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Intestinal lymphangiectasia (IL) is a common complication in dogs. Since nitric oxide (NO) is known to relax the lymphatic vessel, we evaluated inducible NO synthase (iNOS) expression using immunohistochemistry in 13 dogs with lymphoplasmacytic enteritis (LPE) with or without IL. The duodenal iNOS expressing cells were significantly increased in dogs with IL-negative or IL-positive LPE dogs (P=0.025, P=0.007) compared with control dogs. However, there was no significant difference in iNOS expression between IL-positive and IL-negative tissues. Based on these results, there is no clear evidence for the NO overproduction in the pathogenesis of IL in dogs with LPE. Factors other than NO could, thus, contribute to IL in dogs with LPE.

Nonalcoholic fatty liver disease is a hepatic disorder with deposition of fat droplets, and has a high risk of progression to steatosis-related hepatitis and irreversible hepatic cancer. Metronidazole (MNZ) is an antiprotozoal and antimicrobial agent widely used to treat patients infected with anaerobic bacteria and intestinal parasites; however, MNZ has also been shown to induce liver tumors in rodents. To investigate the effects of MNZ on steatosis-related early-stage hepatocarcinogenesis, male rats treated with N-nitrosodiethylamine following 2/3 hepatectomy at week 3 were received a control basal diet, high fat diet (HFD), or HFD containing 0.5% MNZ. The HFD induced obesity and steatosis in liver, accompanied by altered expression of Pparg and Fasn, genes related to lipid metabolism. MNZ increased nuclear translocation of lipid metabolism-related transcription factor peroxisome proliferator-activated receptor gamma in hepatocytes, together with altered liver expression of lipid metabolism genes (Srebf1, Srebf2, Pnpla2). Furthermore, MNZ significantly increased the number of preneoplastic liver foci, accompanied by DNA double-strand breaks and late-stage autophagy inhibition, as reflected by increased levels of γ-H2AX, LC3, and p62. Therefore, MNZ could induce steatosis-related hepatocarcinogenesis by inducing DNA double-strand breaks and modulating autophagy in HFD-fed rats.

Along with nitric oxide (NO), the gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H2S) are emerging as potentially important players in newborn physiology, as mediators of newborn disease, and as new therapeutic modalities. Several recent studies have addressed H2S in particular in animal models of bronchopulmonary dysplasia (BPD), a common complication of preterm birth where oxygen toxicity stunts lung development. In those studies, exogenous H2S attenuated the impact of oxygen toxicity on lung development, and two H2S-generating enzymes were documented to affect pulmonary vascular development. H2S is directly generated endogenously by three enzymes, one of which, 3-mercaptopyruvate sulfurtransferase (MPST), has not been studied in the lung. In a hyperoxia-based animal model of BPD, oxygen exposure deregulated MPST expression during post-natal lung development, where MPST was localized to the smooth muscle layer of the pulmonary vessels in developing lungs. siRNA-mediated abrogation of MPST expression in human pulmonary artery smooth muscle cells in vitro limited baseline cell migration and cell proliferation, without affecting apoptosis or cell viability. In vivo, MPST was dispensable for normal lung development in Mpst-/-mice, and MPST did not contribute to stunted lung development driven by hyperoxia exposure, assessed by design-based stereology. These data demonstrate novel roles for MPST in pulmonary vascular smooth muscle cell physiology. The potential caveats of using Mpst-/- mice to study normal and aberrant lung development are also discussed, highlighting the possible confounding, compensatory effects of other H2S-generating enzymes that are present alongside MPST in the smooth muscle compartment of developing pulmonary vessels.

To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits—tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.

A 30 year-old man with a high fever (37.5°C-40°C), vomiting, slurred speech, and mild cognitive impairment was admitted to our Emergency Department. He had traveled from Spain to the UK on business at the end of February 2020. A nasopharyngeal swab was positive by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but a cerebrospinal fluid (CSF) sample was negative. His neurological abnormalities recovered completely on saline infusion to normalize his low serum sodium level. Although neurological abnormalities in patients with COVID-19 are rare, it is important to distinguish the etiologies including encephalitis, meningitis, or merely electrolyte abnormalities.

[Background] The options for the treatment of nonalcoholic steatohepatitis (NASH) are limited. We examined the effects of ipragliflozin, a sodium-glucose cotransporter 2 inhibitor, on the fatty liver Shionogi (FLS)-ob/ob mice, a non-alcoholic steatohepatitis mouse model. [Methods] FLS-ob/ob male mice were treated with vehicle (n = 10) and ipragliflozin (n = 8). Serum metabolic markers, histopathology of the liver, hepatic cholesterol and triglyceride levels and hepatic mRNA levels related to fibrosis, lipid metabolism and endoplasmic reticulum (ER) stress were compared between the two groups. [Results] The body weight and hepatic cholesterol and triglyceride levels were significantly decreased in the ipragliflozin group compared with the control group. Hepatic steatosis and fibrosis were significantly ameliorated by the treatment with ipragliflozin. Hepatic infiltration of macrophage, expression levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and hepatic mRNA levels of ER stress markers were not significantly modulated by the treatment with ipragliflozin. [Conclusion] Ipragliflozin can be a therapeutic option for patients with NASH. The precise mechanisms of action need to be clarified in future studies.

Wide band gap semiconductors such as ZnO are characterized by unique optoelectronic properties, which have led to numerous applications in the field of sensors and optoelectronics. These components...

