Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Structure-Activity Relationship and In Silico Evaluation of cis- and trans-PCPA-Derived Inhibitors of LSD1 and LSD2.

trans-2-Phenylcycloproylamine (trans-PCPA) has been used as the scaffold to develop covalent-binding inhibitors against lysine-specific demethylase 1 (LSD1/KDM1A), a therapeutic target for several cancers. However, the effects of different structural moieties on the inhibitory activity, selectivity, and reactivity of these derivatives, including the cis isomers, against LSD1 and its paralogue LSD2/KDM1B are not fully understood. Here we synthesized 65 cis- and trans-PCPA derivatives and evaluated their inhibitory activity against LSD1 and LSD2. One of the derivatives, 7c (cis-4-Br-2,5-F2-PCPA; S1024), inhibited LSD1 and LSD2 with K i values of 0.094 µM and 8.4 µM, respectively, and increased the level of dimethylated histone H3 at K4 in CCRF-CEM cells. A machine learning-based regression model (Q 2 = 0.61) to predict LSD1-inhibitory activity was also constructed and showed a good prediction accuracy (R 2 = 0.81) for 12 test-set compounds, including 7c. The present methodology would be useful when designing covalent-binding inhibitors for other enzymes.

Isolation of Caldorazole, a Thiazole-Containing Polyketide with Selective Cytotoxicity under Glucose-Restricted Conditions.

Caldorazole (1) was isolated from the marine cyanobacterium Caldora sp. collected on Ishigaki Island, Okinawa, Japan. Its structure was determined to be a new polyketide that contained two thiazole rings and an O-methylenolpyruvamide moiety. Caldorazole (1) showed strong cytotoxicity toward tumor cells that had been seeded at a high density. Cell death induced by 1 in HeLa and A431 cells was also observed only in the presence of the glycolysis blocker 2-deoxy-d-glucose (2DG). Co-treatment with 1 and 2DG remarkably decreased ATP levels in these cells. Furthermore, 1 selectively inhibited complex I in the mitochondrial respiratory chain. Thus, 1 was demonstrated to exert cytotoxicity toward human tumor cells by blocking mitochondrial respiration.

Inhibition of Lipopolysaccharide-Induced Inflammatory Signaling by Soft Coral-Derived Prostaglandin A2 in RAW264.7 Cells.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria and causes inflammatory diseases. We searched MeOH extracts of collected marine organisms for inhibitors of LPS-induced nitric oxide (NO) production in RAW264.7 cells and identified prostaglandin A2 (PGA2) as an active compound from the MeOH extract of the soft coral Lobophytum sp. PGA2 inhibited the production of NO and reduced the expression of inducible NO synthase (iNOS) in LPS-stimulated RAW264.7 cells. Although short preincubation with PGA2 did not inhibit LPS-induced degradation and resynthesis of IκBα, the suppressive effect of PGA2 was observed only after a prolonged incubation period prior to LPS treatment. In addition, PGA2-inhibited NO production was negated by the addition of the EP4 antagonist L161982. Thus, PGA2 was identified as an inhibitor of LPS-induced inflammatory signaling in RAW264.7 cells.

Antiapoptotic activity of methyl (3R,4E,6Z,15E)-3-hydroxyoctadecatrienoate in human cervical carcinoma HeLa cells.

Inhibitors of thapsigargin-induced cell death in human cervical carcinoma HeLa cells were screened among the metabolites of marine organisms. The MeOH extract of the cyanobacterium Rivularia sp. was found to exhibit inhibitory activity. Column chromatography purification was used to isolate methyl (3R,4E,6Z,15E)-3-hydroxyoctadecatrienoate (MHO) as the active compound. MHO was determined to inhibit apoptotic stimuli-induced cell death in HeLa cells.

Inhibitory effects of biseokeaniamide A against lipopolysaccharide-induced signal transduction.

Lipopolysaccharides (LPS) are associated with various inflammatory diseases; therefore, the inhibition of LPS-induced nitric oxide (NO) production may have extensive therapeutic applications. We searched for inhibitors of NO production in the LPS-stimulated murine macrophage-like cell line RAW264.7 from MeOH extracts of marine organisms. The MeOH extract of the marine cyanobacterium Okeania sp., collected in Okinawa, Japan, showed inhibitory activity. Biseokeaniamide A was isolated from the MeOH extract by chromatographic separation. Biseokeaniamide A inhibited NO production without cytotoxicity. It reduced inducible nitric oxide synthase levels and suppressed the expression of IL-1ß in LPS-stimulated RAW264.7 cells. Biseokeaniamide A did not inhibit IκBα degradation but inhibited IκBα expression. Thus, biseokeaniamide A, a naturally occurring lipopeptide, was identified as a selective inhibitor of LPS signal transduction.

Hypereosinophilia and Multisite Vasculopathy of Fibromuscular Dysplasia: A Case Report.

