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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.)

Gravity-driven microfluidics, which utilizes gravity force to drive liquid flow, offers portability and multi-condition setting flexibility because they do not require pumps or connection tubes to drive the flow. However, because the flow rate decreases with time in gravity-driven microfluidics, it is not suitable for stem cell experiments, which require long-term (at least a day) stability. In this study, gravity-driven microfluidics and a slow-tilting table were developed to culture cells under constant unidirectional perfusion. The microfluidic device was placed on a slow-tilting table, which tilts unidirectionally at a rate of approximately 7° per day to compensate for the reduction in the flow rate. Computational simulations showed that the pulsation of the flow arising from the stepwise movement of the table was less than 0.2%, and the flow was laminar. Hydrophilization of the tanks increased the flow rate, which is consistent with the theoretical values. We showed that vitronectin is better than laminin 511 fragments as a coating material for adhering human induced pluripotent stem cells on a microchamber made of polydimethylsiloxane, and succeeded in culturing the cells for 3 days. It is believed that the system offers easy-to-use cell culture tools, such as conventional multiwell culture vessels, and enables the control of the cell microenvironment., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

IL-26 is a Th17 cytokine, with its gene being absent in rodents. To characterize the in vivo immunological effects of IL-26 in chronic systemic inflammation, we used human IL26 transgenic (hIL-26Tg) mice and human umbilical cord blood mononuclear cells (hCBMC) in mouse allogeneic-graft-versus-host disease (GVHD) and chronic xenogeneic-GVHD model, respectively. Transfer of bone marrow and spleen T cells from hIL-26Tg mice into B10.BR mice resulted in GVHD progression, with clinical signs of tissue damage in multiple organs. IL-26 markedly increased neutrophil levels both in the GVHD-target tissues and peripheral blood. Expression levels of Th17 cytokines in hIL-26Tg mice-derived donor CD4 T cells were significantly increased, whereas IL-26 did not affect cytotoxic function of donor CD8 T cells. In addition, granulocyte-colony stimulating factor, IL-1β, and IL-6 levels were particularly enhanced in hIL-26Tg mice. We also developed a humanized neutralizing anti-IL-26 monoclonal antibody (mAb) for therapeutic use, and its administration after onset of chronic xenogeneic-GVHD mitigated weight loss and prolonged survival, with preservation of graft-versus-leukemia effect. Taken together, our data elucidate the in vivo immunological effects of IL-26 in chronic GVHD models and suggest that a humanized anti-IL-26 mAb may be a potential therapeutic agent for the treatment of chronic GVHD., (© 2022 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Purpose: To evaluate the performance of a new formula of intraocular lens (IOL) power calculation (the O formula) based on ray tracing without commonly used parameters, including ultrasound-compatible axial length, keratometry readings, and A-constant., Setting: Tokyo Medical Center, Tokyo, Japan., Design: Retrospective consecutive case series., Methods: 423 eyes (423 patients) implanted with a single-piece, L-loop, acrylic IOL were enrolled. All biometric data for the O formula were obtained by anterior segment swept-source optical coherence tomography (SS-OCT) and SS-OCT-based biometer. The performance of the O formula was compared with those of the Barrett Universal II (BUII) and Kane formulas at 1 month postoperatively. Statistical analysis was applied according to a heteroscedastic test with SD of prediction errors as the main parameter for formula performance., Results: The SD of the O formula (0.426) was statistically significantly lower than that of the BUII formula (0.464, P = .034) but not statistically significantly different from that of the Kane formula (0.433, P = .601). The percentages of patients with refractive prediction errors within ±0.50 diopter (D) and ±1.00 D of the O, BUII, and Kane formulas were 75.4% and 98.6%, 77.1% and 97.9%, and 76.6% and 98.1%, respectively., Conclusions: The O formula, based on ray tracing using SS-OCT-based devices, is one of the promising approaches for IOL power calculation, although additional larger scale studies are needed. It may be used as an alternative in IOL power calculation because of its independence from commonly used parameters., Competing Interests: Disclosures: N. Maeda has received lecture fees from Tomey Corp. for sponsored seminars unrelated to this article. The coauthors of this article report no additional potential conflicts of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of ASCRS and ESCRS.)

