Protein S-nitrosylation is involved in valproic acid-promoted neuronal differentiation of adipose tissue-derived stem cells.

Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is greatly promoted by valproic acid (VPA) with cAMP elevating agents thorough NO signaling pathways, but its mechanism is not fully understood. In the present study, we investigate the involvement of protein S-nitrosylation in the VPA-promoted neuronal differentiation of ASCs. The whole amount of S-nitrosylated protein was increased by the treatment with VPA alone for three days in ASCs. An inhibitor of thioredoxin reductase (TrxR), auranofin, further increased the amount of S-nitrosylated protein and enhances the VPA-promoted neuronal differentiation in ASCs. On the contrary, another inhibitor of TrxR, dinitrochlorobenzene, inhibited the VPA-promoted neuronal differentiation in ASCs even with cAMP elevating agents, which was accompanied by unexpectedly decreased S-nitrosylated protein. It was considered from these results that increased protein S-nitrosylation is involved in VPA-promoted neuronal differentiation of ASCs. By the proteomic analysis of S-nitrosylated protein in VPA-treated ASCs, no identified proteins could be specifically related to VPA-promoted neuronal differentiation. The identified proteins, however, included those involved in the metabolism of substances regulating neuronal differentiation, such as aspartate and glutamate.

Isolation of Paraphaeoketones: A Plausible Biosynthetic Explanation for Paraphaeolactones via a Benzilic Acid Rearrangement Rather than a Favorskii Rearrangement.

Paraphaeoketones A-C (1-3) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Their structures and relative configurations were determined using spectroscopic analysis and verified through density functional theory (DFT)-based chemical shift calculations. The absolute configurations of these compounds were determined by comparing the experimental electronic circular dichroism (ECD) spectra with those based on DFT calculations. We also propose a plausible biosynthetic route to 1-3. While our prior studies on the isolation and structural elucidation of paraphaeolactones (e.g., 4) led us to suggest a Favorskii rearrangement for their biosynthesis, the isolation of 2 prompted the proposal of an alternative biosynthesis for 4, featuring a benzilic acid rearrangement of 2. Moreover, an in vitro conversion of 2 into 4 was achieved successfully, suggesting that a biosynthetic pathway for paraphaeolactones involving a benzilic acid rearrangement is more plausible than the previously presumed Favorskii rearrangement pathway. Arguments based on DFT calculations for these pathways are also described.

HIV-1 Integrase Inhibitor, Dihydroobionin B, and the Investigation of Its Extraordinary Specific Rotation.

Dihydroobionin B (1), a chiral congener of known obionin B, was isolated from Pseudocoleophoma sp. KT4119, a freshwater fungus collected from a submerged wood block in Kochi Prefecture, Japan, in 2020. The planar structure of 1 was characterized by mass and NMR spectral analysis and confirmed by density functional theory (DFT)-based chemical shift calculations. Its absolute structure was determined by electronic circular dichroism spectral analysis. Notably, 1 exhibited an extraordinarily large specific rotation [[α]20D +1080 (c 0.056, CHCl3)], which was verified by DFT-based specific rotation calculations. However, these calculations indicated that the sign of the specific rotation based on static analysis was insufficient to determine the absolute configuration in this case. Furthermore, Pseudocoleophoma KT4119 produced coleophomapyrones A (2) and B (3) and coleophomaldehyde A (4). While this is the first report of 2 isolated from a natural source, it has also been prepared previously using a synthetic approach. Compound 1 potently inhibited HIV type 1 integrase (IC50 = 0.44 µM) without significant cytotoxicity. Finally, docking experiments were conducted to propose a plausible mechanism for the behavior of 1.

Integrasone Derivatives Isolated from Lepteutypa sp. KT4162 and Their Anti-HIV-1 Integrase Activity.

Five integrasone derivatives, integrasone C (1), isointegrasone C (2), integrasone D1 (3), integrasone D2 (4), and integrasone E (5), were isolated from the culture broth of Lepteutypa sp. KT4162. Neither conventional NMR analyses nor DFT (density functional theory)-based computationally assisted chemical shift discussions were sufficient to elucidate the relative configuration of the 1,4-epoxydiol moiety. A combined analysis using the calculated nJCH values and HMBC spectra was helpful to establish the relative configuration. The absolute configurations of 1-5 were determined using DFT-based ECD (electronic circular dichroism) spectral analysis. Biological assays of these compounds revealed that 2 potently inhibits HIV-1 integrase without cytotoxicity.