[Background] Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease related to metabolic syndrome, which can progress to liver cirrhosis. Standard medication has not been established. Pemafibrate is a selective peroxisome proliferatoractivated receptor (PPAR) α modulator. We retrospectively evaluated the efficacy of pemafibrate in patients with NAFLD. [Methods] We retrospectively enrolled 17 patients (ten men, seven women; median age, 63 years; range, 27?81 years). They were all proven to have fatty liver through imaging and had little or no history of drinking (ethanol consumption of < 20 g/day for women and < 30 g/day for men). They were administered pemafibrate from October 2018 to June 2020. [Results] After administration, serum triglyceride (TG) tended to be decreased (300.5 ± 22.5 to 239.5 ± 34.3 mg/dL, P = 0.06). Serum high-density lipoprotein (HDL) cholesterol and low density lipoprotein (LDL) cholesterol levels did not change. ALT was significantly decreased (-47.4%) for six months (57.5 ± 8.8 to 30.3 ± 5.8 U/L, P < 0.01). The values of serum GGT significantly decreased (-48.7%) for sixth months (63.9 ± 10.3 to 32.8 ± 6.6 U/L, P < 0.01). Aspartate aminotransferase (AST) to platelet ratio (APRI), a fibrosis marker, also was significantly decreased in the sixth month (0.7 ± 0.1 to 0.4 ± 0.1, P < 0.05). Body mass index (BMI) and hemoglobin A1c (HbA1c) showed no significant change. [Conclusion] Pemafibrate dramatically ameliorated the values of liver function tests and APRI in patients with NAFLD.

We report the failure to achieve castrate level of serum testosterone during luteinizing hormone-releasing hormone agonist therapy in a patient with prostate cancer. A 76-year-old man was admitted to our hospital for evaluation of an elevated serum prostate specific antigen (PSA) level (191.10 ng/ml) in August 2011. He was diagnosed with T3aN0M1b prostate adenocarcinoma. A combined androgen blockade using luteinizing hormone-releasing hormone agonist (the 1-month depot of leuprorelin acetate) and antiandrogen was administered. Due to liver dysfunction, antiandrogens, both bicalutamide and flutamide, were stopped. The 1-month depot was switched to the 3-month depot in May 2013, but the patient complained of induration and abscess at the infection site. Leuprorelin acetate was replaced by goserelin acetate. Because no adverse event appeared after injection of the 1-month depot of goserelin acetate, the 3-month depot was administered in October 2013. The PSA level increased gradually, and the testosterone level was greater than 50 ng/dl, that is, above castrate range. The 3-month depot of both leuprorelin acetate and goserelin acetate was not effective for this patient. For this reason, the 1-month depot of leuprorelin acetate was started resulting in a rapid decrease in PSA and testosterone levels. Thereafter, androgen depriving therapy could be continued. Androgen deprivation therapy is the standard treatment for patients with advanced prostate cancer and luteinizing hormone-releasing hormone aims to suppress serum testosterone to castrate range. We recommend assessing the serum testosterone levels during luteinizing hormone-releasing hormone agonist therapy for monitoring treatment efficacy and verifying progression when the PSA level increases.

Metal-halide perovskites show excellent properties for photovoltaic and optoelectronic applications, with power conversion efficiencies of solar cell and LEDs exceeding 20%. Being solution processed, these polycrystalline materials likely contain a large density of defects compared to melt-grown semiconductors. Surprisingly, typical effects from defects (absorption below the bandgap, low fill factor and open circuit voltage in devices, strong nonradiative recombination) are not observed. In this work, we study thin films of metal-halide perovskites CH

Hydrogen sulfide (H2S) is produced by the action of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H2S and pyruvate. H2S is recognized as an endogenous gaseous mediator with multiple regulatory roles in mammalian cells and organisms. In the present study we demonstrate that MPT, the endogenous substrate of 3-MST, acts also as endogenous H2S donor. Colorimetric, amperometric and fluorescence based assays demonstrated that MPT releases H2S in vitro in an enzyme-independent manner. A functional study was performed on aortic rings harvested from C57BL/6 (WT) or 3-MST-knockout (3-MST−/−) mice with and without endothelium. MPT relaxed mouse aortic rings in endothelium-independent manner and at the same extent in both WT and 3-MST−/− mice. N5-(1-Iminoethyl)- l -ornithine dihydrochloride (L-NIO, an inhibitor of endothelial nitric oxide synthase) as well as 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor) did not affect MPT relaxant action. Conversely, hemoglobin (as H2S scavenger), as well as glybenclamide (an ATP-dependent potassium channel blocker) markedly reduced MPT-induced relaxation. The functional data clearly confirmed a non enzymatic vascular effect of MPT. In conclusion, MPT acts also as an endogenous H2S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to increase H2S in conditions where H2S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease.

Yes-associated protein (YAP) positivity indicates a poor prognosis in gastric cancer. Transcriptional co-activator with a PDZ-binding domain (TAZ), a YAP paralog, is highly expressed in gastric signet ring cell carcinoma. Verteporfin (VP), a clinical photosensitizer, was recently shown to inhibit YAP/TAZ. In the present study, the therapeutic potential of VP treatment was explored using two gastric cancer cell lines: MKN-45 (TAZ-dominant) and MKN-74 (YAP-dominant). Cell proliferation was evaluated by MTS assay. Vascular mimicry was evaluated by the tube formation assay. Gene and protein expression levels of YAP/TAZ downstream effectors [such as Survivin, Cysteine-rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF)] were measured. YAP or TAZ localization was evaluated by immunofluorescence. Cell death was assessed by immunofluorescent staining of Annexin V. YAP and TAZ expression were knocked down by small interfering RNA. The current results demonstrate that MKN-45, a poorly differentiated TAZ-dominant gastric cancer cell line, was more sensitive to VP than MKN-74, a moderately differentiated YAP-dominant gastric cancer cell line. VP changed the localization of YAP/TAZ, promoted its degradation and significantly decreased the protein level of Survivin in both cell lines. Cell death was induced by VP treatment in a dose-dependent manner. Vascular mimicry was inhibited in both cell lines. Proliferation in both cell lines decreased in response to YAP/TAZ knockdown. The present study indicated that VP has potential as a therapeutic agent in YAP- and TAZ-dominant gastric cancers due to its ability to suppress the anti-apoptotic protein Survivin via inhibition of YAP and TAZ.

Electrochemical reduction of amides was achieved by using a hydrosilane without any toxic or expensive metals. The key reactive ketyl radical intermediate was generated by cathodic reduction. Continuous reaction with anodically generated silyl radicals or zinc bromide resulted in chemoselective deoxygenation to give the corresponding amines.