The impact of blood disorders on fibromuscular dysplasia is unknown, and cardiovascular surgery results are also unclear. Furthermore, there are only a few case reports about the association between fibromuscular dysplasia and blood disorders. We report a case of a coronary bypass surgery and an aortic root replacement for a patient who is hypereosinophilic with multisite vasculopathy of fibromuscular dysplasia, including that of the coronary artery and saphenous vein, which was diagnosed by a histopathologic examination after an autopsy was performed 5 months after surgery. The outcome of cardiovascular surgery can be unfavorable for fibromuscular dysplasia. Blood disorders may also have an impact on the outcome.

[Adult Cor Triatriatum Found by Chance at the Onset of Myocardial Infarction].

Cor triatriatum sinister is a rare congenital heart disease in which the left atrium is divided into 2 chambers by a membrane, causing resistance to the blood flow to the left ventricle. The onset of symptoms depends upon the effective size of the orifice in the membrane and the associated heart disease. Our case is a 67-year-old woman with cor triatriatum found by chance at the onset of myocardial infarction. Coronary artery bypass surgery and excision of the membrane were successfully performed. Preoperative left ventricular dysfunction was improved after surgery. Imaging examinations such as enhanced computed tomography(CT) and magnetic resonance imaging(MRI) were useful for selecting surgical approach.

PFG acted as an inducer of premature senescence in TIG-1 normal diploid fibroblast and an inhibitor of mitosis in the HeLa cells.

Our previous work has reported an anti-proliferative compound from moutan cortex, paeoniflorigenone which can induce cancer-selective apoptosis. However, its anti-proliferative mechanism is still unknown. According to morphology changes (hypertrophy and flattening), we hypothesized that PFG can induce senescence or inhibit cell mitosis. Here we show that PFG can induce cellular senescence, evidenced by the expression of senescence-associated ß-galactosidase, G0/G1 cell cycle arrest and permanent loss of proliferative ability, in normal TIG-1 diploid fibroblast but not cancerous HeLa cells. In cancerous HeLa cells, PFG inhibited proliferation by inducing S and G2/M cell cycle arrest and mitosis inhibition. DNA damage response was activated by PFG, interestingly the reactive oxygen species level was suppressed instead of escalated. To sum up, we report 3 new roles of PFG as, 1. inducer of premature senescence in normal TIG-1 cells, 2. inhibitor of mitosis in cancerous HeLa cells, 3. ROS scavenger. Abbreviations: PFG: Paeoniflorigenone; ROS: reactive oxygen species; ATM: ataxia telangiectasia mutated; t-BHP: tert-butyl hydroperoxide; SA-ß-gal: senescence-associatedß-galactosidase; DNA-PKcs: DNA-dependent protein kinase; γ-H2AX: H2AX phosphoryla-tion at Ser-139.

Unified Total Synthesis, Stereostructural Elucidation, and Biological Evaluation of Sarcophytonolides.

Sarcophytonolides are cembranolide diterpenes isolated from the soft corals of genus Sarcophyton. Unified total synthesis of sarcophytonolides C, E, F, G, H, and J and isosarcophytonolide D was achieved. The synthetic routes feature NaHMDS- or SmI2-mediated fragment coupling, alkoxycarbonylallylation, macrolactonization, and transannular ring-closing metathesis. These total syntheses led to the absolute configurational confirmation of sarcophytonolide H, elucidation of sarcophytonolides C, E, F, and G, and revision of sarcophytonolide J and isosarcophytonolide D. We also evaluated the antifouling activity and toxicity of the synthetic sarcophytonolides H and J and their analogues as well as the cytotoxicity of the synthetic sarcophytonolides and the key synthetic intermediates.

Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production without inhibiting indoleamine 2,3-dioxygenase activity.

Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-dioxygenase (IDO). The abrogation of kynurenine production is considered a promising therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-Cl-benzimidazole derivative 2b-6, which inhibited both recombinant human IDO (rhIDO) activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent inhibitor of cellular kynurenine production 2i (IC50 = 0.34 µM), which unexpectedly exerted little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1 expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be a promising starting point for a novel approach to immunological cancer treatment.

Direct oral anticoagulant therapy as an alternative to surgery for the treatment of a patient with a floating thrombus in the ascending aorta and pulmonary embolism.

A floating thrombus in the ascending aorta was incidentally discovered in a patient with a descending thoracic aortic aneurysm and a history of alcoholism. The patient developed deep venous thrombosis and pulmonary embolism. However, he refused to undergo surgical excision of the thrombus in the ascending aorta. Therefore, treatment with rivaroxaban was administered for 3 months, and it completely dissolved the thrombus. Anticoagulant therapy may be an alternative treatment when surgery cannot be performed.

