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Abstract We report on the feasibility of Fourier transform holography in the hard X-ray regime using a Free Electron Laser source. Our study shows successful single and multi-pulse holographic reconstructions of the nanostructures. We observe beam-induced heating of the sample exposed to the intense X-ray pulses leading to reduced visibility of the holographic reconstructions. Furthermore, we extended our study exploring the feasibility of recording holographic reconstructions with hard X-ray split-and-delay optics. Our study paves the way towards studying dynamics at sub-nanosecond timescales and atomic lengthscales.

Abstract A intermediate multidomain state and large crystallographic tilting of 1.78° for the (hh0)pc planes of a (001)pc-oriented single-domain Mn-doped BiFeO3 (BFMO) thin film were found when an electric field was applied along the [110]pc direction. The anomalous crystallographic tilting was caused by ferroelastic domain switching of the 109° domain switching. In addition, ferroelastic domain switching occurred via an intermediate multidomain state. To investigate these switching dynamics under an electric field, we used in situ fluorescent X-ray induced Kossel line pattern measurements with synchrotron radiation. In addition, in situ inverse X-ray fluorescence holography (XFH) experiments revealed that atomic displacement occurred under an applied electric field. We attributed the atomic displacement to crystallographic tilting induced by a converse piezoelectric effect. Our findings provide important insights for the design of piezoelectric and ferroelectric materials and devices.

Abstract Mitochondrial diseases are mainly caused by dysfunction of mitochondrial respiratory chain complexes and have a variety of genetic variants or phenotypes. There are only a few approved treatments, and fundamental therapies are yet to be developed. Leigh syndrome (LS) is the most severe type of progressive encephalopathy. We previously reported that apomorphine, an anti- “off” agent for Parkinson’s disease, has cell-protective activity in patient-derived skin fibroblasts in addition to strong dopamine agonist effect. We obtained 26 apomorphine analogs, synthesized 20 apomorphine derivatives, and determined their anti-cell death effect, dopamine agonist activity, and effects on the mitochondrial function. We found three novel apomorphine derivatives with an active hydroxy group at position 11 of the aporphine framework, with a high anti-cell death effect without emetic dopamine agonist activity. These synthetic aporphine alkaloids are potent therapeutics for mitochondrial diseases without emetic side effects and have the potential to overcome the low bioavailability of apomorphine. Moreover, they have high anti-ferroptotic activity and therefore have potential as a therapeutic agent for diseases related to ferroptosis.

Abstract In this study, we investigated the composition of free amino acids and lactate (Lac) in polychaetes in river estuaries and inner bays using chromatographic techniques. Both l-amino acids and d-amino acids (d-asparagine, d-alanine (d-Ala), d-serine, d-aspartic acid, and d-proline (d-Pro)) were detected, indicating that polychaetes contain some d-amino acids. Some polychaete species exhibited notable amino acid levels, such as glycine in Capitellidae sp. and Thelepus sp., d-Pro in Glycera sp., and β-Ala in Scoletoma nipponica and Scoletoma sp.. High d-Lac levels were detected in Tylorrhynchus osawai and Hediste diadroma, (691 and 797 μmol/100 g-wet, respectively), with the d-form exceeding 98%. T. osawai was dominant in the upper tidal-sensitive zone, wherein other organisms were less abundant because of low salinity (3–8 PSU). Seasonal differences in the concentrations of components in T. osawai were observed, particularly a significant increase in d-Lac in the reproductive period. Notably, the d-Lac concentrations of T. osawai were higher upstream than downstream. Thus, d-Lac might be involved in strategies underlying adaptations to low salinity and reproductive activity. These results suggest that both the d-form of Lac and amino acids may play certain physiological roles in the life of polychaetes.

Abstract Originally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of PTSG2 and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors.

Abstract Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene, and female carriers are usually asymptomatic. We describe a 7-month-old female patient with severe intellectual disability, epilepsy, and low levels of serum copper and ceruloplasmin. While heterozygous deletion of exons 16 and 17 of the ATP7A gene was detected in the proband, her mother, and her grandmother, only the proband suffered from Menkes disease clinically. Intriguingly, X chromosome inactivation (XCI) analysis demonstrated that the grandmother and the mother showed skewing of XCI toward the allele with the ATP7A deletion and that the proband had extremely skewed XCI toward the normal allele, resulting in exclusive expression of the pathogenic ATP7A mRNA transcripts. Expression bias analysis and recombination mapping of the X chromosome by the combination of whole genome and RNA sequencing demonstrated that meiotic recombination occurred at Xp21-p22 and Xq26-q28. Assuming that a genetic factor on the X chromosome enhanced or suppressed XCI of its allele, the factor must be on either of the two distal regions derived from her grandfather. Although we were unable to fully uncover the molecular mechanism, we concluded that unfavorable switching of skewed XCI caused Menkes disease in the proband.

