3-Amino-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carbonitrile: A fluorescent molecule that induces differentiation in PC12 cells.

Neural differentiation is triggered by the activation of multiple signaling pathways initiated by various neurotrophic factors. An elucidation of these mechanisms is anticipated to facilitate the prevention of diseases and the development of novel therapeutic approaches. Alternative small-molecule inducers for neuroscience studies are required instead of protein-based reagents for more efficient and convenient experiments. We demonstrated that small molecules of thieno[2,3-b]pyridine derivatives that induce neural differentiation, compounds 3a and 9a in particular, exhibited significant neuritogenic activity in rat pheochromocytoma (PC12) cells. Moreover, 3a displayed pronounced fluorescence and a discernible Stokes shift. Furthermore, the outcome of the experiment conducted on the NGF-insensitive clones of rat PC12 cells, and the results of the intercellular uptake analyses suggested that the 3a-mediated activation of neural differentiation occurred independently of the TrkA receptor. Therefore, 3a portrays potential applicability both as a small molecule reagent to replace novel neurotrophic factors and as a potent fluorescent reagent for various techniques, including bioimaging.

Tejas functions as a core component in nuage assembly and precursor processing in Drosophila piRNA biogenesis.

PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown. Here, we conduct a detailed analysis of the subcellular localization of fluorescently tagged nuage proteins and behavior of piRNA precursors. Our results demonstrate that Tej functions as a core component that recruits Vasa (Vas) and Spindle-E (Spn-E) into nuage granules through distinct motifs, thereby assembling nuage and engaging precursors for further processing. Our study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, we propose that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating the mobility of nuage components.

Identification and mechanistic investigation of ellagitannins from Osbeckia octandra that attenuate liver fibrosis via the TGF-ß/SMAD signaling pathway.

Fibrosis is a major problem in chronic liver disease with limited treatment options due to its complex nature. Herbal medicines are often used as an alternative. The aim of this study was to investigate the therapeutic potential of Osbeckia octandra and to identify its active compounds and regulatory pathways. The effects of crude leaf suspension and boiled leaf extract were investigated in an animal model, and the extract was found to be the more effective treatment. Three major bioactive compounds, pedunculagin, casuarinin, and gallic acid, were isolated from the extract using the hepatic stellate cell line, LX-2-based antifibrotic effect evaluation system. The results showed that all these compounds ameliorated LX-2 in fibrotic state. This inhibitory mechanism was confirmed through the TGF-ß/SMAD signaling pathway. Collectively, the presence of these compounds in O. octandra suggests its potential as a treatment for liver fibrosis.

miRNA/siRNA-directed pathway to produce noncoding piRNAs from endogenous protein-coding regions ensures Drosophila spermatogenesis.

PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.

Improving lipophilicity of 5-(1-acetyl-5-phenylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid increases its efficacy to activate hypoxia-inducible factors.

Hypoxia-inducible factor (HIF) activators aid the treatment of renal anemia and ischemia. Recently, PyrzA (5-(1-acetyl-5-phenylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid), a HIF activator by PHD inhibition without a 2-oxoglutarate moiety was reported. However, PyrzA has low lipophilicity, and it was necessary to improve its solubility by synthesizing derivatives. In this study, we synthesized and evaluated a higher lipophilic derivative of PyrzA and found that it exhibited higher HIF activity and stabilizing ability at low concentrations compared to Roxadustat, a commercially available HIF activator.

Correction to "Prolyl Hydroxylase Domain Protein Inhibitor Not Harboring a 2-Oxoglutarate Scaffold Protects against Hypoxic Stress".

[This corrects the article DOI: 10.1021/acsptsci.2c00002.].

Prolyl Hydroxylase Domain Protein Inhibitor Not Harboring a 2-Oxoglutarate Scaffold Protects against Hypoxic Stress.

