The highly developed symbiotic system between the solar-powered nudibranch Pteraeolidia semperi and Symbiodiniacean algae.

The intricate coexistence of Symbiodiniacean algae with a diverse range of marine invertebrates underpins the flourishing biodiversity observed within coral reef ecosystems. However, the breakdown of Symbiodiniaceae-host symbiosis endangers these ecosystems, necessitating urgent study of the symbiotic mechanisms. The symbiosis between nudibranchs and Symbiodiniaceae has been identified as an efficacious model for examining these mechanisms, yet a comprehensive understanding of their histological structures and cellular processes remains elusive. A meticulous histological exploration of the nudibranch Pteraeolidia semperi, employing optical, fluorescence, and electron microscopy, has revealed fine tubules extending to the body surface, with associated epithelial cells having been shown to adeptly encapsulate Symbiodiniaceae intracellularly. By tracing the stages of the "bleaching" in nudibranchs, it was inferred that algal cells, translocated via the digestive gland, are directly phagocytosed and expelled by these epithelial cells. Collectively, these insights contribute substantially to the scholarly discourse on critical marine symbiotic associations.

Orphan gene expressed in flame cone cells uniquely found in seahorse epithelium.

The seahorse is one of the most unique teleost fishes in its morphology. The body is surrounded by bony plates and spines, and the male fish possess a brooding organ, called the brood pouch, on their tail. The surfaces of the brood pouch and the spines are surrounded by characteristic so-called flame cone cells. Based on our histological observations, flame cone cells are present in the seahorse Hippocampus abdominalis, but not in the barbed pipefish Urocampus nanus or the seaweed pipefish Syngnathus schlegeli, both of which belong to the same family as the seahorse. In the flame cone cells, we observed expression of an "orphan gene" lacking homologs in other lineages. This gene, which we named the proline-glycine rich (pgrich) gene, codes for an amino acid sequence composed of repetitive units. In situ hybridization and immunohistochemical analyses detected pgrich-positive signals from the flame cone cells. Based on a survey of the genome sequences of 15 teleost species, the pgrich gene is only found from some species of Syngnathiformes (namely, the genera Syngnathus and Hippocampus). The amino acid sequence of the seahorse PGrich is somewhat similar to the sequence deduced from the antisense strand of elastin. Furthermore, there are many transposable elements around the pgrich gene. These results suggest that the pgrich gene may have originated from the elastin gene with the involvement of transposable elements and obtained its novel function in the flame cone cells during the evolution of the seahorse.

BAY61-3606 Alters snRNP Composition and Enhances Usage of Suboptimal Splice Acceptor Site.

Intron recognition by the spliceosome mainly depends on conserved intronic sequences such as 5' splice sites, 3' splice sites, and branch sites. Therefore, even substitution of just a single nucleotide in a 5' or 3' splice site abolishes the splicing at the mutated site and leads to cryptic splice site usage. A number of disease-causative mutations have been found in 5' and 3' splice sites, but the genes with these mutations still maintain the correct protein-coding sequence, so recovery of splicing at the mutated splice site may produce a normal protein. Mutations in the spliceosome components have been shown to change the balance between the conformational transition and disassembly of the spliceosome, which affects the decision about whether the reaction of the incorporated substrate will proceed. In addition, the lower disassembly rate caused by such mutations induces splicing of the mutated splice site. We hypothesized that small compounds targeting the spliceosome may include a compound mimicking the effect of those mutations. Thus, we screened a small-compound library and identified a compound, BAY61-3606, that changed the cellular small nuclear ribonucleoprotein composition and also showed activity of enhancing splicing at the mutated 3' splice site of the reporter gene, as well as splicing at the suboptimal 3' splice site of endogenous cassette exons. These results indicate that further analysis of the mechanism of action of BAY61-3606 could enable modulation of the fidelity of splicing.

Effects of Pharmacist-Led Dementia Care Round Interventions on the Use of Sleep Medications.