Graphical abstract Extracellular ATP as a purinergic signaling molecule, together with ATP receptor, are playing an important role in tumor growth, therapy resistance, and host immunity suppression. Meanwhile ATP is a crucial indicator for cellular energy status and viability, thus a vital variable for tissue regeneration and in vitro tissue engineering. Most recent studies on COVID-19 virus suggest infection caused ATP deficit and release as a major characterization at the early stage of the disease and major causes for disease complications. Thus, imaging ATP molecule in both cellular and extracellular contexts has many applications in biology, engineering, and clinics. A sensitive and selective fluorescence “signal-on” probe for ATP detection was constructed, based on the base recognition between a black hole quencher (BHQ)-labeled aptamer oligonucleotide and a fluorophore (Cy5)-labeled reporter flare. The probe was able to detect ATP in solution with single digit µM detection limit. With the assistance of lipofectamine, this probe efficiently entered and shined in the model cells U2OS within 3 h. Further application of the probe in specific scenery, cardio-tissue engineering, was also tested where the ATP aptamer complex was able to sense cellular ATP status in a semi-quantitative manner, representing a novel approach for selection of functional cardiomyocytes for tissue engineering. At last a slight change in probe configuration in which a flexible intermolecular A14 linker was introduced granted regeneration capability. These data support the application of this probe in multiple circumstances where ATP measurement or imaging is on demand. Supplementary Information The online version contains supplementary material available at 10.1007/s00604-021-05012-8.

The large amount of waste derived from coupling reagents is a serious drawback of peptide synthesis from a green chemistry viewpoint. To overcome this issue, we report an electrochemical peptide synthesis in a biphasic system. Anodic oxidation of triphenylphosphine (Ph3P) generates a phosphine radical cation, which serves as the coupling reagent to activate carboxylic acids, and produces triphenylphosphine oxide (Ph3P Created by potrace 1.16, written by Peter Selinger 2001-2019 O) as a stoichiometric byproduct. In combination with a soluble tag-assisted liquid-phase peptide synthesis, the selective recovery of desired peptides and Ph3PO was achieved. Given that methods to reduce Ph3PO to Ph3P have been reported, Ph3PO could be a recyclable byproduct unlike byproducts from typical coupling reagents. Moreover, a commercial peptide active pharmaceutical ingredient (API), leuprorelin, was successfully synthesized without the use of traditional coupling reagents., The large amount of waste derived from coupling reagents is a serious drawback of peptide synthesis from a green chemistry viewpoint.

Ridaifen (RID)-B is an analog derived from tamoxifen (TAM). TAM has an antitumor effect by acting as an antagonist to estrogen receptor (ER). However, TAM is known to also induces apoptosis in cancer cells that do not have ER. We clarified that RID-B induces cell death at a lower concentration than TAM, and causes ER-independent apoptosis and autophagy. Based on the results of previous studies, we assumed that RID-B had a unique target different from ER and examined structural activity correlation to determine what kinds of structural features are related to RID-B activity. As a result, we found there was activity even without one of phenyl groups (Ar3) in RID-B and revealed that two pyrrolidine side chains peculiar to RID-B are related to the action. Furthermore, analogs with shorter alkyl side chains induced autophagy, but analogs with certain length of alkyl side chains induced apoptosis. Also, although there is no doubt that RID-B induces apoptosis by causing mitochondrial injury, our results suggested that such injury induced mitochondria-selective autophagy. We revealed that RID-B induce mitophagy and that this mitophagy is a defense mechanism against RID-B. Our results suggest that autophagy was induced against apoptosis caused by mitochondrial dysfunction in RID-B, so the combination of autophagy inhibitor and anticancer-drug can be effective for cancer treatment.

We examined the effect of the timing of removing cumulus cells surrounding porcine oocytes from small follicles (SFs, < 3 mm in diameter) and medium follicles (MFs; 3-6 mm in diameter) on the meiotic and developmental competence of the oocytes. Cumulus-oocyte complexes (COCs) were collected from SFs and MFs, and the oocytes were denuded at 0, 20, and 44 h after the start of in vitro maturation (IVM), and the meiotic progression of the oocytes was assessed at the end of the IVM period. The incidence of mature oocytes was significantly affected by both the origin of the COCs and the time when the oocytes were denuded. Although the percentage of mature oocytes was always higher when the COCs were collected from MFs than that when the COCs were collected from SFs, the maturation rate was significantly higher when the oocytes were denuded at 20 h than when they were denuded at 44 h after the start of IVM. When the mature oocytes were activated electrically, the developmental competence of the oocytes denuded at 20 and 44 h to reach the blastocyst stage did not differ, whereas the competence of the MF-derived oocytes was significantly higher than that of SF-derived oocytes. When the intracellular cAMP and cGMP levels in SF-derived oocytes were examined at 24 h of IVM, the levels of both were significantly decreased only in the oocytes denuded at 20 h. In conclusion, denuding oocytes at 20 h of IVM caused a significant reduction in ooplasmic cAMP and cGMP levels and increased the meiotic competence of the oocytes without any reduction in blastocyst formation, even in the case of SF-derived oocytes.

This prospective study examined the impact of genetic polymorphisms on the pharmacokinetics and clinical efficacy and safety of lenvatinib, a substrate of ATP-binding transporters, in a cohort of 48 Japanese patients with hepatocellular carcinoma (HCC). Pharmacokinetic studies were performed at the start of lenvatinib therapy (day 1) and on day 15. The coefficients of variation in AUC0–24h of lenvatinib on days 1 and 15 were 44.0% and 52.4%, respectively. Although the ABCB1 3435C > A, 1236C > T, and 2677G>T/A polymorphisms did not influence pharmacokinetic parameters, the AUC0–24h values on days 1 and 15 of the ABCG2 C/A or A/A group were approximately 1.1-fold and 1.4-fold that in the ABCG2 C/C group (P = 0.164 and 0.024). There were no significant differences in AUC0–24h on days 1 and 15 between the responders (complete or partial response) and non-responders (stable or progressive disease). The AUC0–24h on day 15 in those developing anorexia of any grade was significantly higher than that without such development (P = 0.017). In multivariate analysis, ABCG2 421C > A C/A or A/A was significantly associated with the development of anorexia (odds ratio 9.009, P = 0.009). ABCG2 421C > A polymorphism could affect exposure to lenvatinib and the development of anorexia.