Hypoxia-inducible factor-α (HIF-α) activation has shown promising results in the treatment of ischemia, such as stroke, myocardial infarction, and chronic kidney disease. A number of HIF-α activators have been developed to improve the symptoms of these diseases. Many feature 2-oxoglutarate (2-OG) scaffolds that interact with the active centers of prolyl hydroxylase domain-containing proteins (PHDs), displacing the coenzyme 2-OG. This stabilizes HIF-α. Therefore, the specificity of the 2-OG analogs is not high. Here, we identified 5-(1-acetyl-5-phenylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (PyrzA) among over 10 000 compounds as a novel HIF activator that does not contain a 2-OG scaffold. In cultured cells, PyrzA enhanced HIF-α stability and upregulated the expression of HIF target genes. Interestingly, PyrzA decreased HIF-1α prolyl hydroxylation, suggesting that PyrzA may activate HIF to prevent the degradation of HIF-α. These results indicate that PyrzA stabilizes HIF via a novel mechanism and could be a potential HIF activator candidate.

The Tudor Domain-Containing Protein, Kotsubu (CG9925), Localizes to the Nuage and Functions in piRNA Biogenesis in D. melanogaster.

Silencing of transposable elements (TEs) by Piwi-interacting RNAs (piRNAs) is crucial for maintaining germline genome integrity and fertility in animals. To repress TEs, PIWI clade Argonaute proteins cooperate with several Tudor domain-containing (Tdrd) proteins at membraneless perinuclear organelles, called nuage, to produce piRNAs to repress transposons. Here, we identify and characterize Kotsubu (Kots), one of the Drosophila Tudor domain-containing protein-1 (Tdrd1) orthologs, encoded by the CG9925 gene, that localizes to the nuage in gonads. We further show the dynamic localization of Kots in the male germline, where it shows perinuclear signals in spermatogonia but forms large cytoplasmic condensates in the spermatocytes that overlap with components of piNG-body, a nuage-associated organelle. The loss of kots results in a notable upregulation of stellate and a corresponding reduction in the suppressor of stellate piRNAs in the mutants. Furthermore, a moderate yet significant reduction of other piRNAs was observed in kots mutant testes. Taken together, we propose that Kots functions in the piRNA pathway, predominantly in the male germline by forming discrete cytoplasmic granules.

The molecular mechanism of phytosphingosine binding to FFAR4/GPR120 differs from that of other fatty acids.

Free fatty acid receptor 4 (FFAR4)/GPR120 comprises a receptor for medium- and long-chain fatty acids. We previously identified phytosphingosine (PHS) as a novel ligand of FFAR4. Although many natural FFAR4 ligands have carboxyl groups, PHS does not, thus suggesting that binding to FFAR4 is driven by a completely different mechanism than other natural ligands such as α-linolenic acid (ALA). To test this hypothesis, we performed docking simulation analysis using a FFAR4 homology model based on a protein model derived from the crystal structure of activated turkey beta-1 adrenoceptor. The docking simulation revealed that the probable hydrogen bonds to FFAR4 differ between various ligands. In particular, binding was predicted between R264 of the FFAR4 and the oxygen of the carboxylate group in ALA, as well as between E249 of the FFAR4 and the oxygen of the hydroxy group at the C4-position in PHS. Alanine substitution at E249 (E249A) dramatically reduced PHS-induced FFAR4 activation but demonstrated a weaker effect on ALA-induced FFAR4 activation. Kinetic analysis and Km values clearly demonstrated that the E249A substitution resulted in reduced affinity for PHS but not for ALA. Additionally, we observed that sphingosine, lacking a hydroxyl group at C4-position, could not activate FFAR4. Our data show that E249 of the FFAR4 receptor is crucial for binding to the hydroxy group at the C4-position in PHS, and this is a completely different molecular mechanism of binding from ALA. Because GPR120 agonists have attracted attention as treatments for type 2 diabetes, our findings may provide new insights into their development.

Transition-Metal-Catalyzed Diarylation of Isocyanides with Triarylbismuthines for the Selective Synthesis of Imine Derivatives.