[Effective Anticoagulation Therapy Prior to Surgical Excision of an Aortic Valve Papillary Fibroelastoma Diagnosed after a Transient Cerebral Ischemic Attack;Report of a Case].

Cardiac papillary fibroelastoma is reported to be the 2nd most common cardiac tumor following myxoma. Owing to the risk of embolism, early surgical excision is the treatment of choice. We report a case of effective anticoagulation therapy prior to surgical excision of an aortic valve papillary fibroelastoma. A 78-year-old man was admitted to our hospital because of transient cerebral ischemic attack. The symptom was relieved in a short period. Echocardiography revealed a tumor at the aortic valve. Cardiac computed tomography revealed a sea-anemone-like appearance of the tumor. Cardiac papillary fibroelastoma was suspected on close examination. The operation was postponed because of gingivitis that required draining. During 3 months awaiting the operation, he continued receiving anticoagulation therapy, which successfully prevented thromboembolism. Administration of anticoagulation therapy may be considered, unless early surgical excision can be performed.

Apoptosis-inducing activity and antiproliferative effect of Paeoniflorigenone from moutan cortex.

Ninety samples from the extracts of plants from traditional Chinese medicines were screened for antitumor activity. Paeoniflorigenone (PFG) was isolated as an active ingredient from the root of moutan cortex, which showed the strongest activity. In addition, our data indicated that PFG was cytotoxic and induced apoptosis selectively in the cancer cell lines. These effects were cancelled by the addition of caspase inhibitor Z-VAD-FMK, suggesting that it was mediated by caspase-3 activation.

Kanamienamide, an Enamide with an Enol Ether from the Marine Cyanobacterium Moorea bouillonii.

Kanamienamide, an enamide with an enol ether, was isolated from the marine cyanobacterium Moorea bouillonii. The gross structure was established by spectroscopic analyses, and the relative stereochemistry was elucidated on the basis of the analyses of NOESY correlations and 1H-1H coupling constants. The absolute configuration was determined on the basis of the chiral HPLC analysis of the N-Me-Leu derived from kanamienamide. This is the first report of a natural product that possesses an N-Me-enamide adjacent to an enol ether. Kanamienamide showed growth-inhibitory activity toward HeLa cells with an IC50 value of 2.5 µM and induced apoptosis-like cell death.

Janadolide, a Cyclic Polyketide-Peptide Hybrid Possessing a tert-Butyl Group from an Okeania sp. Marine Cyanobacterium.

Janadolide, a new cyclic polyketide-peptide hybrid possessing a tert-butyl group, was isolated from an Okeania sp. marine cyanobacterium. The gross structure was elucidated by spectroscopic analyses, and the absolute configurations of the amino acid moieties were determined by acid hydrolysis and chiral-phase HPLC analyses. The absolute configuration of the two stereogenic centers in the polyketide moiety was elucidated based on a combination of degradation reactions and spectroscopic analyses including the phenyl-glycine methyl ester method. Janadolide showed potent antitrypanosomal activity with an IC50 value of 47 nM without cytotoxicity against human cells at 10 µM.

Isolation and Structure of Kurahyne B and Total Synthesis of the Kurahynes.

Kurahyne B (2), a new analogue of kurahyne (1), was isolated from the marine cyanobacterium Okeania sp. Its gross structure was elucidated based on spectroscopic analyses, and the absolute configuration was established by total synthesis. Kurahyne B (2) inhibited the growth of both HeLa and HL60 cells, with IC50 values of 8.1 and 9.0 µM, respectively. The growth-inhibitory activity of kurahyne B was the same as kurahyne (1). In parallel, the first total synthesis of kurahyne (1) was also achieved.

Identification of a molecular target of kurahyne, an apoptosis-inducing lipopeptide from marine cyanobacterial assemblages.

In 2014, we isolated kurahyne, an acetylene-containing lipopeptide, from a marine cyanobacterial assemblage of Lyngbya sp. Kurahyne exhibited growth-inhibitory activity against human cancer cells, and induced apoptosis in HeLa cells. However, its mode of action is not yet clear. To elucidate its mode of action, we carried out several cell-based assays, and identified the intracellular target molecule of kurahyne as sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). In addition, we found that kurahyne inhibited the differentiation of macrophages into osteoclasts.

Synthesis and structure-activity relationships for cytotoxicity and apoptosis-inducing activity of (+)-halichonine B.

Halichonine B is a sesquiterpene alkaloid isolated from the marine sponge Halichondria okadai Kadota. Halichonine B has exhibited cytotoxicity against mammalian cancer cells and induced apoptosis in the human leukemia cell line HL60. Here we established a practical route for the synthesis of halichonine B and its analogues, and we evaluated their biological activities. It was revealed that the secondary amino groups in the side chain portion are important for the strong cytotoxicity of halichonine B and that the N(11)-prenyl group is unimportant. Halichonine B and its analogues were also observed to induce apoptosis in HL60 cells.