Purpose: To compare the change in intraocular lens (IOL) axial movement, corneal power, and postoperative refraction of eyes implanted with two different single-piece, open loop, acrylic foldable IOLs with planar-haptic design: one IOL with hinges vs. one IOL without hinges. The role of IOL axial movement on short-term refractive shift after cataract surgery was also evaluated., Methods: This retrospective comparative study enrolled consecutive patients who had phacoemulsification with aspheric IOL implantation. The IOL depth (the distance from corneal endothelium to IOL surface) and corneal power were measured via anterior-segment optical coherence tomography at 4 days and 1 month postoperatively. The changes in axial movement of the IOL, corneal power, and manifest refractive spherical equivalent (MRSE) were compared among groups, and the correlations between each lens were evaluated., Results: IOL with hinges was implanted in 42 eyes of 42 patients and IOL without hinges was implanted in 42 eyes of 42 patients. The change in axial movement between 4 days and 1 month was significantly smaller in the IOL with hinges group than in the IOL without hinges group (p < 0.001). The axial movement of IOL with hinges did not correlate with the MRSE change; however, the forward shift of IOL without hinges correlated with the myopic refractive change (Pearson r = 0.62, p < 0.001)., Conclusion: The postoperative axial movement of IOL was more stable in the IOL with hinges group than the IOL without hinges group between 4 days and 1 month after cataract surgery. Even though the two study IOLs with planar-haptic design are made of similar acrylic materials, other characteristics such as hinge structure may affect IOL stability in the bag., Competing Interests: SG is supported by Grants Manpei Suzuki Diabetic Foundation and Japan Society for the Promotion of Science Overseas Research Fellowships that are not related to the submitted work. SG, NM, and TN received honoraria for speaking outside the submitted work from Alcon Japan Ltd. These do not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.

The general anticoagulant evaluation requires high expense equipment, reagents, and space. Therefore, not all laboratories can perform research related to anticoagulant. In this study, we propose a novel simple method "in vitro thrombus-growth model" that can evaluate anticoagulant ability by measuring weight. The in vitro thrombus-growth model is prepared by creating a "growth-clot" with citrate plasma, calcium chloride, and thrombin, and then pouring new citrate plasma onto it. The prepared growth-clots were increased in volume in citrated human plasma, including surpluses calcium chloride, which was released slowly, leading to clot coagulation around the plasma. As a result of evaluating the anticoagulant ability of direct thrombin inhibitor using this in vitro thrombus-growth model, it was confirmed that clot growth was suppressed in a concentration-dependent manner. Therefore, this thrombus-growth model is useful as a primary anticoagulant test that can to discover compounds with anticoagulant activity perform in any laboratory.

In the context of doping control, conventional direct chemical testing detects only a limited scope of target substances in equine biological samples. To expand the ability to detect doping agents and their detection windows, metabolomics has recently become a common approach for monitoring alteration of biomarkers caused by doping agents in relevant metabolic pathways. In horse racing, remarkable changes in metabolic profiles between the rest state and racing are likely to affect the identification of doping biomarkers. Previously, we reported a limited number of significantly upregulated metabolites after racing, based on a non-targeted metabolomics approach using out-of-competition and post-race equine plasma samples. In this study, we performed a more thorough analysis of the data set, using pathway analysis to establish a post-race biomarkers database (PBD) that includes upregulated and downregulated metabolites, their fold changes, and relevant pathways, with the main objective of improving our understanding of changes in physiological status related to horse racing. Statistical analysis of the PBD revealed that two peak combinations of pentadecanoyl carnitine/galactosylglycerol (P/G) and heptadecanoyl carnitine/galactosylglycerol (H/G) could be used as potential biomarkers for the discrimination of the rest and post-race groups. To demonstrate the applicability of the PBD, we validated the post-race biomarkers P/G and H/G (highly involved in lipid metabolism) by a single-blind test. This strategy, which combines establishment of a biomarker database with pathway analysis, represents a powerful tool for discovering potential doping biomarkers in the future., (© 2021 John Wiley & Sons, Ltd.)