Pseudo-Enantiomeric Paraphaeolactones and Their Biosynthetic Derivatives from Paraphaeosphaeria sp. KT4192: A Proposition of a Favorskii Rearrangement for Their Biosynthesis.

Paraphaeolactones A1, A2, B1, and B2 (1-4, respectively), known arthropsadiol D (5), massariphenone (6) and its positional isomer 7, and massarilactones E (8) and G (9) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Although the structural resemblance between 1 and 2 implies that these comprised a diastereomeric pair at the C-2 stereogenic center, electronic circular dichroism (ECD) spectral analyses revealed that they were pseudo-enantiomers possessing the common (2R)-configuration. Paraphaeolactones B1 and B2 (3 and 4) were the derivatives of 2, which equipped the 3-(1-hydroxy-2-oxopropyl)-4-methylcatechol moiety via an acetal bond at C-10. The relative configurations of their acetal carbons were elucidated by NOE experiments, and those of C-8' were deduced independently by ECD spectral analysis. The present study disclosed that 1-5, 8, and 9 contain a methylcyclohexene substructure with the same absolute configuration. This prompted us to reinvestigate the absolute configurations of known structurally related fungal metabolites, allowing us to conclude that the methylcyclohexene moieties of these natural products have the same absolute configuration despite the variety of configurations of other stereogenic centers. The plausible biosynthetic routes for 1-9 are discussed on the basis of the above conclusion. We propose a Favorskii rearrangement as the key transformation for biosyntheses of 1-4.

Valproic acid promotes differentiation of adipose tissue-derived stem cells to neuronal cells selectively expressing functional N-type voltage-gated Ca2+ channels.

The differentiation of adipose tissue-derived stem cells (ASCs) to neuronal cells is greatly promoted by valproic acid (VPA), and is synergistically enhanced by the following treatment with neuronal induction medium (NIM) containing cAMP-elevating agents. In the present study, we investigated the synergism between VPA and NIM in neuronal differentiation of ASCs, assessed by the expression of neurofilament medium polypeptide (NeFM), with respect to Ca2+ entry. VPA (2 mM) treatment for 3 days followed by NIM for 2 h synergistically increased the incidence of neuronal cells differentiated from ASCs to an extent more than VPA alone treatment for 6 days, shortening the time required for the differentiation. VPA increased intracellular Ca2+ and the mRNAs of voltage-gated Ca2+ channels, Cacna1b (Cav2.2) and Cacna1h (Cav3.2), in ASCs. Inward currents through Ca2+ channels were evoked electrophysiologically at high voltage potential in ASCs treated with VPA. NIM reduced the mRNAs of NeFM and Cacna1b in VPA-promoted neuronal differentiation of ASCs. It was concluded that functional N-type voltage-gated Ca2+ channels (Cav2.2) are selectively expressed in VPA-promoted neuronal differentiation of ASCs. NIM seems to enhance the mRNA translation of molecules required for the differentiation. Neuronal cells obtained from ASCs by this protocol will be used as a cell source for regenerative therapy of neurological disorders associated with altered Cav2.2 activity.

Hydrogen sulfide potently promotes neuronal differentiation of adipose tissue-derived stem cells involving nitric oxide-mediated signaling cascade with the aid of cAMP-elevating agents.

