Discovery of Novel Bicyclic Pyrazoles as Potent PIP5K1C Inhibitors.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is generated by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) from phosphatidylinositol 4-phosphate (PI4P). Structurally diverse and selective inhibitors against PIP5Ks are required to further elucidate the therapeutic potential for PIP5K inhibition, although the effects of PIP5K inhibition on various diseases and their symptoms, such as cancer and chronic pain, have been reported. Our medicinal chemistry efforts led to novel and potent PIP5K1C inhibitors. Compounds 30 and 33 not only showed potent activity but also demonstrated low total clearance in mice and high levels of kinase selectivity. These compounds might serve as tools to further elucidate the complex biology and therapeutic potential of PIP5K inhibition.

8-Prenyl daidzein and 8-prenyl genistein from germinated soybean modulate inflammatory response in activated macrophages.

Soy isoflavones have been shown to have anti-inflammatory properties; however, the anti-inflammatory effects of isoflavone metabolites produced during soybean germination remain unclear. We found that the daidzein and genistein derivatives, 8-prenyl daidzein (8-PD) and 8-prenyl genistein (8-PG), demonstrated a more potent effect than daidzein and genistein on repressing inflammatory responses in macrophages. Although IkB protein levels were unaltered, 8-PD and 8-PG repressed nuclear factor kappa B (NF-κB) activation, which was associated with reduced ERK1/2, JNK, and p38 MAPK activation and suppressed mitogen- and stress-activated kinase 1 phosphorylation. Inflammatory responses induced by the medium containing hypertrophic adipocyte secretions were successfully suppressed by 8-PD and 8-PG treatment. In the ex vivo study, 8-PD and 8-PG significantly inhibited proinflammatory C-C motif chemokine ligand 2 (CCL2) secretion from the adipose tissues of mice fed a long-term high-fat diet. The data suggest that 8-PD and 8-PG could regulate macrophage activation under obesity conditions.

Upregulation of Piezo2 in the mesangial, renin, and perivascular mesenchymal cells of the kidney of Dahl salt-sensitive hypertensive rats and its reversal by esaxerenone.

The recent discovery of mechanosensitive ion channels has promoted mechanobiological research in the field of hypertension and nephrology. We previously reported Piezo2 expression in mouse mesangial and juxtaglomerular renin-producing cells, and its modulation by dehydration. This study aimed to investigate how Piezo2 expression is altered in hypertensive nephropathy. The effects of the nonsteroidal mineralocorticoid receptor blocker, esaxerenone, were also analyzed. Four-week-old Dahl salt-sensitive rats were randomly assigned to three groups: rats fed a 0.3% NaCl diet (DSN), rats fed a high 8% NaCl diet (DSH), and rats fed a high salt diet supplemented with esaxerenone (DSH + E). After six weeks, DSH rats developed hypertension, albuminuria, glomerular and vascular injuries, and perivascular fibrosis. Esaxerenone effectively decreased blood pressure and ameliorated renal damage. In DSN rats, Piezo2 was expressed in Pdgfrb-positive mesangial and Ren1-positive cells. Piezo2 expression in these cells was enhanced in DSH rats. Moreover, Piezo2-positive cells accumulated in the adventitial layer of intrarenal small arteries and arterioles in DSH rats. These cells were positive for Pdgfrb, Col1a1, and Col3a1, but negative for Acta2 (αSMA), indicating that they were perivascular mesenchymal cells different from myofibroblasts. Piezo2 upregulation was reversed by esaxerenone treatment. Furthermore, Piezo2 inhibition by siRNA in the cultured mesangial cells resulted in upregulation of Tgfb1 expression. Cyclic stretch also upregulated Tgfb1 in both transfections of control siRNA and Piezo2 siRNA. Our findings suggest that Piezo2 may have a contributory role in modulating the pathogenesis of hypertensive nephrosclerosis and have also highlighted the therapeutic effects of esaxerenone on salt-induced hypertensive nephropathy. Mechanochannel Piezo2 is known to be expressed in the mouse mesangial cells and juxtaglomerular renin-producing cells, and this was confirmed in normotensive Dahl-S rats. In salt-induced hypertensive Dahl-S rats, Piezo2 upregulation was observed in the mesangial cells, renin cells, and notably, perivascular mesenchymal cells, suggesting its involvement in kidney fibrosis.

