Search

In somatic cell nuclear transfer (SCNT), maturation promoting factor (MPF) is believed to be one of the factors involved with nuclear envelope breakdown and chromatin condensation of the transferred nucleus. Although MPF activity is high both in metaphase-I or -II oocytes (M-I and M-II, respectively), only M-II oocytes have been used exclusively as recipient cytoplasts in SCNT. In this study, we examined the effect of different onset of (1) enucleation of recipient oocytes at the M-I and M-II stages, and (2) fusion and activation of the couplets on their developmental ability to the blastocyst stage in pigs. The primary cultured cumulus cells were used as donor karyoplasts, and recipient cytoplasts were prepared by enucleation of in vitro-matured oocytes using gradient centrifugation in percoll solution. A karyoplast and a cytoplast were fused by 2 DC pulses of 1.5 kV cm-1 for 20 s, and then the couplets were activated by 2 DC pulses of 0.8 kV cm-1 for 30 s. The reconstructed embryos were cultured according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) except for the addition of 5% FCS to NCSU-37 during Days 2–7 (Day 0 is the day of SCNT) of embryo culture using the WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 258–264). Some of the embryos were fixed at 1, 10, and 24 h after activation and examined for morphology of nuclei. After 30 h of IVM, oocytes (mainly at the M-I stage) were enucleated. Then the couplets were fused immediately (Group A) or at 48 h after the onset of IVM (Group B); activation was conducted at 48 h of IVM (Group A) or at 1 h after fusion (Group B). As a control group, oocytes were enucleated after 48 h of IVM and then the couplets were fused and activated. None of the embryos in Group B developed to the blastocyst stage. However, a few of the embryos [2/117 (1.7%)] in Group A developed to the blastocyst stage; however, the rate was significantly lower than that of the control group [10/112 (8.9%); chi-square; P = 0.03]. The rates of embryos undergoing premature chromosome condensation (PCC) in Group B at 1 h and 10 h after activation were significantly lower than those in Group A [1 h: 51/69 (73.9%) vs. 76/76 (100%); 10 h: 24/76 (31.6%) vs. 45/91 (49.5%), respectively); some of them had pseudo-pronuclei. By 24 h after activation there were no detectable differences in the rates of cleavage [2/70 (2.9%) vs. 2/61 (3.3%)]; however, the rates were significantly lower than that of the control group [23/90 (25.6%); chi-square; P < 0.05]. These results suggest that MPF activity might be changed in oocytes without nucleus during the maturation culture. Thus, a specific nucleus-associated factor(s) that may present in the cytoplasm seems to be essential for the successful remodeling of the transferred nucleus and the development of SCNT embryos to the blastocyst stage. [ABSTRACT FROM AUTHOR]

