Predictor of the Postoperative Swelling After Craniotomy for Spontaneous Intracerebral Hemorrhage: Sphericity Index as a Novel Parameter.

Objective: Spontaneous intracerebral hemorrhage is a serious type of stroke with high mortality and disability rates. Surgical treatment options vary; however, predicting edema aggravation is crucial when choosing the optimal approach. We propose using the sphericity index, a measure of roundness, to predict the aggravation of edema and guide surgical decisions. Methods: We analyzed 56 cases of craniotomy and hematoma evacuation to investigate the correlation between the sphericity index and patient outcomes, including the need for salvage decompressive craniectomy (DC). Results: The patients included 35 (62.5%) men and 21 (37.5%) women, with a median age of 62.5 years. The basal ganglia was the most common location of hemorrhage (50.0%). The mean hematoma volume was 86.3 cc, with 10 (17.9%) instances of hematoma expansion. Cerebral herniation was observed in 44 (78.6%) patients, intraventricular hemorrhage in 34 (60.7%), and spot signs in 9 (16.1%). Salvage DC was performed in 13 (23.6%) patients to relieve intracranial pressure. The median follow-up duration was 6 months, with a mortality rate of 12.5%. The sphericity index was significantly correlated with delayed swelling and hematoma expansion but not salvage DC. Conclusions: The sphericity index is a promising predictor of delayed swelling and hematoma expansion that may aid in the development of surgical guidelines and medication strategies. Further large-scale studies are required to explore these aspects and establish comprehensive guidelines.

Comprehensive Lipid Profiling Recapitulates Enhanced Lipolysis and Fatty Acid Metabolism in Intimal Foamy Macrophages From Murine Atherosclerotic Aorta.

Lipid accumulation in macrophages is a prominent phenomenon observed in atherosclerosis. Previously, intimal foamy macrophages (FM) showed decreased inflammatory gene expression compared to intimal non-foamy macrophages (NFM). Since reprogramming of lipid metabolism in macrophages affects immunological functions, lipid profiling of intimal macrophages appears to be important for understanding the phenotypic changes of macrophages in atherosclerotic lesions. While lipidomic analysis has been performed in atherosclerotic aortic tissues and cultured macrophages, direct lipid profiling has not been performed in primary aortic macrophages from atherosclerotic aortas. We utilized nanoflow ultrahigh-performance liquid chromatography-tandem mass spectrometry to provide comprehensive lipid profiles of intimal non-foamy and foamy macrophages and adventitial macrophages from Ldlr-/- mouse aortas. We also analyzed the gene expression of each macrophage type related to lipid metabolism. FM showed increased levels of fatty acids, cholesterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylinositol, and sphingomyelin. However, phosphatidylethanolamine, phosphatidic acid, and ceramide levels were decreased in FM compared to those in NFM. Interestingly, FM showed decreased triacylglycerol (TG) levels. Expressions of lipolysis-related genes including Pnpla2 and Lpl were markedly increased but expressions of Lpin2 and Dgat1 related to TG synthesis were decreased in FM. Analysis of transcriptome and lipidome data revealed differences in the regulation of each lipid metabolic pathway in aortic macrophages. These comprehensive lipidomic data could clarify the phenotypes of macrophages in the atherosclerotic aorta.

"Fruiting Liquid" of Mushroom-Forming Fungi, A Novel Source of Bioactive Compounds - Fruiting-Body Inducer and HIF and Axl Inhibitors.

In general, mushroom-forming fungi secrete liquid on the surface of mycelia just before fruiting-body formation. However, no researchers in mushroom science have paid attention to the liquid until now. We formulated a hypothesis that the liquid plays an important role(s) in the formation of the fruiting body and produces various bioactive compounds and named it the "fruiting liquid (FL)". Four novel compounds (1-4) were isolated from FL of Hypholoma lateritium and Hericium erinaceus. The structures of 1-4 except for their stereochemistry were determined by interpretation of MS and NMR data. The absolute configurations of compounds 1-4 were determined by quantum chemical calculation of the ECD spectrum, by single-crystal X-ray diffraction analyses, or by chemical syntheses. Compounds 1, 3, and 4 induced fruiting body formation of Flammulina velutipes. Compound 4 inhibited the activity of hypoxia-inducible factor, and compounds 2-4 suppressed receptor tyrosine kinase (Axl) expression.

Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.

Roles of Vascular Smooth Muscle Cells in Atherosclerotic Calcification.

