Elucidation of Substituent-Responsive Reactivities via Hierarchical and Asymmetric Assemblies in Crystalline p-Quinodimethane Derivatives.

We propose a mechanism for substituent-responsive reactivities of p-quinodimethane derivatives with four ester groups through their hierarchical and asymmetric assembly modes. Four asymmetric 7,8,8-tris(methoxycarbonyl)-p-quinodimethanes with a 7-positioned ethoxycarbonyl (2 a(H)), 2'-fluoroethoxycarbonyl (2 b(F)), 2'-chloroethoxycarbonyl (2 c(Cl)), or 2'-bromoethoxycarbonyl (2 d(Br)) were synthesized and crystallized. 2 a(H), 2 b(F) and 2 d(Br) afforded only one shape crystal, while 2 c(Cl) did two polymorphic 2 c(Cl)-α and 2 c(Cl)-ß. UV-irradiation induced topochemical polymerization for 2 a(H), no reactions for 2 b(F) and 2 c(Cl)-α, and [6+6] photocycloaddition dimerization for 2 c(Cl)-ß and 2 d(Br). Such substituent-responsive reactivities and crystal structures were compared with those of the known symmetric 7,7,8,8-tetrakis(alkoxycarbonyl)-p-quinodimethanes such as 7,7,8,8-tetrakis(methoxycarbonyl)- (1 a(Me)-α and 1 a(Me)-ß), 7,7,8,8-tetrakis(ethoxycarbonyl)- (1 b(Et)), and 7,7,8,8-tetrakis(bromoethoxycarbonyl)- (1 c(BrEt)). The comparative study clarified that the reactivities and crystal structures are classified into four types that link to each other. This linkage is understandable when we analyze the crystal structures through the following hierarchical and asymmetric assemblies; conformers, dimers, one dimensional (1D)-columns, two dimensional (2D)-sheets, and three dimensional (3D)-stacked sheets (3D-crystals). This supramolecular viewpoint is supported by intermolecular interaction energies among neighbored molecules with the density functional theory (DFT) calculation. Such research enables us to elucidate the substituent-responsive reactivities of the crystals, and reminds us of the selection of the right path in a so-called "maze game".

Complete genome sequence of a new orthotospovirus associated with ringspot in Fatsia japonica.

Fatsia japonica is an evergreen shrub native to Japan. For decades, virus-like ringspot symptoms have been observed on leaves of F. japonica in Japan; however, previous attempts to identify the causal agents have been unsuccessful. In this study, we detected an orthotospovirus-like sequence in symptomatic F. japonica plants using RNA sequencing analysis. The complete nucleotide sequences of the L, M, and S segments of the virus were determined using conventional sequencing strategies. The virus had a typical orthotospovirus genome structure, and the putative nucleocapsid protein showed the highest sequence identity to that of groundnut chlorotic fan-spot virus, with 83.7% identity at the amino acid level (which is below the 90% species demarcation cutoff for the genus Orthotospovirus). Although we could not confirm the pathogenicity of the virus in F. japonica due to difficulties associated with mechanical inoculation, its association with the observed symptoms was suggested by the fact that the virus was detected only in symptomatic leaf areas. Based on these results, we consider this virus, which we have named "Fatsia japonica ringspot-associated virus" (FjRSaV), to be the first representative of a new orthotospovirus species, for which we propose the binomial "Orthotospovirus fatsiae".

Complete genome sequence of viola mottle virus, revealing its synonymous relationship to tulip virus X.

Viola mottle virus (VMoV) was discovered in Viola odorata showing symptoms of reduced growth, leaf mottling, and whitish stripes on flowers in northern Italy in 1977. This virus has been provisionally classified as a member of the genus Potexvirus based on its morphological, serological, and biological characteristics. However, since genetic information of VMoV has never been reported, the taxonomic status of this virus is unclear. Here, we report the first complete genome sequence of VMoV to clarify its taxonomic position. Its genomic RNA is 6,052 nucleotides long, excluding the 3'-terminal poly(A) tail, and has five open reading frames (ORFs) typical of potexviruses. Among potexviruses, VMoV showed the most similarity to tulip virus X (TVX) with 81.1-81.2% nucleotide and 90.4-90.7% amino acid sequence identity in ORF1 and 82.9-83.5% nucleotide and 93.2-95.2% amino acid sequence identity in ORF5. These values are much higher than the species demarcation threshold for the genus. Phylogenetic analysis also indicated that VMoV is nested within the clade of TVX isolates. These data demonstrate that VMoV and TVX are members of the same species.

Ecdysteroid ingestion suppresses carbohydrate hydrolysis in larvae of the silkworm Bombyx mori.