Background: Cutibacterium modestum was named in 2020. C. modestum was previously called Propionibacterium humerusii. Several implant-associated infections caused by Cutibacterium species have been previously reported, but native vertebral osteomyelitis due to these bacteria has rarely been reported., Case Presentation: A 72-year-old man, who had previously received several nerve block injections for low back pain, was referred to our hospital for deterioration in back pain in the last 1 month. MRI findings were suggestive of L5-S1 vertebral osteomyelitis. Blood cultures and bone biopsy culture revealed the presence of Gram-positive bacilli. The isolate was identified as C. modestum by 16SrRNA gene sequencing. A diagnosis of vertebral osteomyelitis caused by C. modestum was made. Minocycline followed by oral amoxicillin was administered for 3 months. His symptom improved and did not recur after treatment completion., Conclusion: A case of vertebral osteomyelitis caused by C. modestum was encountered. Although C. modestum is very similar to C. acnes, it could be accurately identified by 16SrRNA gene sequencing. This case represents the first documented C. modestum infection in humans., (© 2022. The Author(s).)

Visualization of the iridocorneal angle, which contains the aqueous humor circulatory system and controls intraocular pressure, is important for diagnosing and managing glaucoma; however, the presence of keratoconus, keratoglobus, or severe myopia may enable direct angle visualization without gonioscopy contact lenses or applying a coupling gel. We present the first report of a case in which the iridocorneal angle was viewed directly in an eye with keratoconus using the RetCam without applying gel to the cornea. This method overcame the inability to view the angle directly in a normal eye because of the total internal reflection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kawashima, Matsushita, Ohnuma, Maeda, Koh and Nishida.)

Background: Mechanical alloknesis (or innocuous mechanical stimuli-evoked itch) often occurs in dry skin-based disorders such as atopic dermatitis and psoriasis. However, the molecular and cellular mechanisms underlying mechanical alloknesis remain unclear. We recently reported the involvement of CD26 in the regulation of psoriatic itch. This molecule exhibits dipeptidyl peptidase IV (DPPIV) enzyme activity and exerts its biologic effects by processing various substances, including neuropeptides., Objective: The aim of the present study was to investigate the peripheral mechanisms of mechanical alloknesis by using CD26/DPPIV knockout (CD26KO) mice., Methods: We applied innocuous mechanical stimuli to CD26KO or wild-type mice. The total number of scratching responses was counted as the alloknesis score. Immunohistochemical and behavioral pharmacologic analyses were then performed to examine the physiologic activities of CD26/DPPIV or endomorphins (EMs), endogenous agonists of μ-opioid receptors., Results: Mechanical alloknesis was more frequent in CD26KO mice than in wild-type mice. The alloknesis score in CD26KO mice was significantly reduced by the intradermal administration of recombinant DPPIV or naloxone methiodide, a peripheral μ-opioid receptor antagonist, but not by that of mutant DPPIV without enzyme activity. EMs (EM-1 and EM-2), selective ligands for μ-opioid receptors, are substrates for DPPIV. Immunohistochemically, EMs were located in keratinocytes, fibroblasts, and peripheral sensory nerves. Behavioral analyses revealed that EMs preferentially provoked mechanical alloknesis over chemical itch. DPPIV-digested forms of EMs did not induce mechanical alloknesis., Conclusion: The present results suggest that EMs induce mechanical alloknesis at the periphery under the enzymatic control of CD26/DPPIV., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