Neuronal differentiation of adipose tissue-derived stem cells (ASCs) is potently promoted by valproic acid (VPA) through a gaseous signaling molecule, nitric oxide (NO). Here, we investigated the involvement of hydrogen sulfide (H2S), another gaseous signaling molecule, in neuronal differentiation of ASCs. VPA-promoted neuronal differentiation of ASCs was accompanied by increased intracellular H2S and sulfane sulfur with increased mRNA expression of enzymes synthesizing sulfane sulfur including cystathionine ß-synthase (CBS), of which inhibition reduced the differentiation efficiency. H2S donors, GYY4137 (GYY) or NaHS, potently promoted neuronal differentiation of ASCs when cAMP-elevating agents, dibutyryl cyclic adenosine monophosphate and isobutyl methyl-xanthine, were added as neuronal induction medium (NIM). Neuronal differentiation of ASCs promoted by NaHS or GYY was accompanied by Ca2+ entry and increased mRNA expression of voltage-gated Ca2+ channels. NaHS or GYY also increased mRNA expression of enzymes of the NO-citrulline cycle including inducible NO synthase (iNOS). It was concluded from these results that H2S potently promoted differentiation of ASCs into neuronal cells expressing functional voltage-gated Ca2+ channels with the aid of cAMP-elevating agents, involving NO-mediated signaling cascade. These effects of H2S were also considered as a partial mechanism for the VPA-promoted neuronal differentiation of ASCs.

Valproic acid up-regulates the whole NO-citrulline cycle for potent iNOS-NO signaling to promote neuronal differentiation of adipose tissue-derived stem cells.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.

Cyclohelminthol CPs: Scope and Limitations of Density Functional Theory-Based Structural Elucidation of Natural Products.

The effectiveness and limitations of density functional theory (DFT) calculations in the structural determination of complexed and conformationally flexible natural products were demonstrated using the cyclohelminthols CP-1 (1) CP-2 (2), CP-3 (3), and CP-4 (4) newly isolated from Helminthosporium velutinum yone96. Prior to DFT calculations, the structures were tentatively assigned using conventional spectroscopic analyses. The structures were verified with reference to DFT-derived 13C and 1H NMR chemical shifts, 3JHH and nJCH values, and electronic circular dichroism (ECD) spectra. The 13C chemical shift calculations were very effective for verifying the ring-structure moieties but less effective for verifying the geometry of the side chain in which the juncture asymmetric carbon (C-16) was apart from the ring-structure moiety. However, 1H chemical shift calculations compensated for the imperfection of the latter. ECD spectral calculations were used to determine the absolute configurations. Calculations for virtual simple model molecules enabled us to evaluate the reliability of the ECD spectral calculation and derive the chiral torsion responsible for the characteristic Cotton effects.

Isolation and structural elucidation of novel fusicoccan dehydroxypericonicin A from Roussoella sp.

A potent antifungal fusicoccane dehydroxypericonicin A (1) was isolated from Roussoella sp. along with known allantofuranone (2). The relative structure of 1 was fully elucidated by NMR spectroscopic manner. The suggested relative structure was confirmed by density functional theory (DFT)-based 13C NMR chemical shift calculations. The absolute configuration was investigated by a spectral comparison of the experimental electronic circular dichroism spectrum with that based on the DFT calculations.

Genetic Variation in Y-Chromosome Genes of Sika Deer (Cervus nippon) in Japan.

Sika deer (Cervus nippon) in Japan are classified into southern and northern groups. However, previous studies primarily relied on maternally inherited mitochondrial DNA (mtDNA). The paternally inherited Y-chromosome is useful for analyzing the contribution of males to the population genetic history of sika deer. In total, approximately 16 kb of partial sequences of four Y-chromosomal genes, Y-linked, sex-determining region Y, DEAD-box helicase 3 Y-linked, and Zinc finger protein Y-linked, were sequenced to investigate intraspecific variation. As a result, we identified nine intronic single nucleotide polymorphisms (SNPs) in 478 sika deer samples collected over the entire Japanese archipelago from Hokkaido to Kyushu. SNP genotyping revealed 10 distinct haplotypes (SYH1-SYH10). The most common haplotype (SYH1) was present in all populations and was the most abundant haplotype, identified in 80.3% of the sampled individuals. The remaining haplotypes were unique to a single locality. SYH1 was also central to all other haplotypes that diverged by a SNP, resulting in this haplotype being the core of a star-like cluster topography. We found that contrary to mtDNA patterns, there was no clear differentiation of Y-chromosome markers between the southern and the northern populations. Due to the female philopatry of sika deer, mtDNA may provide a highly structured differentiation of populations. On the other hand, the male-biased gene flow may provide a reduced differentiation of populations. Our findings revealed that the genetic structure of the Japanese sika deer is more complex than previously thought based on mtDNA-based phylogeographic studies.