Background/aim No biomarkers that predict the benefit from anti-vascular endothelial growth factor (VEGF) antibodies have been identified. It is necessary to discover biomarkers that can identify patients who are more likely to benefit from bevacizumab-containing treatment, especially those who are more likely to benefit from treatment with bevacizumab beyond progression (BBP). Levels of serum lactate dehydrogenase (LDH), reported to be an indirect marker of hypoxia and angiogenesis, may be a useful marker for monitoring the efficacy of suppression of angiogenesis. Patients and methods The clinical data of 91 patients with unresectable metastatic colorectal cancer who were treated with bevacizumab-containing chemotherapy as first-line treatment were collected and studied. Results In the second-line treatment, the bevacizumab plus chemotherapy group showed significantly better progression-free survival (PFS) in comparison to the chemotherapy-alone group in patients with low post-first-line-treatment serum LDH levels. On the other hand, no significant differences in the PFS rate were observed between the two groups in patients with high post-first-line-treatment serum LDH levels. Conclusion The post-first-line-treatment serum LDH levels may, therefore, be useful marker for predicting the efficacy of treatment with BBP.

Fractones, specialized extracellular matrix structures found in the subventricular zone (SVZ) neurogenic niche, can capture growth factors, such as basic fibroblast growth factor, from the extracellular milieu through a heparin-binding mechanism for neural stem cell (NSC) presentation, which promotes neurogenesis. During aging, a decline in neurogenesis correlates with a change in the composition of heparan sulfate (HS) within fractones. In this study, we used antibodies that recognize specific short oligosaccharides with varying sulfation to evaluate the HS composition in fractones in young and aged brains. To further understand the conditions that regulate 6-O sulfation levels and its impact on neurogenesis, we used endosulfatase Sulf1 and Sulf2 double knockout (DKO) mice. Fractones in the SVZ of Sulf1/2 DKO mice showed immunoreactivity for the HS epitope, suggesting higher 6-O sulfation. While neurogenesis declined in the aged SVZ of both wild-type and Sulf1/2 DKO mice, we observed a larger number of neuroblasts in the young and aged SVZ of Sulf1/2 DKO mice. Together, these results show that the removal of 6-O-sulfation in fractones HS by endosulfatases inhibits neurogenesis in the SVZ. Our findings advance the current understanding regarding the extracellular environment that is best suited for NSCs to thrive, which is critical for the design of future stem cell therapies.

Breast cancer susceptibility gene 1 (BRCA1)-associated protein 2 (BRAP2) is a novel protein that binds to BRCA1 and is located in the cytoplasm. BRAP2 has been demonstrated to bind to regulators of the Ras-Raf-MEK and PI3K/Akt pathways, both of which are involved in carcinogenesis. This suggests that BRAP2 may be capable of regulating both pathways. In the present study, the role of BRAP2 in both pathways was clarified during apoptosis and cell proliferation in a leukemia cell line. A BRAP2-deficient leukemia cell line was generated using CRISPR/Cas9, the BRAP2-deficient and parental cells were treated with a Ras, pan-Raf or PI3K inhibitor, and the changes in signal transduction, apoptosis and cell proliferation were evaluated. BRAP2 knockout attenuated the inhibition of signal transduction of the Ras-Raf-MEK and PI3K/Akt pathways by the Ras, pan-Raf or PI3K inhibitor. BRAP2 deletion also suppressed the cytotoxic and apoptotic effects of the Ras and pan-Raf inhibitors. However, the loss of BRAP2 did not suppress the cytotoxicity of the PI3K inhibitor but did suppress the PI3K inhibitor-induced inhibition of cell proliferation. The present results indicated that BRAP2 induces apoptosis and the inhibition of cell proliferation via regulating the Ras-Raf-MEK and PI3K/Akt pathways. In leukemia cells, because the Ras-Raf-MEK and PI3K/Akt pathways are activated aberrantly, the simultaneous inhibition of both pathways is desired. The current results indicated that enhancement of the function of BRAP2 may represent a new target in leukemia treatment.

Retinoids are a family of vitamin A-derived molecules and include the biologically active metabolite, retinoic acid (RA). RA acts as a specific modulator of neuronal differentiation and proliferation. However, teratogenicity and a large excess of RA have been found in animal studies. Thus, development of effective and stable retinoids is desirable. In this study, we showed that treatment with novel synthetic retinobenzoic acids promotes neurite outgrowth in a selected subpopulation of the human neuroblastoma cell line SK-N-SH. Furthermore, we found that, although acting via a different mechanism, retinobenzoic acids have the same neurite outgrowth-inducing effect as RA. Retinoids, including RA, bind to nuclear retinoic acid receptors (RARs). Therefore, we examined the expression of RARs in retinobenzoic acid-treated cells. Similar to already known retinoids, novel synthetic retinobenzoic acids promote the upregulation of RARβ and have no effect on RARα or γ. These results suggest that retinobenzoic acids act via RARβ during neurite outgrowth. Moreover, stimulation with RA or retinobenzoic acids significantly increased the phosphorylation levels of both ERK1/2 and mTOR. ERK1/2 and mTOR inhibition blocked the retinobenzoic acid-induced increase in neurite outgrowth, suggesting that retinobenzoic acids promoted neurite outgrowth by activating the ERK1/2 and mTOR signaling pathways. Notably, the RA-induced increase in neurite outgrowth was blocked by the ERK1/2 inhibitor U0126, but not by the mTOR inhibitor rapamycin. In addition, ERK1/2 inhibition blocked the upregulation of RARβ promoted by RA and retinobenzoic acids. In contrast, mTOR inhibition had no effect on upregulation of RARβ. Our results show that novel synthetic retinobenzoic acids induce neurite outgrowth by a different mechanism than RA. These findings suggest that activation of both ERK1/2, which results in downstream regulation of RARβ, and mTOR, are responsible for the novel synthetic retinobenzoic acid-induced neurite outgrowth in human neuroblastoma cells.