While several metallic implants with bioactive coatings have been developed thus far for treating bone deformations or deterioration, a multifunctional coating with the desired mechanical and antibacterial properties has not been demonstrated. This study aimed to reveal the effect of the composition of hydroxyapatite (HAp)/gray titania coatings on the mechanical and antibacterial properties for biomedical applications. Suspension plasma spray (SPS) aided successful deposition of HAp/gray titania coatings on the surface of titanium substrates. The microstructure of coatings with different compositions was then characterized using scanning electron microscopy, X-ray diffraction, and Raman spectroscopy to identify the crystal structure. All results consistently demonstrated that SPS could transform Ti 2 O 3 into TiO 2 with mixed Magneli phases, such as Ti 4 O 7 and Ti 3 O 5 , which could typically demonstrate photocatalytic activity. Hardness, Young's modulus, and interfacial strength of composite coatings commonly increased with an increase in the weight percentage of TiO 2 . A multi-modal damage assessment combining acoustic emission (AE), infrared ray camera (IR), and digital-image-correlation (DIC) was performed to monitor the damage process of HAp composite coating, which successfully revealed initiations of microcracks and nonlinear deformation at interface until fracture. Antibacterial test performed for examining the cytotoxic effects against E. coli under LED light irradiation conditions revealed that SPS HAp/gray titania coating could significantly enhance the antibacterial properties. Enhanced antibacterial properties can be attributed to an increase in the number of Magneli phases and better bacterial adhesion was attributed to hydrophilic properties conferred by submicron-sized particles. Hence, SPS can help fabricate visible light-responsive antibacterial coating, which can be used for medical devices., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Auto/paracrine factors secreted from cells affect differentiation of human pluripotent stem cells (hPSCs). However, the molecular mechanisms underlying the role of secreted factors are not well known. We previously showed that pattern formation in hPSCs induced by BMP4 could be reproduced by a simple reaction-diffusion of BMP and Noggin, a cell-secreted BMP4 inhibitor. However, the amount of Noggin secreted is unknown. In this study, we measured the concentration of Noggin secreted during the differentiation of hPSCs induced by BMP4. The Noggin concentration in the supernatant before and after differentiation was constant at approximately 0.69 ng/mL, which is approximately 50-200 times less than expected in the model. To explain the difference between the experiment and model, we assumed that macromolecules such as heparan sulfate proteoglycan on the cell surface act as a diffusion barrier structure, where the diffusion slows down to 1/400. The model with the diffusion barrier structure reduced the Noggin concentration required to suppress differentiation in the static culture model. The model also qualitatively reproduced the pattern formation, in which only the upstream but not the downstream hPSCs were differentiated in a one-directional perfusion culture chamber, with a small change in the amount of secreted Noggin resulting in a large change in the differentiation position. These results suggest that the diffusion barrier on the cell surface might enhance the auto/paracrine effects on monolayer hPSC culture., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Cutibacterium modestum is a new species coined in 2020 as the fifth species of genus Cutibacterium , which includes Cutibacterium acnes . The species is predicted as a minor but common member of skin microbiome and includes a group tentatively named as " Propionibacterium humerusii ". The description of the species has been provided only with a single strain. To establish the characteristics of C. modestum and search for possible disease-related subtypes, we investigated the biochemical characteristics of eight live strains and performed in silico comparison of nine genomes. The common features, which included the morphology of Gram-stain positive short rods, the negativity of phenylalanine arylamidase, and several unique MALDI-TOF MS spectral peaks, were considered useful in laboratory identification. Pairwise comparisons of the genomes by in silico DNA-DNA hybridization showed similarity values of 98.1% or larger, which were far higher than the subspecies cutoff of 79-80%. The 16S rRNA gene sequences of thirteen isolates and genomes were identical. Their recA gene sequences were identical except for two strains, HM-510 (HL037PA2) and Marseille-P5998, which showed unique one-nucleotide polymorphisms. The biochemical features using API kits were slightly different among the isolates but far closer than those of the nearest other species, C. acnes and Cutibacterium namnetense . Spectra of MALDI-TOF mass spectrometry showed slight differences in the presence of m / z 10,512 (10 kD chaperonin GroS) and three other peaks, further clustering the eight isolates into three subtypes. These results indicated that these isolates did not separate to form subspecies-level clusters, but subtyping is possible by using recA gene sequences or MALDI-TOF mass spectrometry spectra. Moreover, this work has confirmed that a group " P. humerusii " is included in C. modestum .

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) have received increasing attention for their clinical use. Many protocols induce cardiomyocytes at an initial high cell density (confluence) to utilize cell density effects as hidden factors for cardiomyocyte differentiation. Previously, we established a protocol to induce hiPSC differentiation into cardiomyocytes using a defined culture medium and an initial low cell density (1% confluence) to minimize the hidden factors. Here, we investigated the key factors promoting cardiomyocyte differentiation at an initial low cell density to clarify the effects of cell density. Co-culture of hiPSCs at an initial low cell density with those at an initial high cell density showed that signals secreted from cells (auto/paracrine factors) and not cell-cell contact signals, played an important role in cardiomyocyte differentiation. Moreover, although cultures with initial low cell density showed higher expression of anti-cardiac mesoderm genes, earlier treatment with a Wnt production inhibitor efficiently suppressed the anti-cardiac mesoderm gene expression and promoted cardiomyocyte differentiation by up to 80% at an initial low cell density. These results suggest that the main effect of cell density on cardiomyocyte differentiation is inhibition of Wnt signaling at the early stage of induction, through auto/paracrine factors., (© 2021. The Author(s).)