Ecdysteroids are widely found in terrestrial organisms, including insects, crustaceans, fungi, and plants. The function of ecdysteroids has been extensively studied in insects for decades because ecdysteroids regulate metamorphosis. In plants, in contrast, ecdysteroids (called phytoecdysteroids) do not show apparent hormonal activity and their function remains unclear. However, it has been proposed that phytoecdysteroids have an antifeedant function. Ecdysteroid ingestion disrupts insect development and alters behavior to deter insect feeding, resulting in reduced plant damage by the insect. These points of view are generally accepted, but the function of phytoecdysteroids in specific contexts has not been unveiled. In the present study, we used larvae of the silkworm, Bombyx mori, to investigate the biological significance of phytoecdysteroids. To mimic the situation where larvae consume plant leaves that contain phytoecdysteroids, 26 or 30 larvae were fed the diet containing ecdysteroid or the control diet. We show that ecdysteroid ingestion dramatically suppressed carbohydrate processing in the larval midgut to reduce the nutritional value of the ingested diet. Based on the present results, we propose a new explanation of phytoecdysteroid function: ingested ecdysteroids may lead to the erroneous perception that the plant is poor in nutrients and consequently result in cessation of feeding.

Catalytic Asymmetric Iodoesterification of Simple Alkenes.

Catalytic asymmetric iodoesterification of simple alkenes was achieved using a dinuclear zinc-3,3'-(R,S,S)-bis(aminoimino)binaphthoxide (di-Zn) complex. For iodoesterification using p-methoxybenzoic acid, the N-iodonaphthalenimide (NIN)-I2 system was effective for producing iodoesters in a highly enantioselective manner. The synthetic utility of chiral iodo-p-methoxybenzoates was also demonstrated. The quartet of metal ionic bond, hydrogen bond, halogen bond, and π-π stacking is harmonized on the single reaction sphere of di-Zn catalyst for enabling the highly enantioselective catalytic asymmetric iodoesterification of simple alkenes for the first time.

Catalysis Based on C-I⋠⋠⋠π Halogen Bonds: Electrophilic Activation of 2-Alkenylindoles by Cationic Halogen-Bond Donors for [4+2] Cycloadditions.

Homo- and cross-[4+2] cycloadditions of 2-alkenylindoles, catalyzed by cationic halogen-bond donors, were developed. Under mild reaction conditions, 3-indolyl-substituted tetrahydrocarbazole derivatives were obtained in good to excellent yields. Experimental and quantum calculation studies revealed that the electrophilic activation of 2-alkenylindoles was achieved by C-I⋅⋅⋅π halogen bonds.

Chemical Bonding in Polarised Push-Pull Ethylenes.

1,1-Diamino-2,2-bis(triflyl)ethylenes with both twisted and planar structures around the partial "C=C" bond were synthesised. Bonding properties in these compounds were analysed by an experimental approach using high-resolution X-ray diffraction data treated with X-ray wavefunction refinement (XWR). In the twisted compound, a dominant contribution of the charge-separated resonance structure was revealed. On the contrary, the nearly planar compound still showed π-bonding character, however, with a considerable contribution of the charge-separated resonance structure.

Inter-progenitor pool wiring: An evolutionarily conserved strategy that expands neural circuit diversity.

Diversification of neuronal types is key to establishing functional variations in neural circuits. The first critical step to generate neuronal diversity is to organize the compartmental domains of developing brains into spatially distinct neural progenitor pools. Neural progenitors in each pool then generate a unique set of diverse neurons through specific spatiotemporal specification processes. In this review article, we focus on an additional mechanism, 'inter-progenitor pool wiring', that further expands the diversity of neural circuits. After diverse types of neurons are generated in one progenitor pool, a fraction of these neurons start migrating toward a remote brain region containing neurons that originate from another progenitor pool. Finally, neurons of different origins are intermingled and eventually form complex but precise neural circuits. The developing cerebral cortex of mammalian brains is one of the best examples of inter-progenitor pool wiring. However, Drosophila visual system development has revealed similar mechanisms in invertebrate brains, suggesting that inter-progenitor pool wiring is an evolutionarily conserved strategy that expands neural circuit diversity. Here, we will discuss how inter-progenitor pool wiring is accomplished in mammalian and fly brain systems.

Wnt Signaling Specifies Anteroposterior Progenitor Zone Identity in the Drosophila Visual Center.