Background: Bleeding after gastric endoscopic submucosal dissection (ESD) remains problematic, especially in patients receiving antithrombotic therapy. Thus, our study aimed to identify the risk factors and to create a prediction model for post-ESD bleeding in patients receiving antithrombotic therapy. Methods: Patients receiving antithrombotic therapy who underwent gastric ESD at Osaka Medical College Hospital and at 18 referral hospitals all over Japan were enrolled. Risks of post-ESD bleeding were calculated using multivariable logistic regression. The dataset was randomly divided into derivation cohort (70%) and validation cohort (30%). We created a prediction model using derivation cohort. The accuracy of the model was evaluated using the validation cohort. We compared this model with an existing scoring method, using NRI (net reclassification improvement) and IDI (integrated discrimination improvement). Findings: The study enrolled 1207 patients. Post-ESD bleeding occurred in 142 (11·8%) cases. Multivariable analysis yielded an odds ratio of 1·73 for aspirin, 4·18 for P2Y12 receptor antagonist, 1·42 for cilostazol, 0·80 for other antiplatelet agents, 4·12 for warfarin, 3·76 for dabigatran, 5·14 for apixaban, 6·74 for edoxaban, 4·22 for rivaroxaban, and 1·56 for heparin bridging. The prediction model (SAMURAI model) was created for patients receiving antithrombotic therapy, and it had a good predictive ability with a C-statistic of 0·74. The model had a significantly better predictive ability than a previously reported model (NRI=0·41, 0·15–0·66, p=0·002, IDI=0·05, 0·02–0·08, p=0·002). Interpretation: In this study, the risk of post-ESD bleeding for each antithrombotic agent was evaluated. Use of the SAMURAI model will allow the proactive management of post-ESD bleeding risk during antithrombotic therapy. Funding: 2nd Department of Internal Medicine, Osaka Medical College, Japan Declaration of Interest: The authors declare that there are no conflicts of interest associated with this manuscript. Ethical Approval: This study was conducted according to the guidelines of the Declaration of Helsinki. The study was approved by the Ethics Committee of Osaka Medical College (trial number: 693) and by the institutional review board of each participating institution before the recruitment of patients. Informed consent for this study was obtained in the form of opt-out on the website of each participating hospital. All authors had access to the study data and reviewed and approved the final manuscript.

Rationale Hydrogen sulfide (H2S) is a physiological mediator that regulates cardiovascular homeostasis. Three major enzymes contribute to the generation of endogenously produced H2S, namely cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Although the biological roles of CSE and CBS have been extensively investigated in the cardiovascular system, very little is known about that of 3-MST. In the present study we determined the importance of 3-MST in the heart and blood vessels, using a genetic model with a global 3-MST deletion. Results 3-MST is the most abundant transcript in the mouse heart, compared to CSE and CBS. 3-MST was mainly localized in smooth muscle cells and cardiomyocytes, where it was present in both the mitochondria and the cytosol. Levels of serum and cardiac H2S species were not altered in adult young (2–3 months old) 3-MST−/− mice compared to WT animals. No significant changes in the expression of CSE and CBS were observed. Additionally, 3-MST−/− mice had normal left ventricular structure and function, blood pressure and vascular reactivity. Interestingly, genetic ablation of 3-MST protected mice against myocardial ischemia reperfusion injury, and abolished the protection offered by ischemic pre- and post-conditioning. 3-MST−/− mice showed lower expression levels of thiosulfate sulfurtransferase, lower levels of cellular antioxidants and elevated basal levels of cardiac reactive oxygen species. In parallel, 3-MST−/− mice showed no significant alterations in endothelial NO synthase or downstream targets. Finally, in a separate cohort of older 3-MST−/− mice (18 months old), a hypertensive phenotype associated with cardiac hypertrophy and NO insufficiency was observed. Conclusions Overall, genetic ablation of 3-MST impacts on the mouse cardiovascular system in an age-dependent manner. Loss of 3-MST exerts a cardioprotective role in young adult mice, while with aging it predisposes them to hypertension and cardiac hypertrophy.

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and &gamma, polyglutamic acid (&gamma, PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-&alpha, IFN-&gamma, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.

Background Osteoarthritis (OA) is characterized by the formation and deposition of calcium-containing crystals in joint tissues, but the underlying mechanisms are poorly understood. The gasotransmitter hydrogen sulfide (H2S) has been implicated in mineralization but has never been studied in OA. Here, we investigated the role of the H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) in cartilage calcification and OA development. Methods 3-MST expression was analyzed in cartilage from patients with different OA degrees, and in cartilage stimulated with hydroxyapatite (HA) crystals. The modulation of 3-MST expression in vivo was studied in the meniscectomy (MNX) model of murine OA, by comparing sham-operated to MNX knee cartilage. The role of 3-MST was investigated by quantifying joint calcification and cartilage degradation in WT and 3-MST−/− meniscectomized knees. Chondrocyte mineralization in vitro was measured in WT and 3-MST−/− cells. Finally, the effect of oxidative stress on 3-MST expression and chondrocyte mineralization was investigated. Results 3-MST expression in human cartilage negatively correlated with calcification and OA severity, and diminished upon HA stimulation. In accordance, cartilage from menisectomized OA knees revealed decreased 3-MST if compared to sham-operated healthy knees. Moreover, 3-MST−/− mice showed exacerbated joint calcification and OA severity if compared to WT mice. In vitro, genetic or pharmacologic inhibition of 3-MST in chondrocytes resulted in enhanced mineralization and IL-6 secretion. Finally, oxidative stress decreased 3-MST expression and increased chondrocyte mineralization, maybe via induction of pro-mineralizing genes. Conclusion 3-MST-generated H2S protects against joint calcification and experimental OA. Enhancing H2S production in chondrocytes may represent a potential disease modifier to treat OA.