Triple-negative breast cancer (TNBC) has a poor prognosis compared to other breast cancer subtypes. Although epidermal growth factor receptor (EGFR) is overexpressed in TNBC, clinical trials with EGFR inhibitors including tyrosine kinase inhibitors (EGFR-TKI) in TNBC have heretofore been unsuccessful. To develop effective EGFR-targeted therapy for TNBC, the precise mechanisms of EGFR-TKI resistance in TNBC need to be elucidated. In this study, to understand the molecular mechanisms involved in the differences in EGFR-TKI efficacy on TNBC between human and mouse, we focused on the effect of IL-26, which is absent in mice. In vitro analysis showed that IL-26 activated AKT and JNK signaling of bypass pathway of EGFR-TKI in both murine and human TNBC cells. We next investigated the mechanisms involved in IL-26-mediated EGFR-TKI resistance in TNBC. We identified EphA3 as a novel functional receptor for IL-26 in TNBC. IL-26 induced dephosphorylation and downmodulation of EphA3 in TNBC, which resulted in increased phosphorylation of AKT and JNK against EGFR-TKI-induced endoplasmic reticulum (ER) stress, leading to tumor growth. Meanwhile, the blockade of IL-26 overcame EGFR-TKI resistance in TNBC. Since the gene encoding IL-26 is absent in mice, we utilized human IL-26 transgenic (hIL-26Tg) mice as a tumor-bearing murine model to characterize the role of IL-26 in the differential effect of EGFR-TKI in human and mice and to confirm our in vitro findings. Our findings indicate that IL-26 activates the bypass pathway of EGFR-TKI, while blockade of IL-26 overcomes EGFR-TKI resistance in TNBC via enhancement of ER stress signaling. Our work provides novel insights into the mechanisms of EGFR-TKI resistance in TNBC via interaction of IL-26 with its newly identified receptor EphA3, while also suggesting IL-26 as a possible therapeutic target in TNBC.

In recent years, with the development of precise lathe-cutting equipment, special shaped contact lenses (CL) have been crafted. However, while it is possible to manufacture such a lens, its shape evaluation has not been well-established. We conducted a basic optical experiment using special lenses to measure a spherical lens and nonspherical mold. As the measurement sample, a metal ball, special CL, and a toric-shaped mold were adopted. In order to accurately measure those real shapes, we proposed an algorithm in which the probe light is vertically incident to the sample surface within a numerical aperture of the optical probe. For this algorithm, we developed the specialized time-domain optical coherence tomography (TD-OCT), which was designed to conduct circular scanning while maintaining vertical incidence by driving a two-axis (vertical and horizontal) micro-electromechanical system mirror with a phase difference of 90°. The shape, thickness distribution, and curvature radii of both front and back surfaces of a CL were estimated with this OCT signal analysis and sphere fitting. The shape and curvature radius were evaluated by using the simulated data under the same experimental conditions. They were sufficiently accurate based on the resolution of this OCT. Also, a toric-shaped mold was evaluated by comparing the relationship between each coordinate and intensity of the interference signal. As a result, it is confirmed that the experimental result and the simulated matched well.