Objective: To investigate pharmacist-led dementia care rounds (PDRs) and their effect on the use of sleep medications, including the number and content of prescription suggestions during PDRs and use of sleep medications at the time of hospitalization and discharge.Methods: This was a retrospective observational study of inpatients who received PDR intervention at a hospital in Japan from January 1 to December 31, 2020. The PDR team, consisting of a pharmacist and dementia care nurse, made prescription suggestions through the attending nurse, and the attending physician made the decision to change the prescription. Use of sleep medication was investigated by classifying patients into 2 groups: those for whom prescription suggestions from PDRs were accepted and those for whom they were rejected.Results: PDRs were conducted 1,164 times with 418 patients, and prescription suggestions were made 330 times (28.4%) for 173 (41.4%) patients. Of these, 234 (70.9%) prescription suggestions were accepted. At the time of discharge, the percentage of patients using benzodiazepine-based sleep medications was 3.1% in the accepted group and 11.9% in the rejected group. The percentage of patients using non-benzodiazepine-based sleep medications was 22.1% in the accepted group and 9.5% in the rejected group. Further, the percentage of patients using non-γ-aminobutyric acid receptor agonist drugs as sleep medications was 9.2% in the accepted group and 2.4% in the rejected group. The results show that the percentage of patients using benzodiazepine-based sleep medications was significantly lower in the accepted group than in the rejected group (P = .022).Conclusions: PDR intervention contributed to the appropriate use of sleep medications, with nearly 30% of prescription suggestions. PDRs may play an important role in the appropriate use of sleep medications, and active participation of pharmacists in dementia care is necessary.

Discovery of tricyclic HIV-1 integrase-LEDGF/p75 allosteric inhibitors by intramolecular direct arylation reaction.

We have been conducting exploratory research to develop human immunodeficiency virus type-1 (HIV-1) integrase-LEDGF/p75 allosteric inhibitors (INLAIs). Here, we report on a newly designed compound with a tricyclic scaffold that shows promise as an inhibitor. Various scaffolds were synthesized by intramolecular direct arylation reaction to fix the position of a lipophilic side chain required for antiviral activity. Among these, the compound having an N-mesyl dihydrophenanthridine ring showed the best antiviral activity. Compound 42i, prepared by side chain optimization of the C-4 and C-6 positions, exhibited high antiviral activity against wild-type (WT) and the T174I mutant (EC50 (WT) = 4.6 nM, EC50 (T174I) = 83 nM) with a good PK profile. Based on co-crystal structural analysis of compound 42i and WT HIV-1 IN CCD, we discuss the interaction important for high antiviral activity.

Skeletal muscle releases extracellular vesicles with distinct protein and microRNA signatures that function in the muscle microenvironment.

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and are present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish skeletal muscle-derived EVs (SkM-EVs) from others, whether skeletal muscle releases EVs in vivo and how much SkM-EVs account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings confirm previous studies showing that skeletal muscle cells release exosome-like EVs with specific protein and miRNA profiles in vivo and suggest that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

Azoxystrobin amine: A novel azoxystrobin degradation product from Bacillus licheniformis strain TAB7.

Azoxystrobin (AZ) is a broad-spectrum synthetic fungicide widely used in agriculture globally. However, there are concerns about its fate and effects in the environment. It is reportedly transformed into azoxystrobin acid as a major metabolite by environmental microorganisms. Bacillus licheniformis strain TAB7 is used as a compost deodorant in commercial compost and has been found to degrade some phenolic and agrochemicals compounds. In this article, we report its ability to degrade azoxystrobin by novel degradation pathway. Biotransformation analysis followed by identification by electrospray ionization-mass spectrometry (MS), high-resolution MS, and nuclear magnetic resonance spectroscopy identified methyl (E)-3-amino-2-(2-((6-(2-cyanophenoxy)pyrimidin-4-yl)oxy)phenyl)acrylate, or (E)-azoxystrobin amine in short, and (Z) isomers of AZ and azoxystrobin amine as the metabolites of (E)-AZ by TAB7. Bioassay testing using Magnaporthe oryzae showed that although 40 µg/mL of (E)-AZ inhibited 59.5 ± 3.5% of the electron transfer activity between mitochondrial Complexes I and III in M. oryzae, the same concentration of (E)-azoxystrobin amine inhibited only 36.7 ± 15.1% of the activity, and a concentration of 80 µg/mL was needed for an inhibition rate of 56.8 ± 7.4%, suggesting that (E)-azoxystrobin amine is less toxic than the parent compound. To our knowledge, this is the first study identifying azoxystrobin amine as a less-toxic metabolite from bacterial AZ degradation and reporting on the enzymatic isomerization of (E)-AZ to (Z)-AZ, to some extent, by TAB7. Although the fate of AZ in the soil microcosm supplemented with TAB7 will be needed, our findings broaden our knowledge of possible AZ biotransformation products.

Dihydrodibenzothiepine: Promising hydrophobic pharmacophore in the influenza cap-dependent endonuclease inhibitor.