Ranunculus ternatus is a traditional Chinese medicine with an anticancer effect, but its underlying mechanism is unknown. In this study, we demonstrated by MTT assay that ethyl acetate extract (RTE) from R. ternatus exerts cytotoxic effects on human T cell lymphoma Jurkat cells. Then, to test the apoptosis induction ability of RTE to induce apoptosis, we analyzed phosphatidylserine exposure, DNA fragmentation, and caspase cleavage. RTE induced phosphatidylserine exposure and caspase-7 cleavage, but not caspase-3 cleavage. Sub-G1 cells were accumulated but DNA fragmentation was not observed. A pan-caspase inhibitor Z-Asp-CH2-DCB suppressed RTE-induced caspase cleavage and the above-described events. RTE also induced cell death in caspase-3 null human breast cancer MCF-7 cells, indicating that RTE-induced apoptotic-like cell death depends on the activation of one or more caspases, but not caspase-3. Moreover, RTE-induced cell death was not suppressed in Bcl-2 overexpressing Jurkat cells, suggesting that mitochondria were not involved in RTE-induced cell death. In conclusion, RTE-induced cell death was independent of mitochondria and dependent on caspase-7.

We have been studying the general aspects of the functions of H2S and polysulfides, and the enzymes involved in their biosynthesis, for more than 20 years. Our aim has been to elucidate novel physiological and pathological functions of H2S and polysulfides, and unravel the regulation of the enzymes involved in their biosynthesis, including cystathionine β-synthase (EC 4.2.1.22), cystathionine γ-lyase (EC 4.4.1.1), thiosulfate sulfurtransferase (rhodanese, EC 2.8.1.1), and 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2). Physiological and pathological functions, alternative biosynthetic processes, and additional functions of H2S and polysulfides have been reported. Further, the structure and reaction mechanisms of related enzymes have also been reported. We expect this issue to advance scientific knowledge regarding the detailed functions of H2S and polysulfides as well as the general properties and regulation of the enzymes involved in their metabolism. We would like to cover four topics: the physiological and pathological functions of H2S and polysulfides, the mechanisms of the biosynthesis of H2S and polysulfides, the properties of the biosynthetic enzymes, and the regulation of enzymatic activity. The knockout mouse technique is a useful tool to determine new physiological functions, especially those of H2S and polysulfides. In the future, we shall take a closer look at symptoms in the human congenital deficiency of each enzyme. Further studies on the regulation of enzymatic activity by in vivo substances may be the key to finding new functions of H2S and polysulfides.

Administration of the diuretic, spironolactone (SR), can inhibit chronic liver diseases. We determined the effects of SR alone or in combination with the antioxidant α-glycosyl isoquercitrin (AGIQ) on hyperlipidemia- and steatosis-related precancerous lesions in high-fat diet (HFD)-fed rats subjected to a two-stage hepatocarcinogenesis model. Rats were fed with control basal diet or HFD, which was administered with SR alone or in combination with an antioxidant AGIQ in drinking water. An HFD increased body weight, intra-abdominal fat (adipose) tissue weight, and plasma lipids, which were reduced by coadministration of SR and AGIQ. SR and AGIQ coadministration also reduced hepatic steatosis and preneoplastic glutathione S-transferase placental form-positive foci, in association with decrease in NADPH oxidase (NOX) subunit p22phox-positive cells and an increase in active-caspase-3-positive cells in the foci. Hepatic gene expression analysis revealed that the coadministration of SR and AGIQ altered mRNA levels of lipogenic enzymes ( Scd1 and Fasn), antioxidant-related enzymes ( Catalase), NOX component ( P67phox), and anti-inflammatory transcriptional factor ( Pparg). Our results indicated that SR in combination with AGIQ had the potential of suppressing hyperlipidemia- and steatosis-related early hepatocarcinogenesis through the reduced expression of NOX subunits.

Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Especially, sphingoid base-backbones of sphingolipids (sphingosine, sphinganine, and phytosphingosine) and their metabolites N-acyl-sphingoid bases (ceramides) are highly bioactive. In skin, one of the caspases, caspase-14, is expressed predominantly in cornifying epithelia, and caspase-14 plays an important role in keratinocyte differentiation. As ceramides were surrounding lipids in the keratinocytes and ceramides stimulate keratinocyte differentiation, we therefore examined the upregulation of caspase-14 by various sphingoid bases and ceramide. Sphingosine, sphinganine, phytosphingosine, and C2-ceramide treatment at the doses not damaging cells significantly increased caspase-14 mRNA and protein expression in dose-dependent manner on human keratinocyte HaCaT cells. These results indicated that sphingoid bases and ceramide upregulated caspase-14 mRNA to increase intracellular caspase-14 protein level. We next examined the caspase-14 upregulation mechanism by sphingoid bases. We used the most effective sphingoid base, phytosphingosine, and revealed that specific inhibitors of the mitogen-activated protein kinase, p38 and c-jun N-terminal protein kinase (JNK), blocked caspase-14 expression. This indicates that phytosphingosine upregulation of caspase-14 is involved of p38 and JNK activation. Moreover, phytosphingosine induced caspase-14 upregulation in vivo, suggesting that sphingoid bases were involved in keratinocyte differentiation by affecting caspase-14.

Fluorescence tumor imaging using exogenous fluorescent tumor‐targeting agents has potential to improve early tumor detection. The fluorescent contrast agent indocyanine green (ICG) is used in medical diagnostics. The aim of the present study is to investigate the tumor imaging capability and the imaging mechanism of i.v. administered ICG in a mouse model of colitis‐associated colon cancer. To do this, an azoxymethane/dextran sodium sulfate‐induced colon cancer mouse model was used. Ex vivo imaging experiments were carried out 1 hour after i.v. injection of ICG. The ICG fluorescence was observed in the colon tumor tissues, with sufficient tumor to normal tissue ratio, correlating with tumor malignancy. In the tumor tissues, ICG fluorescence was localized in the vascular interstitial tissue. Immunofluorescence microscopy revealed that tumor cells formed tight junctions normally, suggesting an inability of tumor cellular uptake of ICG. In contrast, tumor tissues increased the CD31‐immunoreactive endothelial cell area, and accumulated stromal cells immunoreactive for COX‐2 and tumor cell population immunoreactive for inducible nitric oxide synthase. In vivo vascular permeability assay revealed that prostaglandin E2 promoted the endothelial cell permeability of ICG. In conclusion, our data indicated that fluorescence contrast‐enhanced imaging following i.v. administered ICG can be applied to the detection of colon tumors in a mouse colitis‐associated colon cancer model. The tumor tissue preference of ICG in the present model can be attributed to the enhanced vascular leakage of ICG involving inflammatory mediators, such as COX‐2 and inducible nitric oxide synthase, in conjunction with increased tumor vascularity.