Although pure Ti is nontoxic, alloying elements may be released into the surrounding tissue when Ti alloys are used, and this can cause cytotoxicity. Therefore, this study performed the damage evaluation of hydroxyapatite (HAp)-coated porous Ti components subjected to cyclic compression in a simulated body fluid (SBF). The HAp coating layer was deposited on the surface of porous Ti by electrophoresis, and a dense and homogeneous coating morphology was observed on the surface of the porous Ti. To specify damage types of HAp coating in situ, acoustic emission (AE) measurements and microscopic observations were simultaneously conducted during compressive fatigue loading tests to detect the specific failure mode. Compression tests revealed that the interfacial strength between the HAp coating and porous Ti was higher than the yield strength of the porous body (7-9 MPa). The AE signals were detected only in the plastic deformation stage of porous Ti, which indicated that they were generated because of plastic deformation/fractures in the porous body. Compressive fatigue tests revealed that no significant HAp coating damage occurred when the applied maximum stress was within the elastic limit of porous Ti in air. In contrast, the HAp coating exhibited delamination at the initial stage of cyclic loading at all stress levels in SBF, while the fatigue limit of the coated porous substrate, 2 MPa, was not affected by the SBF medium. Though the delamination of the HAp coating in SBF occurred during the early stages of fatigue loading, the amorphous calcium phosphate layer was recovered partly through re-precipitation from SBF. The AE signals from the delamination of the HAp coating or fracture in porous Ti could be identified using the peak voltage and frequencies. As microscopic observations were limited to certain parts of the porous body, AE signals were clustered according to the types of failure. The clustered AE signals were successfully correlated with the fatigue behavior of porous Ti. Corrosion fatigue was determined to be the primary mechanism for the delamination of the HAp coating on porous Ti in SBF., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The phase I trial of the humanized anti-CD26 monoclonal antibody YS110 for CD26-expressing tumors was conducted recently. The present study identifies a potential prognostic biomarker for CD26-targeted therapy based on the phase I data., Methods: Box and Whisker plot analysis, Scatter plot analysis, Peason product moment correlation/Spearman's rank-difference correlation, Bar graph analysis, and Receiver Operating Characteristics (ROC) were used to examine the correlation between sCD26 titer variation with YS110 administration and tumor volume change, RECIST criteria evaluation and progression free survival (PFS). Mechanism for serum sCD26 titer variation was confirmed by in vitro experimentation., Results: Serum sCD26/DPP4 titer was reduced following YS110 administration and gradually recovered until the next infusion. Serum sCD26/DPP4 titer before the next infusion was sustained at lower levels in Stable Disease (SD) cases compared to Progressive Disease cases. ROC analysis defined the cut-off level of serum sCD26/DPP4 titer variation at day 29 pre/post for the clinical outcome of SD as tumor response or PFS. In vitro experimentation confirmed that YS110 addition reduced sCD26 production from CD26-expressing tumor and non-tumor cells., Conclusions: Our study indicates that serum sCD26/DPP4 titer variation in the early phase of YS110 treatment is a predictive biomarker for evaluating therapeutic efficacy.

Although thalidomide is highly teratogenic, it has been prescribed for treating multiple myeloma and Hansen's disease. However, its mechanism of action is not fully understood. Here, we employed a reverse transcription quantitative PCR array to measure the expression of 84 genes in human induced pluripotent stem cells (hiPSCs) and their mesodermal differentiation. Thalidomide altered the expression of undifferentiated marker genes in both cell types. Thalidomide affected more genes in the mesoderm than in the hiPSCs. Ectoderm genes were upregulated but mesendoderm genes were downregulated by thalidomide during mesoderm induction, suggesting that thalidomide altered mesoderm differentiation. We found that FABP7 (fatty acid binding protein 7) was dramatically downregulated in the hiPSCs. FABP is related to retinoic acid, which is important signaling for limb formation. Moreover, thalidomide altered the expression of the genes involved in TGF-β signaling, limb formation, and multiple myeloma, which are related to thalidomide-induced malformations and medication. In summary, iPSCs can serve as useful tools to elucidate the mechanisms underlying thalidomide malformations in vitro ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

CD26/Dipeptidyl peptidase IV (DPPIV) is a cell surface glycoprotein with numerous roles including glucose metabolism, immunomodulation, and tumorigenesis. CD26/DPPIV is well recognized in diabetes, with DPPIV inhibitors being a class of oral hypoglycemic drugs called gliptins that are commonly used to treat type two diabetes mellitus. Recent work also indicated a potential role for CD26 in infectious diseases, including COVID-19, and immune-mediated disorders such as rheumatoid arthritis, inflammatory bowel disease, and graft-versus-host disease. In cancer, CD26/DPPIV expression has been characterized in numerous tumors such as hematologic malignancies, malignant pleural mesothelioma (MPM), renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), gastrointestinal stromal tumor (GIST), and prostate, lung, colorectal, and ovarian (PLCO) cancer. Hence, CD26 has been frequently studied as a tumor biomarker and therapeutic target. CD26/DPPIV-targeted therapies have been evaluated in various cancers, including the use of anti-CD26 monoclonal antibodies as anticancer treatment in selected neoplasms. This review highlights our current understanding of the role of CD26 in cancer, diabetes, immune-mediated diseases, and infectious diseases. Enhanced understanding of CD26 biology and function may lead to novel therapeutic approaches in multiple human diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2021, Pan et al.)