The transition-metal-catalyzed diarylation of isocyanides with triarylbismuthines was investigated in detail, and rhodium catalysts such as [RhCl(nbd)]2 were found to selectively afford N-alkyl diaryl ketimines. On the other hand, palladium-catalyzed diarylation proceeded with the incorporation of two molecules of isocyanide, preferentially yielding N,N'-dialkyl or N,N'-diaryl α-diimines. In addition, a cascade synthesis of 2,3-diarylquinoxalines starting from the palladium-catalyzed diarylation of isocyanides with triarylbismuthines was successfully achieved.

Highly regio- and stereoselective phosphinylphosphination of terminal alkynes with tetraphenyldiphosphine monoxide under radical conditions.

The homolytic cleavage of the PV(O)-PIII bond in tetraphenyldiphosphine monoxide simultaneously provides both pentavalent and trivalent phosphorus-centered radicals with different reactivities. The method using V-40 as an initiator is successfully investigated for the regio- and stereoselective phosphinylphosphination of terminal alkynes giving the corresponding trans-isomers of 1-diphenylphosphinyl-2-diphenylthiophosphinyl-1-alkenes in good yields. The protocol can be applied to a wide variety of terminal alkynes including both alkyl- and arylalkynes.

Phosphorus-Recycling Wittig Reaction: Design and Facile Synthesis of a Fluorous Phosphine and Its Reusable Process in the Wittig Reaction.

This study shows that phosphorus sources can be recycled using the appropriate fluorous phosphine in the Wittig reaction. The designed fluorous phosphine, which has an ethylene spacer between its phosphorus atom and the perfluoroalkyl group, was synthesized from air-stable phosphine reagents. The synthesized phosphine can be used for the Wittig reaction process to obtain various alkenes in adequate yields and stereoselectivity. The concomitantly formed fluorous phosphine oxide was extracted from the reaction mixture using a fluorous biphasic system. The fluorous phosphine was regenerated by reducing the fluorous phosphine oxide with diisobutylaluminum hydride. Finally, a series of gram scale phosphorus recycling processes were performed, which included the Wittig reaction, separation, reduction, and reuse.

Photoinduced Syntheses and Reactivities of Phosphorus-Containing Interelement Compounds.

The photoinduced reactions of tetraphenyldiphosphine disulfide with a range of organic dichalcogenides successfully afforded a series of phosphorus(V)-chalcogen interelement compounds via a radical process. The relative reactivities of the organic dichalcogenides (i.e., (PhS)2, (PhSe)2, and (PhTe)2) toward the PIII or PV groups in the diphosphine analogues under light were investigated in detail, and a convenient method was developed to form P-S or P-Se interelement compounds from tetraphenyldiphosphine disulfide and (PhS)2 or (PhSe)2 upon photoirradiation. Furthermore, the relative photochemical properties and reactivities of tetraphenyldiphophine (P-P interelement compound) and its analogues toward photoinduced radical addition reactions were also discussed. The formed P-E (E = S, Se) interelement compounds could be utilized for ionic reactions, and they could be transformed into various phosphine reagents via one-pot processes.

Photoinduced Cyclizations of o-Diisocyanoarenes with Organic Diselenides and Thiols that Afford Chalcogenated Quinoxalines.

This study describes the syntheses of 2,3-bis(selanyl)quinoxalines via the photoinduced cyclizations of o-diisocyanoarenes with diaryl or dialkyl diselenides, in addition to providing a detailed discussion of the corresponding mechanism and revealing that the developed procedure can also be applied to prepare 2-thiolated quinoxaline derivatives from o-diisocyanoarenes and thiols. The developed technique does not need the use of additives or metal catalysts and features the advantages of a high conversion, a broad substrate scope, and mild reaction conditions, thereby rendering it a valuable addition to the quinoxaline synthesis toolbox.

Augmented oxidative stress increases 8-oxoguanine preferentially in the transcriptionally active genomic regions.