The accumulation of calcium in atherosclerotic plaques is a prominent feature of advanced atherosclerosis, and it has a strong positive correlation with the total burden of atherosclerosis. Atherosclerotic calcification usually appears first at the necrotic core, indicating that cell death and inflammatory processes are involved in calcification. During atherosclerotic inflammation, various cell types, such as vascular smooth muscle cells, nascent resident pericytes, circulating stem cells, or adventitial cells, have been assumed to differentiate into osteoblastic cells, which lead to vascular calcification. Among these cell types, vascular smooth muscle cells are considered a major contributor to osteochondrogenic cells in the atherosclerotic milieu. In this review, we summarize the molecular mechanisms underlying the osteochondrogenic switch of vascular smooth muscle cells in atherosclerotic plaques.

Role of hypoxanthine-guanine phosphoribosyltransferase in the metabolism of fairy chemicals in rice.

Fairy chemicals (FCs), 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are molecules with many diverse functions in plants. The defined biosynthetic pathway for FCs is a novel purine metabolism in which they are biosynthesized from 5-aminoimidazole-4-carboxamide. Here, we show that one of the purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), recognizes AHX and AOH as substrates. Two novel compounds, AOH ribonucleotide and its ribonucleoside which are the derivatives of AOH, were enzymatically synthesized. The structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and X-ray single-crystal diffraction analysis. This report demonstrates the function of HGPRT and the existence of novel purine metabolism associated with the biosynthesis of FCs in rice.

Bioactive Compounds from the Mushroom-Forming Fungus Chlorophyllum molybdites.

A novel compound (1) along with two known compounds (2 and 3) were isolated from the culture broth of Chlorophyllum molybdites, and three known compounds (4-6) were isolated from its fruiting bodies. The planar structure of 1 was determined by the interpretation of spectroscopic data. By comparing the specific rotation of the compound with that of the analog compound, the absolute configuration of 1 was determined to be R. This is the first time that compounds 2-4 were isolated from a mushroom-forming fungus. Compound 2 showed significant inhibition activity against Axl and immune checkpoints (PD-L1, PD-L2). In the bioassay to examine growth inhibitory activity against the phytopathogenic bacteria Peptobacterium carotovorum, Clavibacter michiganensis and Burkholderia glumae, compounds 2 and 3 inhibited the growth of P. carotovorum and C. michiganensis. In the bioassay to examine plant growth regulatory activity, compounds 1-4 showed a significant regulatory activity on lettuce growth.

The role of xanthine dioxygenase in the biosynthetic pathway of 2-aza-8-oxohypoxanthine of Lepista sordida.

2-Azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), discovered as causal substances of fairy rings are known to be endogenous in the fairy ring-forming Lepista sordida. In this study, we showed that xanthine dioxygenase, an a-ketoglutarate-dependent dioxygenase, might catalyze the conversion of AHX to AOH in the fungus. Furthermore, this enzyme is the first reported molybdopterin-independent protein of hypoxanthine metabolism.

Identification of Biosynthetic and Metabolic Genes of 2-Azahypoxanthine in Lepista sordida Based on Transcriptomic Analysis.

2-Azahypoxanthine was isolated from the fairy ring-forming fungus Lepista sordida as a fairy ring-inducing compound. 2-Azahypoxanthine has an unprecedented 1,2,3-triazine moiety, and its biosynthetic pathway is unknown. The biosynthetic genes for 2-azahypoxanthine formation in L. sordida were predicted by a differential gene expression analysis using MiSeq. The results revealed that several genes in the purine and histidine metabolic pathways and the arginine biosynthetic pathway are involved in the biosynthesis of 2-azahypoxanthine. Furthermore, nitric oxide (NO) was produced by recombinant NO synthase 5 (rNOS5), suggesting that NOS5 can be the enzyme involved in the formation of 1,2,3-triazine. The gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT), one of the major phosphoribosyltransferases of purine metabolism, increased when 2-azahypoxanthine content was the highest. Therefore, we hypothesized that HGPRT might catalyze a reversible reaction between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. We proved the endogenous existence of 2-azahypoxanthine-ribonucleotide in L. sordida mycelia by LC-MS/MS for the first time. Furthermore, it was shown that recombinant HGPRT catalyzed reversible interconversion between 2-azahypoxanthine and 2-azahypoxanthine-ribonucleotide. These findings demonstrate that HGPRT can be involved in the biosynthesis of 2-azahypoxanthine via 2-azahypoxanthine-ribonucleotide generated by NOS5.

RANKL-responsive epigenetic mechanism reprograms macrophages into bone-resorbing osteoclasts.