The optical measurement algorithm for the real front and back surfaces of contact lenses from their center to periphery accurately and simultaneously is proposed. It is an algorithm that makes light incident vertically along the curved surfaces of contact lenses under the condition that the difference of curvature radii between the front and back surfaces is small enough within the NA of the optical probe. For this purpose, we adopted time-domain optical coherence tomography (OCT) with translation and rotation mechanisms. The shape, thickness distribution, and curvature radii of both surfaces were estimated with OCT signal analysis and circular approximation. The measured results were compared with the designed values and the measured data from a conventional shape measurement device. The curved shape of both surfaces and thickness were well matched with the designed values from lens center to periphery. In a curvature radius of the front surface, there was a proportional bias with a limit of agreement of -0.77 % to -2.09 % , and the correlation coefficient was 0.57. On the back surface, there was no systematic bias, and minimal detectable change was 0.178 mm, in a range of 95% confidential interval. The proposed algorithm well visualized the real shape and optical characteristics of the contact lens with enough accuracy to the design.

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos., (© 2020 Japanese Society of Developmental Biologists.)

Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

Purpose: To compare the changes in axial length (AL) obtained after cataract surgery by partial coherence interferometry (PCI), swept-source optical coherence tomography with the composite method (SS-OCT/CM), and SS-OCT with the segmental method (SS-OCT/SM)., Setting: National Hospital Organization, Tokyo Medical Center, Japan., Design: Retrospective case series., Methods: AL was measured preoperatively and 1 month postoperatively by PCI, SS-OCT/CM, and SS-OCT/SM., Results: Two hundred sixty-eight eyes in 139 patients who underwent cataract surgery were included. The mean ALs measured by PCI and SS-OCT/CM decreased significantly from 24.14 ± 1.58 to 24.05 ± 1.58 mm (P < .0001) and from 24.15 ± 1.58 to 24.05 ± 1.59 mm (P < .0001), respectively. The mean differences between the preoperative and postoperative AL were 0.083 ± 0.044 mm (PCI) and 0.096 ± 0.045 mm (SS-OCT/CM), respectively; Bland-Altman analysis revealed fixed bias between the preoperative and postoperative values. The mean preoperative and postoperative ALs measured by SS-OCT/SM were 24.12 ± 1.54 and 24.12 ± 1.54 mm, respectively (P = 0.97). The mean difference between the preoperative and postoperative ALs was 0.00 ± 0.03 mm on SS-OCT/SM; Bland-Altman analysis indicated good agreement between these values., Conclusions: The difference between preoperative and postoperative ALs is less with SS-OCT/SM than with PCI or SS-OCT/CM. Assuming that AL is not altered by cataract surgery, AL measurement is more accurate by SS-OCT/SM than by PCI or SS-OCT/CM.

Tetratricopeptide repeat protein 37 (TTC37) is a causative gene of trichohepatoenteric syndrome (THES). However, little is known about the pathogenesis of this disease. Here, we characterize the phenotype of a Drosophila model in which ski3, a homolog of TTC37, is disrupted. The mutant flies are pupal lethal, and the pupal lethality is partially rescued by transgenic expression of wild-type ski3 or human TTC37. The mutant larvae show growth retardation, heart arrhythmia, triacylglycerol accumulation, and aberrant metabolism of glycolysis and the TCA cycle. Moreover, mitochondrial membrane potential and respiratory chain complex activities are significantly reduced in the mutants. Our results demonstrate that ski3 deficiency causes mitochondrial dysfunction, which may underlie the pathogenesis of THES., (© 2020 Federation of European Biochemical Societies.)

This study compared the optical axial length (AL) obtained by composite and segmental methods using swept-source optical coherence tomography (SS-OCT) devices, and demonstrated its effects on the post-operative refractive errors (RE) one month after cataract surgery. Conventional AL measured with the composite method used the mean refractive index. The segmented-AL method used individual refractive indices for each ocular medium. The composite AL (24.52 ± 2.03 mm) was significantly longer (P < 0.001) than the segmented AL (24.49 ± 1.97 mm) among a total of 374 eyes of 374 patients. Bland-Altman analysis revealed a negative proportional bias for the differences between composite and segmented ALs. Although there was no significant difference in the RE obtained by the composite and segmental methods (0.42 ± 0.38 D vs 0.41 ± 0.36 D, respectively, P = 0.35), subgroup analysis of extremely long eyes implanted with a low power intraocular lens indicated that predicted RE was significantly smaller with the segmental method (0.45 ± 0.86 D) than that with the composite method (0.80 ± 0.86 D, P < 0.001). Segmented AL with SS-OCT is more accurate than composite AL in eyes with extremely long AL and can improve post-operative hyperopic shifts in such eyes.