Delayed tension gastrothorax caused necrosis of stomach and re-expansion pulmonary edema: a case report.

BACKGROUND: Traumatic tension gastrothorax is a rare and potentially fatal condition occurring in patients with congenital or acquired diaphragmatic defects. Traumatic tension gastrothorax leads to acute and severe respiratory distress. Delayed tension gastrothorax that develops late during injury can be more severe. CASE PRESENTATION: An 84-year-old woman was brought to our facility with cardiac arrest and returned to spontaneous circulation after 2 min of cardiopulmonary resuscitation. Computed tomography showed diaphragmatic injury and tension gastrothorax due to trauma because of a fall episode few days earlier. Emergency thoracotomy and laparotomy was performed, because nasogastric tube insertion failed. There was a partially necrotic stomach in the chest cavity. The stomach was retracted from the thoracic cavity into the abdominal cavity and placed in its proper position. There was a 5 cm tear of the diaphragm. The tear was sutured and closed and then the necrotic area of the stomach was resected. Although the surgery relieved the intrathoracic compression, it resulted in re-expansion pulmonary edema immediately after surgery and hypoxemia. The patient was unable to overcome the hypoxemic state and eventually died. CONCLUSIONS: Delayed tension gastrothorax can lead not only to obstructive shock due to intrathoracic compression but also to more severe organ ischemia and re-expansion pulmonary edema due to insufflation.

Piezo2 expression and its alteration by mechanical forces in mouse mesangial cells and renin-producing cells.

The kidney plays a central role in body fluid homeostasis. Cells in the glomeruli and juxtaglomerular apparatus sense mechanical forces and modulate glomerular filtration and renin release. However, details of mechanosensory systems in these cells are unclear. Piezo2 is a recently identified mechanically activated ion channel found in various tissues, especially sensory neurons. Herein, we examined Piezo2 expression and regulation in mouse kidneys. RNAscope in situ hybridization revealed that Piezo2 expression was highly localized in mesangial cells and juxtaglomerular renin-producing cells. Immunofluorescence assays detected GFP signals in mesangial cells and juxtaglomerular renin-producing cells of Piezo2GFP reporter mice. Piezo2 transcripts were observed in the Foxd1-positive stromal progenitor cells of the metanephric mesenchyme in the developing mouse kidney, which are precursors of mesangial cells and renin-producing cells. In a mouse model of dehydration, Piezo2 expression was downregulated in mesangial cells and upregulated in juxtaglomerular renin-producing cells, along with the overproduction of renin and enlargement of the area of renin-producing cells. Furthermore, the expression of the renin coding gene Ren1 was reduced by Piezo2 knockdown in cultured juxtaglomerular As4.1 cells under static and stretched conditions. These data suggest pivotal roles for Piezo2 in the regulation of glomerular filtration and body fluid balance.

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly.

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in RPL5 Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.

Automated evaluation of retinal pigment epithelium disease area in eyes with age-related macular degeneration.

The retinal pigment epithelium (RPE) is essential for the survival and function of retinal photoreceptor cells. RPE dysfunction causes various retinal diseases including age-related macular degeneration (AMD). Clinical studies on ES/iPS cell-derived RPE transplantation for RPE dysfunction-triggered diseases are currently underway. Quantification of the diseased RPE area is important to evaluate disease progression or the therapeutic effect of RPE transplantation. However, there are no standard protocols. To address this issue, we developed a 2-step software that enables objective and efficient quantification of RPE-disease area changes by analyzing the early-phase hyperfluorescent area in fluorescein angiography (FA) images. We extracted the Abnormal region. This extraction was based on deep learning-based discrimination. We scored the binarized extracted area using an automated program. Our program's performance for the same eye from the serial image captures was within 3.1 ± 7.8% error. In progressive AMD, the trend was consistent with human assessment, even when FA images from two different visits were compared. This method was applicable to quantifying RPE-disease area changes over time, evaluating iPSC-RPE transplantation images, and a disease other than AMD. Our program may contribute to the assessment of the clinical course of RPE-disease areas in routine clinics and reduce the workload of researchers.

Different cell imaging methods did not significantly improve immune cell image classification performance.