Abstract Hyperosmotic stress activates in live cells numerous processes and also promotes intracellular protein/RNA aggregation and phase separation. However, the time course and the extent of these changes remain largely uncharacterized. To investigate dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress in live cells, we used fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy (FCS) to quantify changes in the local environment by measuring the fluorescence lifetime and the diffusion of the monomeric enhanced green fluorescent protein (eGFP), respectively. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the acceptor/donor emission ratio using the MMC-sensitive Förster resonance energy transfer sensor (GimRET). This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, eGFP diffusion assessed by FCS indicated higher intracellular viscosity due to increased MMC during hyperosmotic stress. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. Our approach can therefore be used to reveal in live cells short-lived transient states through which MMC builds over time, which could not be observed when measuring changes in other physical properties that occur at slower time scales.

Abstract The post-surgical fluid leakage from the tubular tissues is a critical symptom after gastrointestinal or urinary tract surgeries. Elucidating the mechanism for such abnormalities is vital in surgical and medical science. The exposure of the fluid such as peritonitis due to urinary or gastrointestinal perforation has been reported to induce severe inflammation to the surrounding tissue. However, there have been no reports for the tissue responses by fluid extravasation and assessment of post-surgical and injury complication processes is therefore vital. The current model mouse study aims to investigate the effect of the urinary extravasation of the urethral injuries. Analyses on the urinary extravasation affecting both urethral mesenchyme and epithelium and the resultant spongio-fibrosis/urethral stricture were performed. The urine was injected from the lumen of urethra exposing the surrounding mesenchyme after the injury. The wound healing responses with urinary extravasation were shown as severe edematous mesenchymal lesions with the narrow urethral lumen. The epithelial cell proliferation was significantly increased in the wide layers. The mesenchymal spongio-fibrosis was induced by urethral injury with subsequent extravasation. The current report thus offers a novel research tool for surgical sciences on the urinary tract.

Abstract Triple negative breast cancer (TNBC) is associated with worse outcomes and results in high mortality; therefore, great efforts are required to find effective treatment. In the present study, we suggested a novel strategy to treat TNBC using mesenchymal stem cell (MSC)-derived extracellular vesicles (EV) to transform the behaviors and cellular communication of TNBC cells (BCC) with other non-cancer cells related to tumorigenesis and metastasis. Our data showed that, BCC after being internalized with EV derived from Wharton’s Jelly MSC (WJ-EV) showed the impaired proliferation, stemness properties, tumorigenesis and metastasis under hypoxic conditions. Moreover, these inhibitory effects may be involved in the transfer of miRNA-125b from WJ-EV to BCC, which downregulated the expression of HIF1α and target genes related to proliferation, epithelial-mesenchymal transition, and angiogenesis. Of note, WJ-EV-internalized BCC (wBCC) showed transformed behaviors that attenuated the in vivo development and metastatic ability of TNBC, the angiogenic abilities of endothelial cells and endothelial progenitor cells and the generation of cancer-associated fibroblasts from MSC. Furthermore, wBCC generated a new EV with modified functions that contributed to the inhibitory effects on tumorigenesis and metastasis of TNBC. Taken together, our findings suggested that WJ-EV treatment is a promising therapy that results in the generation of wBCC to interrupt the cellular crosstalk in the tumor environment and inhibit the tumor progression in TNBC.

Abstract Anti-vascular endothelial growth factor treatment for macular edema secondary to branch retinal vein occlusion generally provides good visual acuity (VA) improvement but may require repeated injections for years. To reduce the number of patients who suffer from avoidable VA loss caused by treatment drop-out, providing prospects of the correlation between expected vision improvement and required number of injections at the early stages of treatment may be helpful. In this post hoc analysis of the phase IV, randomized, open-label ZIPANGU study, we investigated the correlation between the data from Month 2 and Month 12 in terms of VA and required ranibizumab injection numbers. Fifty-nine patients were evaluated (ranibizumab monotherapy, 29; combination therapy, 30). In the monotherapy group, patients who received 1 and 3 injections by Month 2 received a mean total of 2.8 and 8.3 injections during the year, respectively. Data from the combination group were similar. The correlation coefficients for VA scores at Months 2 and 12 were 0.60 and 0.51 for the monotherapy and combination groups, respectively (both p

Abstract CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). The CUX2 variants showed abnormal localization in human cell culture analysis. While wild-type CUX2 enhances dendritic arborization in fly neurons, the effect was compromised by some of the variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.

Abstract Herein, we compared the connectivity of resting-state networks between participants with high and low working memory capacity groups. Brain network connectivity was assessed under both resting and working memory task conditions. Task scans comprised dual-task (reading sentences while memorizing target words) and single-task (reading sentences) conditions. The low capacity group showed relatively stronger connectivity during resting-state in most brain regions, and the high capacity group showed a stronger connectivity between the medial prefrontal and posterior parietal cortices. During task performance, the dorsal attention and salience networks were relatively strongly connected in the high capacity group. In the comparison between dual- and single-task conditions, increased coupling between the anterior cingulate cortex and other attentional control-related areas were noted in the high capacity group. These findings suggest that working memory differences are related with network connectivity variations in attentional control-associated regions during both resting and task performance conditions.