Monocyte/macrophage lineage cells are highly plastic and can differentiate into various cells under different environmental stimuli. Bone-resorbing osteoclasts are derived from the monocyte/macrophage lineage in response to receptor activator of NF-κB ligand (RANKL). However, the epigenetic signature contributing to the fate commitment of monocyte/macrophage lineage differentiation into human osteoclasts is largely unknown. In this study, we identified RANKL-responsive human osteoclast-specific superenhancers (SEs) and SE-associated enhancer RNAs (SE-eRNAs) by integrating data obtained from ChIP-seq, ATAC-seq, nuclear RNA-seq and PRO-seq analyses. RANKL induced the formation of 200 SEs, which are large clusters of enhancers, while suppressing 148 SEs in macrophages. RANKL-responsive SEs were strongly correlated with genes in the osteoclastogenic program and were selectively increased in human osteoclasts but marginally presented in osteoblasts, CD4+ T cells, and CD34+ cells. In addition to the major transcription factors identified in osteoclasts, we found that BATF binding motifs were highly enriched in RANKL-responsive SEs. The depletion of BATF1/3 inhibited RANKL-induced osteoclast differentiation. Furthermore, we found increased chromatin accessibility in SE regions, where RNA polymerase II was significantly recruited to induce the extragenic transcription of SE-eRNAs, in human osteoclasts. Knocking down SE-eRNAs in the vicinity of the NFATc1 gene diminished the expression of NFATc1, a major regulator of osteoclasts, and osteoclast differentiation. Inhibiting BET proteins suppressed the formation of some RANKL-responsive SEs and NFATc1-associated SEs, and the expression of SE-eRNA:NFATc1. Moreover, SE-eRNA:NFATc1 was highly expressed in the synovial macrophages of rheumatoid arthritis patients exhibiting high-osteoclastogenic potential. Our genome-wide analysis revealed RANKL-inducible SEs and SE-eRNAs as osteoclast-specific signatures, which may contribute to the development of osteoclast-specific therapeutic interventions.

TXNIP Suppresses the Osteochondrogenic Switch of Vascular Smooth Muscle Cells in Atherosclerosis.

BACKGROUND: The osteochondrogenic switch of vascular smooth muscle cells (VSMCs) is a pivotal cellular process in atherosclerotic calcification. However, the exact molecular mechanism of the osteochondrogenic transition of VSMCs remains to be elucidated. Here, we explore the regulatory role of TXNIP (thioredoxin-interacting protein) in the phenotypical transitioning of VSMCs toward osteochondrogenic cells responsible for atherosclerotic calcification. METHODS: The atherosclerotic phenotypes of Txnip-/- mice were analyzed in combination with single-cell RNA-sequencing. The atherosclerotic phenotypes of Tagln-Cre; Txnipflox/flox mice (smooth muscle cell-specific Txnip ablation model), and the mice transplanted with the bone marrow of Txnip-/- mice were analyzed. Public single-cell RNA-sequencing dataset (GSE159677) was reanalyzed to define the gene expression of TXNIP in human calcified atherosclerotic plaques. The effect of TXNIP suppression on the osteochondrogenic phenotypic changes in primary aortic VSMCs was analyzed. RESULTS: Atherosclerotic lesions of Txnip-/- mice presented significantly increased calcification and deposition of collagen content. Subsequent single-cell RNA-sequencing analysis identified the modulated VSMC and osteochondrogenic clusters, which were VSMC-derived populations. The osteochondrogenic cluster was markedly expanded in Txnip-/- mice. The pathway analysis of the VSMC-derived cells revealed enrichment of bone- and cartilage-formation-related pathways and bone morphogenetic protein signaling in Txnip-/- mice. Reanalyzing public single-cell RNA-sequencing dataset revealed that TXNIP was downregulated in the modulated VSMC and osteochondrogenic clusters of human calcified atherosclerotic lesions. Tagln-Cre; Txnipflox/flox mice recapitulated the calcification and collagen-rich atherosclerotic phenotypes of Txnip-/- mice, whereas the hematopoietic deficiency of TXNIP did not affect the lesion phenotype. Suppression of TXNIP in cultured VSMCs accelerates osteodifferentiation and upregulates bone morphogenetic protein signaling. Treatment with the bone morphogenetic protein signaling inhibitor K02288 abrogated the effect of TXNIP suppression on osteodifferentiation. CONCLUSIONS: Our results suggest that TXNIP is a novel regulator of atherosclerotic calcification by suppressing bone morphogenetic protein signaling to inhibit the transition of VSMCs toward an osteochondrogenic phenotype.

Possible molecular mechanism for acute encephalopathy by angel-wing mushroom ingestion - Involvement of three constituents in onset.