Developments in high-throughput microscopy have made it possible to collect huge amounts of cell image data that are difficult to analyse manually. Machine learning (e.g., deep learning) is often employed to automate the extraction of information from these data, such as cell counting, cell type classification and image segmentation. However, the effects of different imaging methods on the accuracy of image processing have not been examined systematically. We studied the effects of different imaging methods on the performance of machine learning-based cell type classifiers. We observed lymphoid-primed multipotential progenitor (LMPP) and pro-B cells using three imaging methods: differential interference contrast (DIC), phase contrast (Ph) and bright-field (BF). We examined the classification performance of convolutional neural networks (CNNs) with each of them and their combinations. CNNs achieved an area under the receiver operating characteristic (ROC) curve (AUC) of ~0.9, which was significantly better than when the classifier used only cell size or cell contour shape as input. However, no significant differences were found between imaging methods and focal positions.

Tissue Distribution of Orally Administered Prenylated Isoflavones, Glyceollins, in Sprague-Dawley Rats.

Apart from the physiological effects of glyceollins, information regarding their tissue distribution is scarce in the literature. Thus, the aim of this study is to clarify the distribution of glyceollins in rat organs. Glyceollins I and III were orally administered to Sprague-Dawley rats (1.0 mg/kg) with daidzein as control, and their accumulations in organs were investigated by liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS). Glyceollins accumulated in intact and conjugated forms in circulatory organs with a Tmax of 0.5 h, in the following order of descending preference: liver, kidney, heart, lung, soleus muscle, and abdominal aorta. The accumulation of hydrophobic glyceollin I was more than 1.5 times higher than that of III. In contrast, daidzein and hydroxy equol were detected only in the liver and kidneys at lower concentrations (1/100 times) than those of glyceollins. In conclusion, prenylated isoflavones, glyceollins, were preferentially distributed in circulatory organs as intact, sulfated, or glucuronidated forms up to 6 h after the intake.

Metabolome analysis revealed that soybean-Aspergillus oryzae interaction induced dynamic metabolic and daidzein prenylation changes.

Several isoflavonoids are well known for their ability to act as soybean phytoalexins. However, the overall effects of the soybean-Aspergillus oryzae interaction on metabolism remain largely unknown. The aim of this study is to reveal an overview of nutritive and metabolic changes in germinated and A. oryzae-elicited soybeans. The levels of individual nutrients were measured using the ustulation, ashing, Kjeldahl, and Folch methods. The levels of individual amino acids were measured using high-performance liquid chromatography. Low-molecular-weight compounds were measured through metabolome analysis using liquid chromatography-mass spectrometry. Although the levels of individual nutrients and amino acids were strongly influenced by the germination process, the elicitation process had little effect on the change in the contents of individual nutrients and amino acids. However, after analyzing approximately 700 metabolites using metabolome analysis, we found that the levels of many of the metabolites were strongly influenced by soybean-A. oryzae interactions. In particular, the data indicate that steroid, terpenoid, phenylpropanoid, flavonoid, and fatty acid metabolism were influenced by the elicitation process. Furthermore, we demonstrated that not the germination process but the elicitation process induced daidzein prenylation, suggesting that the soybean-A. oryzae interactions produce various phytoalexins that are valuable for health promotion and/or disease prevention.

Visualization Analysis of Glyceollin Production in Germinating Soybeans by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Imaging Technique.

Apart from the physiological functions of soybean phytoalexins, the production sites in soybeans remain unknown. In this study, the dynamic production of phytoalexins, glyceollins, in germinating soybeans inoculated with Aspergillus oryzae was visually investigated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging. During a 3-day sensitization using a fungus, glyceollins I-III were produced in germinating soybeans (from 0.03 mg/g for glyceollin III to 0.96 mg/g for glyceollin I). Imaging analysis provided visual evidence that glyceollins were produced only in the regions of seed coat and germinated root of the soybeans, while no production was observed in other regions, including the cotyledons. In contrast, their precursor, isoflavone, was distributed throughout the soybean. The evidence that the inoculation of the inactivated fungi also caused glyceollin production at the seed coat led us to speculate that glyceollins could be produced in the region of soybean attached to the fungus body.

A Variable Scheduling Maintenance Culture Platform for Mammalian Cells.