This work describes a set of discovery research studies of an influenza cap-dependent endonuclease (CEN) inhibitor with a carbamoyl pyridone bicycle (CAB) scaffold. Using influenza CEN inhibitory activity, antiviral activity and pharmacokinetic (PK) parameters as indices, structure activity relationships (SAR) studies were performed at the N-1 and N-3 positions on the CAB scaffold, which is a unique template to bind two metals. The hydrophobic substituent at the N-1 position is extremely important for CEN inhibitory activity and antiviral activity, and dihydrodibenzothiepine is the most promising pharmacophore. The compound (S)-13i showed potent virus titer reduction over oseltamivir phosphate in an in vivo mouse model. The CAB compound described herein served as the lead compound of baloxavir marboxil with a tricyclic scaffold, which was approved in Japan and the USA in 2018.

Discovery of novel integrase-LEDGF/p75 allosteric inhibitors based on a benzene scaffold.

We report herein the discovery of novel integrase-LEDGF/p75 allosteric inhibitors (INLAIs) based on a benzene scaffold 3. This scaffold can extend substituents from the C1 position unlike the common pyridine scaffolds 2. Structure-activity relationship studies showed that the sulfonamide linker at the C1 position was important for the antiviral activity. Interaction between sulfonamide and Q95 was observed by X-ray crystallography. Compound 31h showed more potent antiviral activity (EC50 (NL432) = 3.9 nM) than BI-224436 (EC50 (NL432) = 56 nM), suggesting the potential of the newly designed scaffold 3.

Synthesis and SAR Study of Carbamoyl Pyridone Bicycle Derivatives as Potent Inhibitors of Influenza Cap-dependent Endonuclease.

The medicinal chemistry and structure-activity relationships (SAR) for a novel series of carbamoyl pyridone bicycle (CAB) compounds as influenza Cap-dependent endonuclease (CEN) inhibitors are disclosed. Substituent effects were evaluated at the C (N)-1, N-3, and C-7 positions of the CAB ring system using a docking study. Submicromolar EC50 values were achieved in the cellular assay with C-7-unsubstituted CAB which possessed a benzhydryl group on either the C-1 or the N-1 position. An N-3 substituent was found to be critical for the plasma protein binding effect in vitro, and the CAB-N analogue 2v exhibited reasonable total clearance (CLtot). More importantly, compound 2v displayed significant efficacy in a mouse model infected with influenza viruses.

Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor.

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.

Comparison of Egg Envelope Thickness in Teleosts and its Relationship to the Sites of ZP Protein Synthesis.

Teleost egg envelope generally consists of a thin outer layer and a thick inner layer. The inner layer of the Pacific herring egg envelope is further divided into distinct inner layers I and II. In our previous study, we cloned four zona pellucida (ZP) proteins (HgZPBa, HgZPBb, HgZPCa, and HgZPCb) from Pacific herring, two of which (HgZPBa and HgZPCa) were synthesized in the liver and two (HgZPBb and HgZPCb) in the ovary. In this study, we raised antibodies against these four proteins to identify their locations using immunohistochemistry. Our results suggest that inner layer I is constructed primarily of HgZPBa and Ca, whereas inner layer II consists primarily of HgZPBa. HgZPBb and Cb were minor components of the envelope. Therefore, the egg envelope of Pacific herring is primarily composed of liver-synthesized ZP proteins. A comparison of the thickness of the fertilized egg envelopes of 55 species suggested that egg envelopes derived from liver-synthesized ZP proteins tended to be thicker in demersal eggs than those in pelagic eggs, whereas egg envelopes derived from ovarian-synthesized ZP proteins had no such tendency. Our comparison suggests that the prehatching period of an egg with a thick egg envelope is longer than that of an egg with a thin egg envelope. We hypothesized that acquisition of liver-synthesized ZP proteins during evolution conferred the ability to develop a thick egg envelope, which allowed species with demersal eggs to adapt to mechanical stress in the prehatching environment by thickening the egg envelope, while pelagic egg envelopes have remained thin.

Discovery of novel 5-hydroxy-4-pyridone-3-carboxy acids as potent inhibitors of influenza Cap-dependent endonuclease.

We report the discovery of a novel series of influenza Cap-dependent EndoNuclease (CEN) inhibitors based on the 4-pyridone-carboxylic acid (PYXA) scaffold, which were found from our chelate library. Our SAR research revealed the lipophilic domain to be the key to CEN inhibition. In particular, the position between the chelate and the lipophilic domain in the derivatives was essential for enhancing the potency. Our study, based on virtual modeling, led to the identification of 2y as a potent CEN inhibitor with an IC50 of 5.12nM.

Replication of partial double-stranded RNAs by Qß replicase.