Oocyte and cumulus cells communicate through an extensive network of gap junctions (GJs), which permit the transfer of small molecules such as cAMP. Gonadotropin strongly enhances the intracellular cAMP concentration in cumulus cells, and induces oocyte meiotic resumption. Enhanced cAMP also triggers a reduction of GJ communications (GJCs) in cumulus-oocyte complexes (COCs), accompanied by cumulus expansion. Intracellular cAMP is modulated by both adenylate cyclase (AC) for synthesizing and phosphodiesterase (PDE) for degrading. Addition of AC to gonadotropin-free medium induces meiotic resumption of bovine oocytes without cumulus expansion, suggesting that maintenance of cAMP at a certain level in COCs may be crucial for either prolonged maintenance of GJCs or the timing of oocyte meiotic resumption. In the present study, we investigated the intracellular cAMP concentrations in porcine COCs or oocytes, and GJCs during in vitro oocyte maturation culture using PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Porcine COCs obtained from prepubertal gilts were cultured for 20 h (1st culture) using M199 containing 10% FCS (basic medium, BM group) with FSH (FSH group) or IBMX (IBMX group). Following this, the COCs were transferred into the basic medium containing FSH and LH, and cultured for another 24 h (2nd culture). At 6, 12, and 20 h of the 1st culture, intracellular cAMP in COCs or oocytes was measured. To determine GJCs in each COC, Lucifer Yellow fluorescent dye was microinjected into cumulus-enclosed oocytes at 6 or 12 h of the 1st culture, and the ability of dye transfer, which is related to the GJCs, from the oocyte to the surrounding cumulus cells was observed. At the end of the 1st culture, 30.8 6.0% of the oocytes in the FSH group underwent germinal vesicle breakdown (GVBD), whereas only a few oocytes in the BM group (8.6 2.4%) and the IBMX group (5.8 3.0%) achieved GVBD (P < 0.05). In contrast, ratios of metaphase-II (M-II) stage oocytes at the end of the 2nd culture did not differ between the FSH group (75.7 3.9%) and the IBMX group (68.2 6.8%), although a few oocytes in BM group (10.1 3.7%) reached the M-II stage (P < 0.05). Concentrations of cAMP in COCs and oocytes increased drastically in the FSH group compared to those of the BM and IBMX groups (P < 0.05). In addition, the concentration of cAMP in IBMX group oocytes was also higher than that in the BM group, with a significant difference detected at 20 h (P < 0.05). The GJCs in the FSH group were gradually closed, depending on the length of time in culture (54.9 3.7% of COCs closed their GCJs at 12 h of the 1st culture). In contrast, in the IBMX group, only 23.0 3.7% of COCs closed their GJCs at 12 h of the 1st culture, which was significantly different from that of the other two groups (P < 0.05). These results suggest that treatment with IBMX during the first half of IVM culture can induce subsequent meiotic resumption of porcine oocytes, and that a moderate increase of cAMP concentration in COCs or oocytes prolongs GJCs during the treatment. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 2.3, 39.3 1.7, and 42.5 2.7, respectively) was significantly higher (P < 0.01) than those in the control (33.4 1.6) and DPI-0.5 (32.7 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P < 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA. [ABSTRACT FROM AUTHOR]

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P < 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P < 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P < 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P < 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P < 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes.This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02). [ABSTRACT FROM AUTHOR]

The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ∼80% of the analytes were less than or equal to 50 pg ml -1 , with the lowest LoD (1 pg ml -1 ) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.

Glucose transporter 1 deficiency syndrome (GLUT1DS), caused by impaired glucose transport at the blood-brain barriers, leads to various central nervous system dysfunctions. A comprehensive understanding of the underlying disease pathogenesis is still lacking. In this study, we have generated GLUT1DS-specific human induced pluripotent stem cells (hiPSCs) derived from two patients. These established GLUT1DS-specific hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift or missense mutations in the responsible SLC2A1 gene. These novel cell resources provide new avenues for understanding disease mechanisms and developing new therapies for GLUT1DS., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Satoru Takahashi reports financial support was provided by Japan Agency for Medical Research and Development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

We retrospectively reviewed patients with Corynebacterium striatum bacteremia treated with daptomycin. All 11 isolates were initially susceptible to daptomycin, but the emergence of daptomycin nonsusceptibility during treatment and clinical failure occurred in 36% and 45% of patients, respectively., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

