed fluorescent probe for lysosomal Fe(ii), revealed that the intracellular distribution balance of Fe(ii) ions between the Golgi apparatus and lysosomes is normally Golgi-dominant, whereas the lysosome-specific elevation of Fe(ii) ions was observed in cells with induced dysfunction of VPS35, a member of the retromer complex. Treatment of cells with dysfunctional VPS35 with R55, a molecular chaperone, resulted in the restoration of the subcellular distribution of Fe(ii) ions to the Golgi-dominant state. These results indicate that the impairment of the DMT1 traffic machinery affects subcellular iron homeostasis, promoting Fe(ii) leakage at the Golgi and lysosomal accumulation of Fe(ii) through missorting of DMT1.
Manual segmentation of the petals of flower computed tomography (CT) images is time-consuming and labor-intensive because the flower has many petals. In this study, we aim to obtain a ...three-dimensional (3D) structure of Camellia japonica flowers and propose a petal segmentation method using computer vision techniques. Petal segmentation on the slice images fails by simply applying the segmentation methods because the shape of the petals in CT images differs from that of the objects targeted by the latest instance segmentation methods. To overcome these challenges, we crop two-dimensional (2D) long rectangles from each slice image and apply the segmentation method to segment the petals on the images. Thanks to cropping, it is easier to segment the shape of the petals in the cropped images using the segmentation methods. We can also use the latest segmentation method for the task because the number of images used for training is augmented by cropping. Subsequently, the results are integrated into 3D to obtain 3D segmentation volume data. The experimental results show that the proposed method can segment petals on slice images with higher accuracy than the method without cropping. The 3D segmentation results were also obtained and visualized successfully.
A soluble form of human intestinal lactoferrin receptor (shLFR) is identical to human intelectin-1 (hITLN-1), a galactofuranose-binding protein that acts as a host defense against invading pathogenic ...microorganisms. We found that recombinant shLFR, expressed in mammalian cells (CHO DG44, COS-1, and RK13), binds tightly to Sepharose 4 Fast Flow (FF)-based matrices in a Ca2+-dependent manner. This binding of shLFR to Sepharose 4 FF-based matrices was inhibited by excess D-galactose, but not by D-glucose, suggesting that shLFR recognizes repeating units of α-1,6-linked D-galactose in Sepharose 4 FF. Furthermore, shLFR could bind to both Sepharose 4B- and Sepharose 6B-based matrices that were not crosslinked in a similar manner as to Sepharose 4 FF-based matrices. Therefore, shLFR (hITLN-1) binds to Sepharose-based matrices in a Ca2+-dependent manner. This binding property is most likely related to the ability, as host defense lectins, to recognize sepharose (agarobiose)-like structures present on the surface of invading pathogenic microorganisms.
Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens ...enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151
population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151
population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151
population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.
Abstract only
Introduction:
In conventional cardiac differentiation methods, a heterogeneous cardiac population is generated from human induced pluripotent stem cells (hiPSCs). Purifying ventricular- ...or atrial cardiomyocytes (VCMs or ACMs, respectably) is valuable for drug discovery for region-specific heart diseases. Previously, we have identified a novel marker, CD151, which can separate advanced specified subtypes; CD151
high
VCMs in ventricular inducing condition (VIC) and CD151
low
ACMs in atrial inducing condition (AIC). Although CD151 is known as a cell adhesion molecule, the function in cardiac subtype specification is unclear. In this study, we addressed the mechanisms of how CD151 distinguishes cardiomyocyte subtypes in cardiac differentiation from hiPSCs.
Methods:
We performed RNA sequencing in VIC- and AIC- CD151
high/low
CMs. Pathway analysis using differential expression genes between CD151
high/low
CMs was conducted to identify prominent pathways in subtype specification.
Results:
In VIC, mitosis-related genes were enriched in CD151
high
VCMs. To clarify whether those genes affect cell proliferation or multi-nucleation, we performed the flow cytometric analysis of CD151
high/low
VCMs with KI-67 proliferation marker and Hoechst staining. Although the percentage of KI67-positive cells was almost the same, binucleated cells were significantly more in CD151
high
VCMs than in CD151
low
VCMs. In AIC, transcriptome analysis suggested that Notch signaling was activated in CD151
high
ACMs. To reveal that Notch signaling inhibits atrial specification, we analyzed ACMs treated with LY411575, a Notch signaling inhibitor. LY411575 induced atrial marker gene expression and increased the number of cells showing action potentials of ACMs. CD151
low
ACMs after LY411575 treatment contained ACMs with the highest efficiency.
Conclusion:
Our results demonstrate that the CD151 expression indicates mitotic activity in VCMs. CD151
high
VCMs are more advanced specified VCMs with binucleation without cell proliferation. On the other hand, in ACMs, CD151 works as an indicator of Notch signaling, which suppresses atrial specification. Our study provides a novel approach to generating and purifying hiPSCs-ACMs by Notch inhibition and CD151 separation.