Qß replicase, an RNA-dependent RNA polymerase of bacteriophage Qß, uses single-stranded RNA as a template to synthesize the complementary strand. A single-stranded RNA template may contain rigid secondary structures, such as long stems, intermolecular double-stranded RNA regions. Presently, the effect of the size of such double-stranded regions on the replication of RNA by Qß replicase is unknown. In this study, we prepared RNA templates hybridized with complementary RNA or DNA strands of various sizes and analyzed their replication by Qß replicase. We found that Qß replicase synthesizes the complementary strand as long as the template RNA is hybridized with no more than 200 nt fragments, although the replication amounts were decreased. This is important information to evaluate processivity of Qß replicase.

Glypican-5 Increases Susceptibility to Nephrotic Damage in Diabetic Kidney.

Type 2 diabetes mellitus is a leading health issue worldwide. Among cases of diabetes mellitus nephropathy (DN), the major complication of type 2 diabetes mellitus, the nephrotic phenotype is often intractable to clinical intervention and demonstrates the rapid decline of renal function to end-stage renal disease. We recently identified the gene for glypican-5 (GPC5), a cell-surface heparan sulfate proteoglycan, as conferring susceptibility for acquired nephrotic syndrome and additionally identified an association through a genome-wide association study between a variant in GPC5 and DN of type 2 diabetes mellitus. In vivo and in vitro data showed a progressive increase of GPC5 in type 2 DN along with severity; the excess was derived from glomerular mesangial cells. In this study, diabetic kidney showed that accumulation of fibroblast growth factor (Fgf)2 strikingly induced progressive proteinuria that was avoided in Gpc5 knockdown mice. The efficacy of Gpc5 inhibition was exerted through expression of the Fgf receptors 3 and 4 provoked in the diabetic kidney attributively. Extraglomerular Fgf2 was pathogenic in DN, and the deterrence of Gpc5 effectively inhibited the glomerular accumulation of Fgf2, the subsequent increase of mesangial extracellular matrix, and the podocytes' small GTPase activity. These findings elucidate the pivotal role of GPC5, identified as a susceptible gene in the genome-wide association study, in hyperglycemia-induced glomerulopathy.

Molecular events in adaptive evolution of the hatching strategy of ovoviviparous fishes.

Ovoviviparous fish, whose embryonic development and hatching take place in the maternal body, is one of the good model organisms for studying adaptive evolution. Using genome database of the ovoviviparous platy Xiphophorus maculatus, we tried to search hatching enzyme genes (high choriolytic enzyme HCE and low choriolytic enzyme LCE) and egg envelope protein genes (choriogenin H, Hm, and L). Analysis of genes co-localized with them confirmed that shared synteny was found between platy and medaka genome. Both hatching enzyme genes HCE and LCE were pseudogenized in platy. In addition, one of the three choriogenin genes Hm was completely lost from the genome, the other two genes H and L encoded functional proteins. On the other hand, the expression of H and L was very low as compared to oviparous fishes, and the platy egg envelope was extremely thinner. Considering that ovoviviparous fish embryos are protected in the maternal body, an importance of egg envelope for protection of egg/embryo would be reduced in the ovoviviparous fishes. Platy embryos would escape from their thin egg envelope without help of hatching enzymes. In another ovoviviparous fish, black rockfish belonging to different order from the platy, one of the hatching enzyme genes has been reported to be pseudogenized, that is, the embryo of black rockfish can escape from egg envelope by only one hatching enzyme HCE. Adaptive evolution of the hatching strategy of ovoviviparous teleosts may be established by pseudogenization of hatching enzyme genes and/or lowering of expression and/or pseudogenization of hatching enzyme and egg envelope genes.

A split luciferase-based reporter for detection of a cellular macromolecular complex.

The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling U1, U2, U4, U5, and U6 small nuclear RNPs (snRNPs). To process large numbers of introns with a limited number of snRNPs, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage. However, the mechanism that maintains cellular snRNP levels is unknown. Molecules that modulate cellular snRNP levels may help to define this mechanism but are not available. Therefore, the goal of the current study was to develop a reporter for snRNP levels using split luciferase based on proteomic analysis of snRNPs. We constructed an expression library of a luciferase fragment fused to core components of U5 snRNP and used it to isolate pre-mRNA processing factor 6 (PRPF6) and small nuclear ribonucleoprotein 40 kDa (U5-40K) that specifically reconstitute luciferase activity in the U5 snRNP complex. Here we show that this reporter detects the effects of small molecules on the levels of the U5 snRNP reporter protein complex. Our approach provides an alternative assay to discover small molecules targeting a macromolecular complex when the structure of the complex is not precisely identified.