CD26 is a human T cell costimulatory molecule as well as a T cell subset marker, and increase of CD26+ T cells in inflamed tissues and peripheral blood has been reported in diverse autoimmune diseases. In contrast, our group has previously shown that levels of circulating CD26+ T cells are decreased in patients with systemic lupus erythematosus (SLE), although the role of reduced CD26 T cell surface expression in SLE pathology remains to be elucidated. In the present study, we conducted CD26-based T cell subset analyses utilizing peripheral blood mononuclear cells from 57 SLE patients and 31 healthy adult volunteers. We show that the increase in CD26(-) T cell population reflects the abnormal expansion of CD26(-)CD28(-) cytotoxic subsets of both CD8 T cells and CD4 T cells in SLE patients. Single cell RNA sequencing analysis of the CD26(-)CD28(-) CD4 and CD8 T cell populations reveals unique characteristics with similarities to natural killer T cells. In addition, the level of CD26(-)CD28(-) T cells is increased in some active stage SLE patients with renal manifestation. Meanwhile, effect of prednisolone treatment on these populations varies from patient to patient, with levels of these cytotoxic effector populations still being elevated in some inactive stage SLE patients. Taken together, our data suggest that analysis of these populations in SLE may be a useful tool to classify this markedly heterogeneous condition., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Satoru Takahashi reports financial support was provided by Japan Agency for Medical Research and Development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To investigate the difference between the segmented axial length (AL) and the composite AL on a swept-source optical coherence tomography biometer and to evaluate the subsequent effects on artificial intelligence intraocular lens (IOL) power calculations: the Kane and Hill-RBF 3.0 formulas compared with established vergence formulas., Setting: National Hospital Organization, Tokyo Medical Center, Japan., Design: Retrospective case series., Methods: Consecutive patients undergoing cataract surgery with a single-piece IOL were reviewed. The prediction accuracy of the Barrett Universal II, Haigis, Hill-RBF 3.0, Hoffer Q, Holladay 1, Kane, and SRK/T formulas based on 2 ALs were compared for each formula. The heteroscedastic test was used with the SD of prediction errors as the endpoint for formula performance., Results: The study included 145 eyes of 145 patients. The segmented AL (24.83 ± 1.89) was significantly shorter than the composite AL (24.88 ± 1.96, P < .001). Bland-Altman analysis revealed a negative proportional bias for the differences between the segmented AL and the composite AL. The SD values obtained by Hoffer Q, Holladay 1, and SRK/T formulas based on the segmented AL (0.52 diopters [D], 0.54 D, and 0.50 D, respectively) were significantly lower than those based on the composite AL (0.57 D, 0.60 D, and 0.52 D, respectively, P < .01)., Conclusions: The segmented ALs were longer in short eyes and shorter in long eyes than the composite ALs. The refractive accuracy can be improved in the Hoffer Q, Holladay 1, and SRK/T formulas by changing the composite ALs to the segmented ALs., (Copyright © 2024 Published by Wolters Kluwer on behalf of ASCRS and ESCRS.)

Gastrulation is the first major differentiation process in animal embryos. However, the dynamics of human gastrulation remain mostly unknown owing to the ethical limitations. We studied the dynamics of the mesoderm and endoderm cell differentiation from human pluripotent stem cells for insight into the cellular dynamics of human gastrulation. Human pluripotent stem cells have properties similar to those of the epiblast, which gives rise to the three germ layers. The mesoderm and endoderm were induced with more than 75% purity from human induced pluripotent stem cells. Single-cell dynamics of pluripotent stem cell-derived mesoderm and endoderm cells were traced using time-lapse imaging. Both mesoderm and endoderm cells migrate randomly, accompanied by short-term directional persistence. No substantial differences were detected between mesoderm and endoderm migration. Computer simulations created using the measured parameters revealed that random movement and external force, such as the spread out of cells from the primitive streak area, mimicked the homogeneous discoidal germ layer formation. These results were consistent with the development of amniotes, which suggests the effectiveness of human pluripotent stem cells as a good model for studying human embryogenesis., (© 2024. The Society for In Vitro Biology.)

Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation and increased permeability of lung microvascular cells. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein that is expressed in several cell types and mediates multiple pleiotropic effects. We previously reported that DPP4 inhibition by sitagliptin attenuates lipopolysaccharide (LPS)-induced lung injury in mice. The current study characterized the functional role of CD26/DPP4 expression in LPS-induced lung injury in mice, isolated alveolar macrophages, and cultured lung endothelial cells. In LPS-induced lung injury, inflammatory responses [bronchoalveolar lavage fluid (BALF) neutrophil numbers and several proinflammatory cytokine levels] were attenuated in Dpp4 knockout ( Dpp4 KO) mice. However, multiple assays of alveolar capillary permeability were similar between the Dpp4 KO and wild-type mice. TNF-α and IL-6 production was suppressed in alveolar macrophages isolated from Dpp4 KO mice. In contrast, in cultured mouse lung microvascular endothelial cells (MLMVECs), reduction in CD26/DPP4 expression by siRNA resulted in greater ICAM-1 and IL-6 expression after LPS stimulation. Moreover, the LPS-induced vascular monolayer permeability in vitro was higher in MLMVECs treated with Dpp4 siRNA, suggesting that CD26/DPP4 plays a protective role in endothelial barrier function. In summary, this study demonstrated that genetic deficiency of Dpp4 attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential functional roles of CD26/DPP4 expression in resident cellular components of the lung. CD26/DPP4 may be a potential therapeutic target for ARDS and warrants further exploration to precisely identify the multiple functional effects of CD26/DPP4 in ARDS pathophysiology. NEW & NOTEWORTHY We aimed to clarify the functional roles of CD26/DPP4 in ARDS pathophysiology using Dpp4-deficient mice and siRNA reduction techniques in cultured lung cells. Our results suggest that CD26/DPP4 expression plays a proinflammatory role in alveolar macrophages while also playing a protective role in the endothelial barrier. Dpp4 genetic deficiency attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential roles of CD26/DPP4 expression in the resident cellular components of the lung.

The comparative analysis between humans and non-human primates is an instrumental approach for elucidating the evolutional traits and disease propensity of humans. However, in primates, cross-species analyses of their developmental events have encountered constraints because of the ethical and technical limitations in available sample collection, sequential monitoring, and manipulations. In an endeavor to surmount these challenges, in recent years, induced pluripotent stem cells (iPSCs) have garnered escalating interest as an in vitro tool for cross-species analyses between humans and non-human primates. Meanwhile, compared to humans, there is less information on in vitro differentiation of non-human primate iPSCs, and their genetic diversity including subspecies may cause different eligibility to in vitro differentiation methods. Therefore, antecedent to embarking on a comparative analysis to humans, it is a prerequisite to develop the efficacious methodologies for in vitro differentiation regardless of the intraspecies genetic background in non-human primates. In this study, we executed the in vitro differentiation of cardiomyocytes from four chimpanzee iPSC lines with different subspecies and individual backgrounds. To induce cardiomyocytes from chimpanzee iPSCs, we evaluated our methodology for in vitro cardiac differentiation of human iPSCs. Eventually, with minor alterations, our cardiac differentiation method was applicable to all chimpanzee iPSC lines tested as assessed by the expression of cardiac marker genes and the beating ability. Hence, our in vitro differentiation method will advance iPSC-based research of chimpanzee cardiac development and also hold possible utility to cross-species analyses among primate species., (© 2024. The Society for In Vitro Biology.)

Pulmonary hypertension (PH) with interstitial lung diseases (ILDs) often causes intractable conditions. CD26/Dipeptidyl peptidase-4 (DPP4) is expressed in lung constituent cells and may be related to the pathogenesis of various respiratory diseases. We aimed to clarify the functional roles of CD26/DPP4 in PH-ILD, paying particular attention to vascular smooth muscle cells (SMCs). Dpp4 knockout ( Dpp4 KO) and wild type (WT) mice were administered bleomycin (BLM) intraperitoneally to establish a PH-ILD model. The BLM-induced increase in the right ventricular systolic pressure and the right ventricular hypertrophy observed in WT mice were attenuated in Dpp4 KO mice. The BLM-induced vascular muscularization in small pulmonary vessels in Dpp4 KO mice was milder than that in WT mice. The viability of TGFβ-stimulated human pulmonary artery SMCs (hPASMCs) was lowered due to the DPP4 knockdown with small interfering RNA. According to the results of the transcriptome analysis, upregulated genes in hPASMCs with TGFβ treatment were related to pulmonary vascular SMC proliferation via the Notch, PI3K-Akt, and NFκB signaling pathways. Additionally, DPP4 knockdown in hPASMCs inhibited the pathways upregulated by TGFβ treatment. These results suggest that genetic deficiency of Dpp4 protects against BLM-induced PH-ILD by alleviating vascular remodeling, potentially through the exertion of an antiproliferative effect via inhibition of the TGFβ-related pathways in PASMCs.