•The first report of cloning, expression and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida.•The amino acid sequence of Ef-Man showed similarity with endo-1,4- β – mannanases ...from invertebrate.•The Ef-Man gene was expressed in Pichia pastoris.•Ef-Man requires at least six subsites for efficient hydrolysis.•Overall structure of recombinant Ef-Man is similar to those of GH5 family proteins.
The endo-1,4-β-mannanases (Ef-Man) gene from Eisenia fetida was determined to consist of 1131 bp and encode a 377 amino acid protein. The amino acid sequence showed similarity with the endo-1,4-β-mannanases of Daphnia pulex (62%), Cryptopygus antarcticus (64%), Crassostrea gigas (61%), Mytilus edulis (60%), and Aplysia kurodai (58%). The gene encoding mature Ef-Man was expressed in Pichia pastoris (GS115 strain). Based on SDS-PAGE analysis, the molecular mass of the purified recombinant Ef-Man (rEf-Man) was estimated to be 39 kDa. All catalytically important residues of endo-1,4-β-mannanases in the glycoside hydrolase (GH) family 5 were conserved in Ef-Man. The optimal temperature for rEf-Man was identified as 60 °C. HPLC and HPAEC analyses suggest that Ef-Man requires at least six subsites for efficient hydrolysis and is capable of performing transglycosylation reactions. The overall structure of rEf-Man is similar to those of GH5 family proteins, and tertiary structures around the active site are conserved among endo-1,4-β-mannanase families. X-ray crystallographic analysis supports the hydrolysis and transglycosylation reaction mechanism determined by HPLC and HPAEC analyses.
The earthworm Eisenia fetida possesses several cold‐active enzymes, including α‐amylase, β‐glucanase and β‐mannanase. E. fetida possesses two isoforms of α‐amylase (Ef‐Amy I and II) to digest raw ...starch. Ef‐Amy I retains its catalytic activity at temperatures below 10°C. To identify the molecular properties of Ef‐Amy I, X‐ray crystal structures were determined of the wild type and of the inactive E249Q mutant. Ef‐Amy I has structural similarities to mammalian α‐amylases, including the porcine pancreatic and human pancreatic α‐amylases. Structural comparisons of the overall structures as well as of the Ca2+‐binding sites of Ef‐Amy I and the mammalian α‐amylases indicate that Ef‐Amy I has increased structural flexibility and more solvent‐exposed acidic residues. These structural features of Ef‐Amy I may contribute to its observed catalytic activity at low temperatures, as many cold‐adapted enzymes have similar structural properties. The structure of the substrate complex of the inactive mutant of Ef‐Amy I shows that a maltohexaose molecule is bound in the active site and a maltotetraose molecule is bound in the cleft between the N‐ and C‐terminal domains. The recognition of substrate molecules by Ef‐Amy I exhibits some differences from that observed in structures of human pancreatic α‐amylase. This result provides insights into the structural modulation of the recognition of substrates and inhibitors.
X‐ray crystal structures were determined of the wild type and an inactive mutant of α‐amylase from the earthworm Eisenia fetida. Structural analyses reveal the molecular properties that are responsible for catalytic activity at low temperatures and substrate recognition.
Iron is involved in numerous physiologically essential processes in our body. However, excessive iron is a pathogenic factor in neurodegenerative diseases, causing aberrant oxidative stress. Divalent ...metal transporter 1 (DMT1) acts as a primary transporter of Fe(
ii
) ions. The intracellular delivery of DMT1 toward the cellular membrane
via
the trans-Golgi network during the endocytotic process is partially regulated by a retromer-mediated protein-sorting system comprising vacuolar protein-sorting proteins (VPSs). Thus, together with DMT1, the Golgi-apparatus acts as a hub organelle in the delivery system for intracellular Fe(
ii
) ions. Dysfunction of the VPS-relevant protein sorting system can induce the abnormal delivery of DMT1 toward lysosomes concomitantly with Fe(
ii
) ions. To explore this issue, we developed a fluorescent probe, Gol-SiRhoNox, for the Golgi-specific detection of Fe(
ii
) ions by integrating our original
N
-oxide-based Fe(
ii
)-specific chemical switch, a new Golgi-localizable chemical motif, and polarity-sensitive fluorogenic scaffold. Our synchronous imaging study using Gol-SiRhoNox and LysoRhoNox, a previously developed fluorescent probe for lysosomal Fe(
ii
), revealed that the intracellular distribution balance of Fe(
ii
) ions between the Golgi apparatus and lysosomes is normally Golgi-dominant, whereas the lysosome-specific elevation of Fe(
ii
) ions was observed in cells with induced dysfunction of VPS35, a member of the retromer complex. Treatment of cells with dysfunctional VPS35 with R55, a molecular chaperone, resulted in the restoration of the subcellular distribution of Fe(
ii
) ions to the Golgi-dominant state. These results indicate that the impairment of the DMT1 traffic machinery affects subcellular iron homeostasis, promoting Fe(
ii
) leakage at the Golgi and lysosomal accumulation of Fe(
ii
) through missorting of DMT1.
Fluctuation of labile Fe(
ii
) at Golgi apparatus was specifically detected by a new fluorescent probe.
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