Aluminum (Al) is neurotoxic to adults and also to infants. In this study, we investigated the developmental exposure effect of AlCl3 on postnatal hippocampal neurogenesis. Pregnant mice were administered 0-, 900-, or 1800-ppm AlCl3 via drinking water from gestational day 6 to postnatal day (PND) 21, with their offspring examined on PND 21 and PND 77. On PND 21, GFAP-immunoreactive (+) neural stem cells (NSCs) and p21Cip1/Waf1+ cells were decreased in number in the subgranular zone at 900 and ≥900 ppm, respectively. Pcna transcript level examined at 1800 ppm was decreased in the dentate gyrus. These results suggest induction of compromised cell quiescence that caused impaired self-renewal capacity of NSCs accompanying slowing down of cell cycling, which ultimately resulted in exhaustion of the NSC pool. At 1800 ppm, Reelin+ hilar GABAergic interneurons were also decreased, suggesting a contribution to the NSC reduction. At this dose, TBR2+ or DCX+ progenitor and immature granule cells and PVALB+ interneurons were increased. Moreover, COX-2+ granule cells were increased at ≥900 ppm. These results suggest facilitation of transient progenitor cell proliferation and differentiation during exposure. Moreover, TUNEL+ or Morin-stained granule cells were increased, together with Casp12 transcript upregulation, suggesting induction of Al accumulation-related endoplasmic reticulum stress-mediated granule cell apoptosis. Transcript expression changes on cholinergic and glutamatergic signals and synaptic plasticity suggested contribution to disruptive neurogenesis. The NSC-targeting effects sustained through the adult stage despite no sustained Al-accumulation. These results suggest that developmental AlCl3-exposure irreversibly affects postnatal hippocampal neurogenesis involving multiple functions in mice.

Cross talk between inflammation and coagulation plays important roles in acute or subacute progressive pulmonary fibrosis characterized by diffuse alveolar damage. Thrombomodulin is a physiological inhibitor of high-mobility group box 1 (HMGB1), and thrombin and may be effective for this condition. This study investigated the roles of HMGB1 and thrombin in the pathophysiology of bleomycin-induced pulmonary fibrosis and the efficacy of recombinant human soluble thrombomodulin (rhTM). Pulmonary fibrosis was induced in wild-type C57BL/6 mice by intratracheal instillation of bleomycin. We first assessed HMGB1, thrombin, transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) levels in bronchoalveolar lavage fluid and lung tissue sections over time. Expression of HMGB1 and thrombin was elevated before that of TGF-β1 and α-SMA and remained high during the fibrotic phase after bleomycin instillation. We next examined whether in vitro stimulation with HMGB1 and thrombin induced expression of TGF-β1 and α-SMA in cultured alveolar macrophages and lung fibroblasts, respectively, by performing quantitative PCR, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence analyses. HMGB1 and thrombin stimulation induced TGF-β1 production by alveolar macrophages, and thrombin stimulation also induced α-SMA expression in lung fibroblasts. Finally, we evaluated the effect of rhTM on bleomycin-induced pulmonary fibrosis. Compared with the vehicle control, both early and late-phase administration of rhTM suppressed the fibrotic process. Our results suggest that HMGB1 and thrombin were involved in the pathophysiology of pulmonary fibrosis via production of profibrotic proteins and that rhTM attenuated bleomycin-induced pulmonary fibrosis. rhTM may be a therapeutic option for acute or subacute pulmonary fibrosis.

It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPH = 1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H2Sn revealed that H2S2 first appeared, and then H2S and H2S3 did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis.

BACKGROUND [color=black]Bowel preparation is an important factor for an optimal outcome of colonoscopy. Recently, polyethylene glycol (PEG) solution has been in common use for bowel cleansing for colonoscopy, but some patients are intolerant of PEG because of taste or volume. A low-volume PEG administered with ascorbic acid solution (PEG-Asc) was designed to improve tolerability, but the administration of this method is more complex than that with PEG alone. This study aimed to compare bowel cleansing efficacy, safety, and tolerability of 1 L PEG-Asc with a 2 L PEG preparation with use of sennosides and mosapride.[/color] MATERIAL AND METHODS [color=black]This was a prospective, single-center, non-inferiority trial that included 112 patients (PEG-Asc group, 68; PEG group, 44). The primary endpoint was the efficacy of colon cleansing assessed by endoscopists using a validated 4-point scale according to the Aronchick scale and was verified by a blinded investigator. Acceptability, tolerability, and adenoma detection rate (ADR) of these 2 regimens were secondary endpoints.[/color][color=black] [/color] RESULTS [color=black]We found no statistically significant differences between the groups in colon-cleansing efficacy or in the adenoma detection rate (ADR). Moreover, overall, patients significantly favored PEG-Asc over PEG, reflecting better acceptance of PEG-Asc. Additionally, more patients favored PEG-Asc over PEG for a hypothetical future colonoscopy. [/color] CONCLUSIONS [color=black]The alternate 1 L PEG-Asc regimen and standard 2 L PEG regimen were clinically equivalent with respect to cleansing efficacy, safety, and ADR, and more patients favored PEG-Asc than PEG. This alternate regimen may improve patient compliance and acceptance of surveillance colonoscopy.[/color].