Single-chain monellin (SCM) is an engineered protein that links the two chains of monellin, a naturally sweet-tasting protein. This protein is an attractive candidate for use as a sugar replacement in food and beverages and has numerous other applications. Therefore, generating SCM mutants with improved stability is an active area of research to broaden the range of its potential applications. In this study, we focused on the Cys41 residue of SCM, which is a single cysteine residue present at a structurally important position. This residue is often substituted with Ser. However, this substitution may destabilize SCM because Cys41 is buried in the hydrophobic core of the protein. Therefore, we designed mutants that substituted Ala, Val, and Leu for this residue, namely C41A, C41V, and C41L. We characterized these three mutants, SCM C41S, and wild type (WT). Differential scanning fluorimetric analysis revealed that substituting Cys41 with Ala or Val increased the thermal stability of SCM, while substitution with Ser or Leu decreased its stability. Determination of the crystal structures of SCM C41A and C41V mutants revealed that the overall structures and main chain structures around the 41st residue of both mutants were almost identical to the WT. On the other hand, the orientations of the amino acid side chains near the 41st residue differed among the SCM variants. Taken together, our results indicate that substituting Cys41 with Ala or Val increases the stability of SCM and provide insight into the structural basis of this improvement., (© 2023. The Author(s).)

Purpose: To evaluate the performance of traditional vergence formulas with segmented axial length (AL) compared to traditional composite AL in extremely long eyes, and to determine whether the segmented AL can be extended to the new-generation formulas, including the Barrett Universal II, Emmetropia Verifying Optical 2.0 (EVO2), Hill-RBF 3.0 (Hill3), Kane, and Ladas Super formula (LSF) formulas in extremely long eyes., Setting: National Hospital. Organization, Tokyo Medical Center, Japan., Design: Retrospective case series., Methods: Consecutive patients who underwent uncomplicated cataract surgery implanted with a three-piece intraocular lens between December 2015 and March 2021 were retrospectively reviewed. The composite AL was measured with a swept-source optical coherence tomography (SS-OCT) biometer using a mean refractive index. The segmented AL was calculated by summing the geometric lengths of the ocular segments (cornea, aqueous, lens, and vitreous) using multiple specific refractive indices based on the data obtained by the SS-OCT-based biometer. When refraction was measured at three months postoperatively, the median absolute errors (MedAEs) were calculated with two ALs for each formula., Results: The study included 31 eyes of 22 patients. The segmented AL (30.45 ± 1.23 mm) was significantly shorter than the composite AL (30.71 ± 1.28 mm, p < 0.001). The MedAEs were significantly reduced when using segmented AL for SRK/T, Haigis, Hill3, and LSF, compared to those obtained using composite AL (0.38 vs. 0.62, 0.48 vs. 0.79, 0.50 vs. 0.90, 0.34 vs. 0.61, p < 0.001 for all formulas, respectively). On the contrary, the MedAE obtained by Kane with segmented AL was significantly worse compared to the one with composite AL (0.35 vs. 0.27, p = 0.03)., Conclusion: In extremely high myopic eyes, the segmented AL improves the performance of SRK/T, Haigis, Hill3, and LSF formulas compared to the composite AL, while the segmented AL worsens the prediction accuracy of the Kane formula.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are a promising cell source for regenerative medicine and drug discovery. However, the use of animal models for studying human cardiomyocytes derived from hiPSCs in vivo is limited and challenging. Given the shared properties between humans and zebrafish, their ethical advantages over mammalian models, and their immature immune system that is rejection-free against xenografted human cells, zebrafish provide a suitable alternative model for xenograft studies. We microinjected fluorescence-labeled cardiac lineage cells derived from hiPSCs, specifically mesoderm or cardiac mesoderm cells, into the yolk and the area proximal to the outflow tract of the linear heart at 24 hours post-fertilization (hpf). The cells injected into the yolk survived and did not migrate to other tissues. In contrast, the cells injected contiguous with the outflow tract of the linear heart migrated into the pericardial cavity and heart. After 1 day post injection (1 dpi, 22-24 hpi), the injected cells migrated into the pericardial cavity and heart. Importantly, we observed heartbeat-like movements of some injected cells in the zebrafish heart after 1 dpi. These results suggested successful xenografting of hiPSC-derived cardiac lineage cells into the zebrafish embryo heart. Thus, we developed a valuable tool using zebrafish embryos as a model organism for investigating the molecular and cellular mechanisms involved in the grafting process. This is essential in developing cell transplantation-based cardiac therapeutics as well as for drug testing, notably contributing to advancements in the field of cardio-medicine., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Kiyoshi Ohnuma reports financial support was provided by Japan Society for the Promotion of Science. Chinmoy Patra reports financial support was provided by Japan Society for the Promotion of Science. Mika Takahi reports financial support was provided by Nagaoka University of Technology Development Education Research Promotion Association., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Embryo contour extraction is the initial step in the quantitative analysis of embryo morphology, and it is essential for understanding the developmental process. Recent developments in light-sheet microscopy have enabled the in toto time-lapse imaging of embryos, including zebrafish. However, embryo contour extraction from images generated via light-sheet microscopy is challenging owing to the large amount of data and the variable sizes, shapes, and textures of objects. In this report, we provide a workflow for extracting the contours of zebrafish blastula and gastrula without contour labeling of an embryo. This workflow is based on the edge detection method using a change point detection approach. We assessed the performance of the edge detection method and compared it with widely used edge detection and segmentation methods. The results showed that the edge detection accuracy of the proposed method was superior to those of the Sobel, Laplacian of Gaussian, adaptive threshold, Multi Otsu, and k-means clustering-based methods, and the noise robustness of the proposed method was superior to those of the Multi Otsu and k-means clustering-based methods. The proposed workflow was shown to be useful for automating small-scale contour extractions of zebrafish embryos that cannot be specifically labeled owing to constraints, such as the availability of microscopic channels. This workflow may offer an option for contour extraction when deep learning-based approaches or existing non-deep learning-based methods cannot be applied., (© 2023 Japanese Society of Developmental Biologists.)