Valproic acid promotes mature neuronal differentiation of adipose tissue-derived stem cells through iNOS-NO-sGC signaling pathway.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing ßIII-tubulin protein, a neuronal marker, by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2 h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400 W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as ßIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS-NO-sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.

Isolation of Peribysins O, P, and Q from Periconia macrospinosa KT3863 and Configurational Reinvestigation of Peribysin E Diacetate from Periconia byssoides OUPS-N133.

Peribysins O (1), P (3), and Q (4) were isolated from Periconia macrospinosa KT3863. The relative configuration of the 6,7-epoxide of 1 was elucidated by performing quantitative NOE experiments. The structure of 2, which is a tautomer of 1 present in CDCl3 solutions in 5% abundance, was also fully characterized by NMR analysis. Their absolute configurations were independently determined by the modified Mosher's method (for 1 and 3), the electronic circular dichroism (ECD) exciton coupling theory after conversion into dibenzoate 9 (for 3), and theoretical ECD calculations (for 1, 3, and 4). The obtained relative structures 1, 3, and 4 were verified by calculating their 13C chemical shifts using density functional theory (DFT). Although the established (4 S)-enantiomer for 1-4 is in accordance with that of other peribysins isolated from the related fungus Periconia byssoides OUPS-N133, Danishefsky's total synthesis of peribysin E (5) led to the subsequent revision of the (2 R,4 S,5 R,6 S,7 S,8 R,10 S)-enantiomer to the (2 S,4 R,5 S,6 R,7 R,8 S,10 R)-enantiomer. This discordance led us to reinvestigate the configuration using time-dependent DFT-based ECD spectral calculations, which supported the original (4 S)-enantiomer.

Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification.

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140-170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.

Identification and antifungal sensitivity of two new species of Diaporthe isolated.

Diaporhte species are plant pathogens rarely involved in human diseases, especially eye diseases. We report our findings in two undescribed Diaporhte species. Both were identified by their morphological characteristics and by DNA sequence analyses. In Case 1, an 81-year-old male farmer who had pterygium surgery 7 years earlier developed keratitis and the causal fungus was identified as a new species of Diaporthe, D. oculi. This species can be distinguished from the closely related D. limonicola on Citrus limon (Rutaceae) by the ITS, tef1, and TUB (515/520 = 99.0% in ITS, 315/324 = 97.2% in tef1, and 601/614 = 97.9% in TUB). The isolate from Case 2, a 68-year-old man with a rose thorn injury, was also identified as a new Diaporthe species, D. pseudooculi. Phylogenetically, D. pseudooculi is different from the closely related D. podocarpi-macrophylli by the ITS, tef1, and TUB (525/531 = 98.9% in ITS, 314/333 = 94.3% in tef1, and 436/442 = 98.6% in TUB). We report on the identification, drug sensitivity, and treatment outcomes for these two new species of Diaporthe, D. oculi and D. pseudooculi.

Cyclohelminthols Y1-Y4 Metabolites Possessing Two Spirocyclopropanes in Their Structure.

Cyclohelminthols Y1-Y4 (1-4) were isolated from the culture broth of Helminthosporium velutinum yone96. These compounds are diastereomers to each other featuring 3-azabicyclo[3.1.0]hexane-6-spirocyclopentane linked with a cyclopentanespirocyclopropane framework. Their planar structures were established via the comparison of their spectra with the simpler analogue cyclohelminthol X as well as analysis of their HMBC spectra. Although the proton-deficient core frameworks of 1-4 prevented us from obtaining configurational information via conventional NMR analysis, their total structures involving the relative and absolute configurations were established using density functional theory (DFT)-based molecular modeling calculations. The present study demonstrates the effectiveness of the comparison between the theoretical and experimental δ13C values for stereochemical analysis by focusing on the carbons that show relatively large δ13C deviations among the isomers. The G-ring of these molecules most likely originates from the cyclopropanation of the C6C7 double bond with the carbene equivalent 6 derived from cyclohelminthol IV (7), which was isolated from the same producer fungus. Preliminary biological experiments revealed the potent cytotoxicity of the (6 S)-isomers against COLO201 cells, whereas the (6 R)-isomers exhibited weak activity. The antifungal assay with Cochiobolus miyabeanus showed a slightly different profile.