Background: Considering the physiological changes in serum procalcitonin (PCT) levels in newborns due to age, we recently established an age-specific percentile-based reference curve for serum PCT level. The present study aimed to determine the best cutoff percentile line using this reference curve for the differentiation between infected and colonized preterm infants., Methods: A total of 52 preterm infants with positive bacterial culture (9 with bacterial infection, 43 with colonization) were enrolled within the study period. The 97.5th, 95.0th, 92.5th, 90.0th, 80.0th, 70.0th, 60.0th, and 50.0th percentile lines were drawn in the reference curve. PCT levels in infected or colonized infants were used, and sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The best cutoff percentile line was determined in the receiver operating characteristic curve analysis., Results: Of the 52 preterm infants, 9 were infected (5 and 4 infants with an onset of < 7 days and ≥ 7 days after birth, respectively), whereas 43 were colonized (6 and 37 infants with an onset of < 7 days and ≥ 7 days after birth, respectively). The best cutoff percentile lines were the 90.0th percentile (sensitivity, 0.800; specificity, 0.833; PPV, 0.800; NPV, 0.833) and 97.5th percentile (sensitivity, 1.00; specificity, 0.973; PPV, 0.800; NPV, 1.00) in infants with an onset of < 7 days and ≥ 7 days after birth, respectively., Conclusions: The age-specific percentile-based reference curve for serum PCT level is clinically applicable as a new tool for diagnosing infections in preterm infants with positive culture results, particularly at ≥ 7 days after birth.

Dipeptidyl peptidases (DPPs) are proteolytic enzymes that are ideal therapeutic targets in human diseases. Indeed, DPP4 inhibitors are widely used in clinical practice as anti-diabetic agents. In this paper, we show that DPP4 inhibitors also induced cell death in multiple human myeloma cells. Among five DPP4 inhibitors, only two of them, vildagliptin and saxagliptin, exhibited apparent cytotoxic effects on myeloma cell lines, without any difference in suppression of DPP4 activity. As these two DPP4 inhibitors are known to have off-target effects against DPP8/9, we employed the specific DPP8/9 inhibitor 1G244. 1G244 demonstrated anti-myeloma effects on several cell lines and CD138+ cells from patients as well as in murine xenograft model. Through siRNA silencing approach, we further confirmed that DPP8 but not DPP9 is a key molecule in inducing cell death induced by DPP8/9 inhibition. In fact, the expression of DPP8 in CD38+ cells from myeloma patients was higher than that of healthy volunteers. DPP8/9 inhibition induced apoptosis, as evidenced by activated form of PARP, caspases-3 and was suppressed by the pan-caspase inhibitor Z-VAD-FMK. Taken together, these results indicate that DPP8 is a novel therapeutic target for myeloma treatment.

Interleukin (IL)-26, known as a Th17 cytokine, acts on various cell types and has multiple biological functions. Although its precise role still remains to be elucidated, IL-26 is suggested to be associated with the pathology of diverse chronic inflammatory diseases such as psoriasis, inflammatory bowel diseases and rheumatoid arthritis. To develop novel neutralizing anti-human IL-26 monoclonal antibodies (mAbs) for therapeutic use in the clinical setting, we immunized mice with human IL-26 protein. Hybridomas producing anti-IL-26 mAbs were screened for various in vitro functional assays, STAT3 phosphorylation and antibiotic assays. Although the IL-20RA/IL-10RB heterodimer is generally believed to be the IL-26 receptor, our data strongly suggest that both IL-20RA-dependent and -independent pathways are involved in IL-26-mediated stimulation. We also investigated the potential therapeutic effect of anti-IL-26 mAbs in the imiquimod-induced psoriasis-like murine model using human IL-26 transgenic mice. These screening methods enabled us to develop novel neutralizing anti-human IL-26 mAbs. Importantly, administration of IL-26-neutralizing mAb did not have an effect on the antimicrobial activity of IL-26. Taken together, our data strongly suggest that our newly developed anti-human IL-26 mAb is a potential therapeutic agent for the treatment of diverse chronic inflammatory diseases including psoriasis.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)