The pathogenesis of pulmonary fibrosis involves complex interplay between cell types and signaling pathways. Recurrent alveolar epithelial injury can occur during pulmonary inflammation, causing dysregulation of epithelial repair. Dysregulated repair interacts with mesenchymal, inflammatory, and endothelial cells to trigger fibroblast-to-myofibroblast activation. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein mediating pleiotropic effect. However, the mechanistic role of CD26/DPP4 in pulmonary fibrosis remains unclear. In this study, we aimed to characterize Dpp4 deficiency in a mouse bleomycin (BLM)-induced pulmonary fibrosis model and in cell culture systems of human lung fibroblasts (HLFs). Dpp4 knockout (Dpp4 KO) mouse lungs exhibited lower Ashcroft scale indices, collagen content, and numbers of fibroblasts and myofibroblasts compared with those in C57BL/6 wild-type (WT) mice. Upregulation of Tgfb1 and Tgfb2 mRNA levels in the lungs after BLM treatment was lower in Dpp4 KO mice compared with those in WT mice. Although TGF-β-driven endothelial-to-mesenchymal transition (EndMT) has been implicated as one of the mechanisms of pulmonary fibrosis, a number of partial EndMT cells in lungs did not differ between Dpp4 KO mice and WT mice. The proliferation capacity and mRNA levels of COL1A1, a collagen deposition-related gene, in cultured HLFs were suppressed in DPP4 small interfering RNA-treated cells. This study indicates that the genetic deficiency of DPP4 has protective effects against BLM-induced pulmonary fibrosis, partly through the reduction in TGF-β expression and inhibition of fibroblast activation in the lung. Our study suggests that CD26/DPP4 inhibition is a potential therapeutic strategy for pulmonary fibrosis., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)