8-Oxoguanine (8-oxoG) is the most common DNA base modification in the mammalian genome, associated with oxidative stress. Here we analysed the alterations in the distribution of 8-oxoG across the entire murine genome, before and after an elevation of oxidative stress by the use of ferric nitrilotriacetate (Fe-NTA) as an oxidative stress inducer in the renal proximal tubules. We isolated DNA fragments containing 8-oxoGs with immunoprecipitation from the murine genome, and amplified them by PCR for a distribution analysis with microarray-based comparative genomic hybridisation. The distribution profiles revealed that frequencies of 8-oxoG fluctuated with a cycle of 1-10 Mb along the chromosomes and the amplitude of the fluctuation was reduced after Fe-NTA administration. The distributions of 8-oxoG along the entire genome in the control and oxidatively stressed conditions were negatively correlated with that of gene density but positively correlated with that of Lamin B1 interaction, which corresponds to lamina-associated domains. These results on the murine genome were consistent with those on the rat genome we previously reported. We further discovered a negative correlation between the distributions of 8-oxoG and transcriptional activity along the genome. Finally, a comparison of the distributions before and after Fe-NTA administration suggested that 8-oxoGs are generated in response to the augmented oxidative stress preferentially in the transcriptionally active genomic regions, where 8-oxoGs have been less accumulated in the control condition.

Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression.

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. Our results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. We propose that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.

Palladium-Catalyzed Diarylation of Isocyanides with Tetraarylleads for the Selective Synthesis of Imines and α-Diimines.

Using tetraaryllead compounds (PbAr4) as arylating reagents, isocyanides undergo selective diarylation in the presence of palladium catalysts such as Pd(OAc)2 or Pd(PPh3)4 to afford imines and/or α-diimines based on the isocyanide employed. With aliphatic isocyanides, imines are obtained preferentially, whereas α-diimines are formed in the case of electron-rich aromatic isocyanides. The differences in imine/α-diimine selectivity can be attributed to the stability of imidoylpalladium intermediates formed in this catalytic reaction. Compared with other arylating reagents, tetraaryllead compounds are excellent candidates for use in the selective transformations to imines and/or α-diimines, especially in terms of inhibiting the oligomerization of isocyanides, which results in a lower product selectivity in many transition-metal-catalyzed reactions of isocyanides.

Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1.

The Keap1-Nrf2 system plays a central role in the oxidative stress response; however, the identity of the reactive oxygen species sensor within Keap1 remains poorly understood. Here, we show that a Keap1 mutant lacking 11 cysteine residues retains the ability to target Nrf2 for degradation, but it is unable to respond to cysteine-reactive Nrf2 inducers. Of the 11 mutated cysteine residues, we find that 4 (Cys226/613/622/624) are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, reveal that Keap1 uses the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. This sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses.

Reductive Rearrangement of Tetraphenyldiphosphine Disulfide To Trigger the Bisthiophosphinylation of Alkenes and Alkynes.

The facile synthesis of organophosphorus compounds is of great importance for the development of new synthetic methods by using air-stable sources of phosphorus. In this respect, a synthetic method that is based on a reductive rearrangement and is capable of converting air-stable pentavalent phosphorus compounds into reactive trivalent phosphorus compounds is a powerful tool. Tetraphenyldiphosphine disulfide, which is a shelf-stable solid, was the focus of this study, and it was shown to undergo reductive rearrangement to trigger the bisthiophosphinylation of a variety of alkenes, such as terminal, cyclic, internal, and branched alkenes, 1,3-dienes, and terminal alkynes when exposed to light without any catalyst, base, or additive.

Synthesis of Bis(phosphanyl)alkane Monosulfides by the Addition of Diphosphane Monosulfides to Alkenes under Light.

Bis-phosphanated compounds are regarded as the most ubiquitous privileged ligand structures in transition-metal catalysis. The development of highly atom economical reactions is of great importance for their syntheses because less atom economical methods often require complicated purification procedures under inert atmospheres to remove excess starting materials and byproducts. Herein, the photoinduced addition reactions of diphosphane monosulfides bearing PV (S)-PIII single bonds to alkenes is disclosed. These reactions require only equimolar amounts of the diphosphane monosulfide relative to the alkene and facilitate highly selective introduction of two different types of phosphorus-containing groups, such as thiophosphoryl and phosphanyl groups, into a variety of alkenes without any catalyst, base, or additive.