Cyclohelminthol X, a Hexa-Substituted Spirocyclopropane from Helminthosporium velutinum yone96: Structural Elucidation, Electronic Circular Dichroism Analysis, and Biological Properties.

Helminthosporium velutinum yone96 produces cyclohelminthol X (1), a unique hexa-substituted spirocyclopropane. Although its molecular formula and NMR spectral data resemble those of AD0157, being isolated from marine fungus Paraconiothyrium sp. HL-78-gCHSP3-B005, our detailed analyses disclosed a totally different structure. Chemical shift calculations and electronic circular dichroism spectral calculations were quite helpful to establish the structure, when those were performed based on density functional theory. The carbon framework of cyclohelminthols I-IV is found at the C1-C8 propenylcyclopentene substructure of 1. Thus, 1 is assumed to be biosynthesized by cyclopropanation between an oxidized form of cyclohelminthol IV and a succinic anhydride derivative 4. Cytotoxicity for two cancer cell lines and proteasome inhibition efficiency are measured.

Roussoella solani causing keratomycosis, with an observed both sexual and asexual morphs.

We describe an 82-year-old male farmer who had diabetes mellitus with no history of ocular trauma by soil or plants and who developed a corneal infection due to a fungus. The organism was identified as Roussoella solani based on both the morphological characteristics and phylogenetic analysis using LSU and ITS nrDNA sequences. The sexual stage of R. solani is described and illustrated for the first time. The patient was treated successfully with a combination of topical and systemic voriconazole and micafungin. This case is the first report of keratomycosis caused by R. solani.

Computation-Assisted Structural Elucidation of Epoxyroussoeone and Epoxyroussoedione Isolated from Roussoella japanensis KT1651.

The structures of epoxyroussoenone (1) and epoxyroussoedione (3) isolated from a culture broth of Roussoella japanensis KT1651 were determined. Although NMR spectra provided insufficient structural information, computation of the theoretical chemical shifts with DFT EDF2/6-31G* enabled us to elucidate not only the planar structure, but also the relative configuration. Their ECD (electric circular dichroism) spectra suggested the absolute configurations, which were confirmed with time-dependent DFT calculations employing BHandHLYP/TZVP. The ECD calculations for other stereoisomers yielded obviously different spectral profiles, thus confirming the relative structures of 1 and 3.

Taxonomic revision of Pseudolachnea and Pseudolachnella and establishment of Neopseudolachnella and Pseudodinemasporium gen. nov.

The taxonomy of Pseudolachnea and Pseudolachnella is controversial. Some authors have regarded them as congeneric, whereas others have considered them to be distinct genera differentiated merely on the number of conidial septa. A total of 26 isolates of Pseudolachnea-like fungi were subjected to morphological examination and phylogenetic analyses of nuc rDNA internal transcribed spacers 1 and 2 and partial 28S sequences and partial sequence of the translation elongation factor 1α gene. The results indicated that our materials should be classified in four genera: Pseudolachnea, Pseudolachnella, and two new genera, Neopseudolachnella and Pseudodinemasporium. Although the monophyly of both Pseudolachnea and Pseudolachnella was confirmed, it was concluded that differences observed in the conidiomatal structure, such as thickness of basal stroma and the excipulum, were more reliable for their circumscription, instead of conidial septation. Neopseudolachnella was similar to Pseudolachnea and Pseudolachnella in conidial morphology but was characterized by the conidiomata lacking an excipulum, unlike members of the latter two genera. Pseudodinemasporium bore conidia morphologically similar to those of Dinemasporium but was differentiated from the latter by the conidiomata, which was composed of a well developed peridial structure. A total of 12 new species, namely three in Neopseudolachnella (N. acutispora, N. magnispora, N. uniseptata), one in Pseudodinemasporium (P. fabiforme) and eight in Pseudolachnella (P. asymmetrica, P. botulispora, P. brevicoronata, P. campylospora, P. complanata, P. falcatispora, P. fusiformis and P. pachyderma) are described and illustrated.