In Japan in 2004, 59 people who had consumed angel-wing mushroom, Pleurocybella porrigens, experienced acute encephalopathy, and of these 17 died. We purified a lethal protein to mice, pleurocybelline (PC), from P. porrigens. Although PC caused no damage to the brain, PC formed a complex with a lectin (PPL) and showed exo-protease activity, degrading substrates from both N- and C-termini. In addition, the presence of an unstable toxic compound, pleurocybellaziridine (PA), in the mushroom was demonstrated. We hypothesized that the complex and PA are involved in disease development and verified that apoptotic cells in the hippocampus were significantly increased by injection of the mixture of PC, PPL, and PA, indicating that these substances might be involved in acute encephalopathy.

Utilization of Corn Steep Liquor for the Production of Fairy Chemicals by Lepista sordida Mycelia.

There are various potential practical uses of fairy chemicals (FCs) in the fields of agriculture, cosmetics, and medicine; however, the production costs of FCs are very high. To enable the practical use of FCs, more efficient and inexpensive methods of culturing the mycelia of FCs-producing fungi and producing FCs need to be developed. The purpose of the present study was to determine methods of reducing the production costs of FCs and mycelia of the FCs-producing fungus Lepista sordida. We investigated the effects of four food industrial by-products, i.e., corn steep liquor (CSL), rice bran, wheat bran, and Japanese liquor lees, as nutritional additives in the liquid culture medium of the fungus. We found that CSL was more effective than the other tested additives in increasing the production of FCs and mycelia. Medium containing 1% CSL was optimal for increasing the mycelial yield while medium containing 6% CSL was optimal for increasing the production of FCs. The reason for this difference in the optimal CSL concentration was considered to be related to the stress on the mycelia caused by the amount of nutrients in the liquid medium. These results are expected to facilitate the practical use of FCs and the mycelia of FCs-producing fungi.

Axl, Immune Checkpoint Molecules and HIF Inhibitors from the Culture Broth of Lepista luscina.

Two compounds 1 and 2 were isolated from the culture broth of Lepista luscina. This is the first time that compound 1 was isolated from a natural source. The structure of compound 1 was identified via 1D and 2D NMR and HRESIMS data. Compounds 1 and 2 along with 8-nitrotryptanthrin (4) were evaluated for their biological activities using the A549 lung cancer cell line. As a result, 1 and 2 inhibited the expression of Axl and immune checkpoint molecules. In addition, compounds 1, 2 and 4 were tested for HIF inhibitory activity. Compound 2 demonstrated statistically significant HIF inhibitory effects on NIH3T3 cells and 1 and 2 against ARPE19 cells.

Estradiol treatment increases M2-like visceral adipose tissue macrophages in obese ovariectomized mice regardless of its anorectic action.

Estradiol (E2) treatment has been known to induce changes in food intake, energy expenditure, and weight gain. However, its direct effects on adipose tissue macrophages (ATM) in vivo are not fully understood. Thus, we aimed to explore this aspect at cellular and molecular levels in ovariectomized obese mice. We examined the changes in ATMs after eight weeks of a high-fat diet (HFD) in male, female, and ovariectomized (OVX) mice. After eight weeks, osmotic pumps were inserted into OVX mice to provide two weeks of E2 treatment. We additionally set up a vehicle Pair-Fed (PF) control group that supplied the same amount of HFD consumed by the E2-treated group. We then investigated the in vivo phenotypic changes of visceral adipose tissue (VAT) macrophages. The percentage of M1-like ATMs decreased by the anorectic effect of E2, while M2-like ATMs increased regardless of the anorexia. E2 treatment increased the expression of anti-inflammatory genes but decreased pro-inflammatory genes in VAT. Monocyte recruitment and local proliferation contributed to M2-like ATMs. Furthermore, M2-like phenotypes were induced by E2 treatment in human macrophages. E2 treatment increases M2-like macrophages and improves the tissue milieu of VAT regardless of the anorectic reaction of E2.

Single-cell transcriptomics reveal cellular diversity of aortic valve and the immunomodulation by PPARγ during hyperlipidemia.