Cell culturing is a basic experimental technique in cell biology and medical science. However, culturing high-quality cells with a high degree of reproducibility relies heavily on expert skills and tacit knowledge, and it is not straightforward to scale the production process due to the education bottleneck. Although many automated culture systems have been developed and a few have succeeded in mass production environments, very few robots are permissive of frequent protocol changes, which are often required in basic research environments. LabDroid is a general-purpose humanoid robot with two arms that performs experiments using the same tools as humans. Combining our newly developed AI software with LabDroid, we developed a variable scheduling system that continuously produces subcultures of cell lines without human intervention. The system periodically observes the cells on plates with a microscope, predicts the cell growth curve by processing cell images, and decides the best times for passage. We have succeeded in developing a system that maintains the cultures of two HEK293A cell plates with no human intervention for 192 h.

Identification of a Unique Type of Isoflavone O-Methyltransferase, GmIOMT1, Based on Multi-Omics Analysis of Soybean under Biotic Stress.

Isoflavonoids are commonly found in leguminous plants. Glycitein is one of the isoflavones produced by soybean. The genes encoding the enzymes in the isoflavone biosynthetic pathway have mostly been identified and characterized. However, the gene(s) for isoflavone O-methyltransferase (IOMT), which catalyzes the last step of glycitein biosynthesis, has not yet been identified. In this study, we conducted multi-omics analyses of fungal-inoculated soybean and indicated that glycitein biosynthesis was induced in response to biotic stress. Moreover, we identified a unique type of IOMT, which participates in glycitein biosynthesis. Soybean seedlings were inoculated with Aspergillus oryzae or Rhizopus oligosporus and sampled daily for 8 d. Multi-omics analyses were conducted using liquid chromatography-tandem mass spectrometry and RNA sequencing. Metabolome analysis revealed that glycitein derivatives increased following fungal inoculation. Transcriptome co-expression analysis identified two candidate IOMTs that were co-expressed with the gene encoding flavonoid 6-hydroxylase (F6H), the key enzyme in glycitein biosynthesis. The enzymatic assay of the two IOMTs using respective recombinant proteins showed that one IOMT, named as GmIOMT1, produced glycitein. Unlike other IOMTs, GmIOMT1 belongs to the cation-dependent OMT family and exhibited the highest activity with Zn2+ among cations tested. Moreover, we demonstrated that GmIOMT1 overexpression increased the levels of glycitein derivatives in soybean hairy roots when F6H was co-expressed. These results strongly suggest that GmIOMT1 participates in inducing glycitein biosynthesis in response to biotic stress.

Intestinal Absorption of Prenylated Isoflavones, Glyceollins, in Sprague-Dawley Rats.

Although prenylated isoflavones or glyceollins elicit physiological effects more potent than those by isoflavones, the bioavailability remains unclear. The present study aimed to clarify the intestinal absorption behavior of glyceollins in Sprague-Dawley rats. Upon oral administration of 1.0 mg/kg glyceollin I or III (daidzein as comparative compound) to the rats, no peaks corresponding to the intact forms of the compounds were detected in plasma by liquid chromatography-time-of-flight/mass spectrometry (LC-TOF/MS) analysis. In contrast, enzymatic deconjugation of plasma resulted in successful MS detection of each glyceollin; glyceollin I absorption was >10 times higher than that of daidzein, given its high log P value. The present study demonstrated for the first time that glyceollins were more absorbable than mother isoflavones due to their high hydrophobicity, and they metabolized to form sulfated, glucuronized, and methylated conjugates during the intestinal absorption process.

Persistent Vegetative State after Diabetic Ketoacidosis Triggered by an Overdose of Psychotropic Agents.

A 28-year-old man with type 1 diabetes mellitus was admitted for shock and coma due to diabetic ketoacidosis. Despite aggressive treatment and management of the patient's underlying clinical issues, the patient remained in a comatose state. Further investigations revealed an excess consumption of psychotropic agents; however, there was no evidence of an insulin overdose. Physicians should be aware that, in patients who are highly dependent upon insulin, an overdose of psychotropic agents can lead to hypoxic-ischemic brain injury.

Endocrine therapy-resistant breast cancer model cells are inhibited by soybean glyceollin I through Eleanor non-coding RNA.