Abstract The Fukushima-Daiichi Nuclear Power Plant (FNPP) accident in March of 2011 released substantial amounts of radionuclides into the environment. We collected 4,957 deciduous teeth formed in children before the Fukushima accident to obtain precise control data for teeth formed after the accident. Radioactivity was measured using imaging plates (IP) and epidemiologically assessed using multivariate regression analysis. Additionally, we measured 90Sr, 137Cs, and natural radionuclides which might be present in teeth. Epidemiological studies of IP showed that the amount of radioactivity in teeth from Fukushima prefecture was similar to that from reference prefectures. We found that artificial radionuclides of 90Sr and 137Cs, which were believed to have originated from past nuclear disasters, and natural radionuclides including 40 K and daughter nuclides in the 238U and 232Th series contributed to the generation of radioactivity in teeth. We also found no evidence to suggest that radionuclides originating from the FNPP accident significantly contaminated pre-existing teeth. This is the first large-scale investigation of radioactivity and radionuclides in teeth. The present findings will be indispensable for future studies of teeth formed after the FNPP accident, which will fall out over the next several years and might be more contaminated with radionuclides.

Abstract The ZIPANGU study assessed the efficacy and safety of ranibizumab as a one loading dose + pro re nata (one + PRN) regimen with/without focal/grid laser among treatment-naïve patients suffering from macular edema (ME) following branch retinal vein occlusion (BRVO). ZIPANGU was a phase IV, prospective, randomized, open-label, active-controlled, 12-month, two-arm, multicenter study. Treatment-naïve patients with visual impairment (19–73 letters) caused by ME, defined as central subfield thickness (CSFT) > 300 µm, due to BRVO were randomly assigned to ranibizumab monotherapy (n = 29) or combination therapy (ranibizumab + focal/grid short-pulse laser, n = 30). The primary endpoint was the number of ranibizumab injections. Secondary endpoints were mean changes in best-corrected visual acuity (BCVA) and CSFT, and safety. There were no statistically significant differences in the mean number of ranibizumab injections between monotherapy (4.3 injections) vs. combination (4.1 injections) therapy, or in CSFT. BCVA improvement in the monotherapy arm (22.0 letters) was better than the combination therapy arm (15.0 letters) (p = 0.035). Overall, both regimens appeared to be safe and well tolerated. One + PRN ranibizumab is safe and efficacious in treatment-naïve patients with ME secondary to BRVO. A conjunctive laser treatment did not lead to better functional outcomes or fewer ranibizumab injections.

Abstract Excessive intake of fat causes accumulation of fat in liver, leading to non-alcoholic fatty liver disease (NAFLD). High-fat diet (HFD) upregulates the expression of Factor D, a complement pathway component, in the liver of mice. However, the functions of Factor D in liver are not well known. Therefore, the current study investigated the relationship between Factor D and hepatic lipid accumulation using CRISPR/Cas9-mediated Factor D knockout (FD-KO) mice. Factor D deficiency downregulated expression of genes related to fatty acid uptake and de novo lipogenesis in the liver. Furthermore, Factor D deficiency reduced the expression of inflammatory factors (Tnf and Ccl2) and fibrosis markers and decreased accumulation of F4/80-positive macrophages. These data suggest that the Factor D deficiency improved hepatic lipid accumulation and hepatic inflammation in HFD-fed mice.

Abstract Recent studies have revealed unique biological characteristics of molecular hydrogen (H2) as an anti-inflammatory agent. We developed a novel haemodialysis (E-HD) system delivering an H2 (30–80 ppb)-enriched dialysis solution by water electrolysis, and conducted a non-randomized, non-blinded, prospective observational study exploring its clinical impact. Prevalent chronic HD patients were allocated to either the E-HD (n = 161) group or the conventional HD (C-HD: n = 148) group, and received the respective HD treatments during the study. The primary endpoint was a composite of all-cause mortality and development of non-lethal cardio-cerebrovascular events (cardiac disease, apoplexy, and leg amputation due to peripheral artery disease). During the 3.28-year mean observation period, there were no differences in dialysis parameters between the two groups; however, post-dialysis hypertension was ameliorated with significant reductions in antihypertensive agents in the E-HD patients. There were 91 events (50 in the C-HD group and 41 in the E-HD group). Multivariate analysis of the Cox proportional hazards model revealed E-HD as an independent significant factor for the primary endpoint (hazard ratio 0.59; [95% confidence interval: 0.38–0.92]) after adjusting for confounding factors (age, cardiovascular disease history, serum albumin, and C-reactive protein). HD applying an H2-dissolved HD solution could improve the prognosis of chronic HD patients.