Valvular inflammation triggered by hyperlipidemia has been considered as an important initial process of aortic valve disease; however, cellular and molecular evidence remains unclear. Here, we assess the relationship between plasma lipids and valvular inflammation, and identify association of low-density lipoprotein with increased valvular lipid and macrophage accumulation. Single-cell RNA sequencing analysis reveals the cellular heterogeneity of leukocytes, valvular interstitial cells, and valvular endothelial cells, and their phenotypic changes during hyperlipidemia leading to recruitment of monocyte-derived MHC-IIhi macrophages. Interestingly, we find activated PPARγ pathway in Cd36+ valvular endothelial cells increased in hyperlipidemic mice, and the conservation of PPARγ activation in non-calcified human aortic valves. While the PPARγ inhibition promotes inflammation, PPARγ activation using pioglitazone reduces valvular inflammation in hyperlipidemic mice. These results show that low-density lipoprotein is the main lipoprotein accumulated in the aortic valve during hyperlipidemia, leading to early-stage aortic valve disease, and PPARγ activation protects the aortic valve against inflammation.

Characteristics of plaque lipid-associated macrophages and their possible roles in the pathogenesis of atherosclerosis.

PURPOSE OF REVIEW: Recent findings from single-cell transcriptomic studies prompted us to revisit the role of plaque foamy macrophages in the pathogenesis of atherosclerosis. In this review, we compared the gene expression profile of plaque foamy macrophages with those of other disease-associated macrophages and discussed their functions in the pathogenesis of atherosclerosis. RECENT FINDINGS: To understand the phenotypes of macrophages in atherosclerotic aorta, many research groups performed single-cell RNA sequencing analysis and found that there are distinct phenotypic differences among intimal foamy, nonfoamy and adventitial macrophages. Especially, the plaque foamy macrophages express triggering receptor expressed on myeloid cells 2 (TREM2), a key common feature of disease-associated macrophages in Alzheimer's disease, obesity, cirrhosis and nonalcoholic steatohepatitis. These TREM2 + macrophages seem to be protective against chronic inflammation. SUMMARY: As the gene expression profile of plaque foamy macrophages is highly comparable to that of lipid-associated macrophages from obesity, we named the plaque foamy macrophages as plaque lipid-associated macrophages (PLAMs). PLAMs have a high level of gene expression related to phago/endocytosis, lysosome, lipid metabolism and oxidative phosphorylation. Considering the protective function of lipid-associated macrophages against adipose tissue inflammation, PLAMs may suppress atherosclerotic inflammation by removing modified lipids and cell debris in the plaque.

Anti-phytopathogenic-bacterial fatty acids from the mycelia of the edible mushroom Agaricus blazei.

Five compounds including a new compound (1) were isolated from mycelia of a mushroom-forming fungus Agaricus blazei. Compound 2 was isolated from nature for the first time. Their structures were determined by the interpretation of spectroscopic data. In the bioassay examining growth inhibitory activity against phytopathogenic bacteria Clavibacter michiganensis, Burkholderia glumae, and Peptobacterium carotovorum, all the compounds showed inhibition effects on C. michiganensis. Compounds 3 and 4 also showed weak inhibitory activity against growth of B. glumae.

CmLec4, a lectin from the fungus Cordyceps militaris, controls host infection and fruiting body formation.

Fungi belonging to the Ascomycete genus Cordyceps are endoparasitoids and parasites, mainly of insects and other arthropods. Cordyceps militaris has been used as a therapeutic drug for cancer patients. However, the infection, parasitism, and fruiting body formation mechanisms of this fungus are still unknown. Based on our hypothesis that lectin(s) is involved in the interaction between the C. militaris fungi and insects, we partially purified and characterized a new lectin from C. militaris, designated CmLec4. In addition, we searched for substance(s) in the infected silkworm extracts that could bind to CmLec4, and succeeded in purifying the sex-specific storage protein 2 as a specific binding target. To examine function of the binding protein during the process of parasitism, we investigated the effect of recombinant CmLec4 on silkworms by inoculating the protein into silkworm pupae, and found that it significantly delayed emergence compared to the control. Furthermore, cmlec4 gene knockout strains constructed in this study produced markedly lower amounts of fruiting body than the wild-type strain. All the results revealed that the lectin CmLec4 produced by C. militaris would be involved in the infection into silkworm and fruiting body formation from the host.

The complete mitochondrial genome sequence of the fairy ring-forming fungus Lepista sordida.

Lepista sordida is a fairy ring-forming fungus that belongs to the family Tricholomataceae and is widely distributed in the Northern Hemisphere. Here, we report the complete mitochondrial genome sequence of L. sordida. The mitochondrial genome (57,375 bp) contained 20 protein-coding genes, 2 ribosomal RNA genes, and 26 transfer RNA genes. Phylogenetic analysis based on 14 conserved protein sequences from L. sordida and 15 related basidiomycetes showed that L. sordida was located on the outermost branch of the Tricholomataceae clade. This study is the first to report the complete mitochondrial genome sequence of a fairy ring-forming fungus belonging to the genus Lepista.