Long-term estrogen deprivation (LTED) of an estrogen receptor (ER) α-positive breast cancer cell line recapitulates cancer cells that have acquired estrogen-independent cell proliferation and endocrine therapy resistance. Previously, we have shown that a cluster of non-coding RNAs, Eleanors (ESR1 locus enhancing and activating non-coding RNAs) formed RNA cloud and upregulated the ESR1 gene in the nuclei of LTED cells. Eleanors were inhibited by resveratrol through ER. Here we prepared another polyphenol, glyceollin I from stressed soybeans, and identified it as a major inhibitor of the Eleanor RNA cloud and ESR1 mRNA transcription. The inhibition was independent of ER, unlike one by resveratrol. This was consistent with a distinct tertiary structure of glyceollin I for ER binding. Glyceollin I preferentially inhibited the growth of LTED cells and induced apoptosis. Our results suggest that glyceollin I has a novel role in LTED cell inhibition through Eleanors. In other words, LTED cells or endocrine therapy-resistant breast cancer cells may be ready for apoptosis, which can be triggered with polyphenols both in ER-dependent and ER-independent manners.

Total Synthesis of the 7,10-Epimer of the Proposed Structure of Amphidinolide N, Part I: Synthesis of the C1-C13 Subunit.

Amphidinolide N, the structure of which has been recently revised, is a 26-membered macrolide featuring allyl epoxide and tetrahydropyran moieties with 13 chiral centers. Due to its challenging structure and extraordinary potent cytotoxicity, amphidinolide N is a highly attractive target of total synthesis. During our total synthesis studies of the 7,10-epimer of the proposed structure of amphidinolide N, we have synthesized the C1-C13 subunit enantio- and diastereoselectively. Key reactions include an l-proline catalyzed enantioselective intramolecular aldol reaction, Evans aldol reaction, Sharpless asymmetric epoxidation and Tamao-Fleming oxidation. To aid late-stage manipulations, we also developed the 4-(N-benzyloxycarbonyl-N-methylamino)butyryl group as a novel ester protective group for the C9 alcohol.

Total Synthesis of the 7,10-Epimer of the Proposed Structure of Amphidinolide N, Part II: Synthesis of C17-C29 Subunit and Completion of the Synthesis.

The total synthesis of 7,10-epimer of the proposed structure of amphidinolide N was accomplished. The requisite chiral C17-C29 subunit was assembled stereoselectively via Keck allylation, Shi epoxidation, diastereoselective 1,3-reduction, and a later oxidative synthesis of the THF framework. The C1-C13 and C17-C29 subunits were successfully coupled using a Enders RAMP "linchpin" as the C14-C16 three carbon unit, thereby controlling the chirality at C14 and C16. The labile allyl epoxy moiety was successfully constructed by Grieco-Nishizawa olefination at a final stage of the synthesis.

Phosphodiesterase inhibitors. Part 6: design, synthesis, and structure-activity relationships of PDE4-inhibitory pyrazolo[1,5-a]pyridines with anti-inflammatory activity.

We previously identified KCA-1490 [(-)-6-(7-methoxy-2-trifluoromethyl-pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone], a dual PDE3/4 inhibitor. In the present study, we found highly potent selective PDE4 inhibitors derived from the structure of KCA-1490. Among them, N-(3,5-dichloropyridin-4-yl)-7-methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridine-4-carboxamide (2a) had good anti-inflammatory effects in an animal model.

Phosphodiesterase inhibitors. Part 5: hybrid PDE3/4 inhibitors as dual bronchorelaxant/anti-inflammatory agents for inhaled administration.

(-)-6-(7-Methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (KCA-1490) exhibits moderate dual PDE3/4-inhibitory activity and promises as a combined bronchodilatory/anti-inflammatory agent. N-alkylation of the pyridazinone ring markedly enhances potency against PDE4 but suppresses PDE3 inhibition. Addition of a 6-aryl-4,5-dihydropyridazin-3(2H)-one extension to the N-alkyl group facilitates both enhancement of PDE4-inhibitory activity and restoration of potent PDE3 inhibition. Both dihydropyridazinone rings, in the core and extension, can be replaced by achiral 4,4-dimethylpyrazolone subunits and the core pyrazolopyridine by isosteric bicyclic heteroaromatics. In combination, these modifications afford potent dual PDE3/4 inhibitors that suppress histamine-induced bronchoconstriction in vivo and exhibit promising anti-inflammatory activity via intratracheal administration.