Rat 3-mercaptopyruvate sulfurtransferase (MPST) is a 32 808 Da simple protein. Cys247 is a catalytic site, and Cys154 and Cys263 are on the enzyme surface. MPST is found in all tissues, particularly in the kidneys, although the localization of its activity differs in each tissue. In this review, four functions of MPST are reviewed: (i) antioxidative function: Cys247 is redox-sensitive and serves as a redox-sensing switch. It is oxidized to cysteine sulfenate, which has a low redox potential, upon which the enzyme is inactivated. Then, reduced thioredoxin (Trx) with a reducing system (Trx reductase and NADPH) reduces the sulfenate to restore activity; meanwhile, Cys154 and Cys263 form an intermolecular disulfide bond, which serves as another redox-sensing switch. Consequently, Trx specifically cleaves the intermolecular disulfide bond by converting it from the inactive form (dimer) to the active form (monomer). (ii) Hydrogen sulfide and polysulfide production: hydrogen sulfide is produced via reduction of the persulfurated sulfur-acceptor substrate by reduced Trx or Trx with a reducing system; as an alternative process, stable polysulfurated or persulfurated Cys247 as a reaction intermediate is reduced by Trx with a reducing system to release hydrogen sulfide and polysulfides. (iii) Possible sulfur oxide production: sulfur oxides (SO, SO2 and SO3 ) can be produced in the redox cycle of sulfane sulfur formed at the catalytic site Cys247 (Cys-SO- , Cys-SO2- and Cys-SO3- ) as reaction intermediates and released by reduced Trx or Trx with a reducing system. (iv) Possible anxiolytic-like effects: MPST-knockout mice exhibited anxiolytic-like effects.

Mineralocorticoid receptor (MR)/NADPH oxidase (NOX) signaling is involved in the development of obesity, insulin resistance, and renal diseases; however, the role of this signaling on steatotic preneoplastic liver lesions is not fully elucidated. We determined the effects of the MR antagonist potassium canrenoate (PC) on MR/NOX signaling in hepatic steatosis and preneoplastic glutathione S-transferase placental form (GST-P)-positive liver foci. Rats were subjected to a two-stage hepatocarcinogenesis model and fed with basal diet or high fat diet (HFD) that was co-administered with PC alone or in combination with the antioxidant alpha-glycosyl isoquercitrin (AGIQ). PC reduced obesity and renal changes (basophilic tubules that expressed MR and p22phox) but did not affect blood glucose tolerance and non-alcoholic fatty liver disease activity score (NAS) in HFD-fed rats. However, the drug increased the area of GST-P-positive liver foci that expressed MR and p22phox as well as increased expression of NOX genes (p22phox, Poldip2, and NOX4). PC in combination with AGIQ had the potential of inhibiting the effects of PC on the area of GST-P-positive liver foci and the effects were associated with increasing expression of an anti-oxidative enzyme (Catalase). The results suggested that MR/NOX signaling might be involved in development of preneoplastic liver foci and renal basophilic changes in HFD-fed rats; however, the impacts of PC were different in each organ.

Development of conductive nanocomposites is critical to realize flexible electronics on diverse substrates. Tuning the electrical and mechanical characteristics of these nanocomposites can enable a spectrum of applications. Herein, we demonstrate a robust microfabrication protocol to implement electrically conductive polymer by doping polydimethylsiloxane (PDMS) with silver fibers, with potential applications in wearable devices, MEMS, and microfluidics. The conductive polymer was made by mixing silver fibers into uncured PDMS matrix. The prepared mixture was micromolded by three dimension (3D) printed master using soft lithography technique. Serpentine and spiral like microstructures were embedded on the nonconductive layer of PDMS. The SEM images show uniform dispersion of silver fibers in the composites. The percolation threshold for the prepared composite was found to be 25 wt%. Resistivity of the conductive elastomeric composite at 25 wt% was 0.013 Ω cm.

Pulmonary fibrosis is a progressive disease with poor prognosis and limited therapeutic options. In this study, we evaluated the potential therapeutic effects of CG223, a novel inhibitor of bromodomain and extra-terminal motif (BET) proteins, on pulmonary fibrosis by focusing on the transforming growth factor-β1 (TGF-β1) pathway. In a murine model of bleomycin-induced pulmonary fibrosis, CG223 attenuated fibrosis while reducing the infiltration of inflammatory cells into the lungs. Fibroblasts expressing BRD4, a member of the BET protein family, were enriched in the tissue regions corresponding to bleomycin-induced fibrotic lesions. Additionally, pulmonary fibroblasts isolated from bleomycin-instilled mice showed a significantly increased association of BRD4 with the promoters of two pro-fibrotic genes linked to the entry into the TGF-β1 autocrine/paracrine loop, thrombospondin 1 (Thbs1) and integrin β3 (Itgb3), as well as with the promoter of a myofibroblast marker gene, actin alpha 2 (Acta2). Subsequent in vitro studies with murine primary lung fibroblasts showed that the mRNA induction of Thbs1, Itgb3, and Acta2 by TGF-β1 can be inhibited by CG223 in a dose-dependent manner. Taken together, CG223-induced BRD4 inhibition suppressed lung fibrogenesis by affecting multiple genes, including those involved in the triggering of the TGF-β1 autocrine/paracrine loop.

AimsTo investigate morphological changes in type VI collagen in the human trabecular meshwork associated with the rho kinase inhibitor ripasudil.MethodsThis cross-sectional study evaluated the effects of ripasudil eye drop administration (RA) or no ripasudil eye drop administration (NRA) in patients with primary open-angle glaucoma (POAG; age range 60–80 years) who underwent conventional outflow reconstruction between December 2015 and September 2016 at Tokai University Hachioji Hospital. The juxtacanalicular tissue was removed and imaged using transmission electron microscopy. Type VI collagen comprises cross-banded aggregates with transverse bands 30 nm apart repeating every 105 nm. The transverse bands are called the outer rod-like region (ORR) and the intervals are called the inner rod-like region (IRR). The waveform intensity in the type VI collagen was analysed in electron micrographs using Fourier transformation to detect the IRR and ORR borders.ResultsTen eyes of 10 patients were included (n=5/group). The baseline characteristics did not differ significantly between groups. ORR width was significantly smaller in the RA group (37.85±3.43 nm) than in the NRA group (50.62±5.23 nm, pConclusionsRipasudil administration in patients with POAG induced morphological changes in type VI collagen. Patients with POAG administered RA had a significantly smaller ORR width and a significantly greater IRR width than patients with POAG not administered RA.