UNLABELLED: During brain development, various types of neuronal populations are produced from different progenitor pools to produce neuronal diversity that is sufficient to establish functional neuronal circuits. However, the molecular mechanisms that specify the identity of each progenitor pool remain obscure. Here, we show that Wnt signaling is essential for the specification of the identity of posterior progenitor pools in the Drosophila visual center. In the medulla, the largest component of the visual center, different types of neurons are produced from two progenitor pools: the outer proliferation center (OPC) and glial precursor cells (GPCs; also known as tips of the OPC). We found that OPC-type neurons are produced from the GPCs at the expense of GPC-type neurons when Wnt signaling is suppressed in the GPCs. In contrast, GPC-type neurons are ectopically induced when Wnt signaling is ectopically activated in the OPC. These results suggest that Wnt signaling is necessary and sufficient for the specification of the progenitor pool identity. We also found that Homothorax (Hth), which is temporally expressed in the OPC, is ectopically induced in the GPCs by suppression of Wnt signaling and that ectopic induction of Hth phenocopies the suppression of Wnt signaling in the GPCs. Thus, Wnt signaling is involved in regionalization of the fly visual center through the specification of the progenitor pool located posterior to the medulla by suppressing Hth expression. SIGNIFICANCE STATEMENT: Brain consists of considerably diverse neurons of different origins. In mammalian brain, excitatory and inhibitory neurons derive from the dorsal and ventral telencephalon, respectively. Multiple progenitor pools also contribute to the neuronal diversity in fly brain. However, it has been unclear how differences between these progenitor pools are established. Here, we show that Wnt signaling, an evolutionarily conserved signaling, is involved in the process that establishes the differences between these progenitor pools. Because ß-catenin signaling, which is under the control of Wnt ligands, specifies progenitor pool identity in the developing mammalian thalamus, Wnt signaling-mediated specification of progenitor pool identity may be conserved in insect and mammalian brains.

eyeless/Pax6 controls the production of glial cells in the visual center of Drosophila melanogaster.

Pax6 is known as a neurogenic factor in the development of the central nervous system and regulates proliferation of neuronal progenitor cells and promotes neuronal differentiation. In addition to neurogenesis, Pax6 is also involved in the specification and maturation of glial cells. Here, we show that Eyeless (Ey), Drosophila homolog of Pax6, regulates the production of glial cells in the brain. In the developing fly visual center, the production of neurons and glial cells are controlled by the temporal transcription factors that are sequentially expressed in neuroblasts (NBs). Among them, NBs of the last temporal window produce astrocyte-like glial cells. Ey is strongly expressed in the middle aged NBs, whose temporal window is earlier compared with glia producing older NBs. Weak Ey expression is also detected in the glia producing NBs. Our results suggest that Ey expression in the middle aged NBs indirectly control gliogenesis from the oldest NBs by regulating other temporal transcription factors. Additionally, weak Ey expression in the NBs of last temporal window may directly control gliogenesis. Ey is also expressed in neurons produced from the NBs of Ey-positive temporal window. Interestingly, neuron-specific overexpression of Ey causes significant increase in glial cells suggesting that neuronal expression of Ey may also contribute to gliogenesis. Thus, Pax6-dependent regulation of astrocyte-like glial development is conserved throughout the animal kingdom.

Characterization of Ocular Iontophoretic Drug Transport of Ionic and Non-ionic Compounds in Isolated Rabbit Cornea and Conjunctiva.

Ocular iontophoresis (IP) in isolated rabbit cornea and conjunctiva was examined in terms of transport enhancement, tissue viability and integrity using electrophysiological parameters by the Ussing-type chamber technique. Lidocaine hydrochloride (LC, a cationic compound), sodium benzoate (BA, anionic compound), and fluorescein isothiocyanate labeled dextran (molecular weight 4400 Da, FD-4, hydrophilic large compound) were used as model permeants. Direct electric current was applied at 0.5-5.0 mA/cm(2) for the cornea and 0.5-20 mA/cm(2) for the conjunctiva for 30 min. LC and BA fluxes across the cornea and conjunctiva were significantly increased by the application of electric current up to 2.3- and 2.5-fold and 4.0- and 3.4-fold, respectively, and returned to their baseline level on stopping the current. Furthermore, a much higher increase by IP application was obtained for the FD-4 transport. The increased FD-4 flux in the conjunctiva returned to baseline on stopping the current, whereas the flux in the cornea was sustained at a higher level after stopping the current. The transepithelial electric resistance of the cornea and conjunctiva was lowered by electric current application but fully recovered after stopping the current up to 2.0 mA/cm(2) for the cornea and 10 mA/cm(2) for the conjunctiva, suggesting that the corneal and conjunctival viability and integrity are maintained even after application of these current densities. These results indicate that ocular IP may be a useful non-invasive technique to achieve drug delivery of hydrophilic large molecules into the eyes.

Waves of differentiation in the fly visual system.

Sequential progression of differentiation in a tissue or in multiple tissues in a synchronized manner plays important roles in development. Such waves of differentiation are especially important in the development of the Drosophila visual system, which is composed of the retina and the optic lobe of the brain. All of the components of the fly visual system are topographically connected, and each ommatidial unit in the retina corresponds to a columnar unit in the optic lobe, which is composed of lamina, medulla, lobula and lobula plate. In the developing retina, the wave of differentiation follows the morphogenetic furrow, which progresses in a posterior-to-anterior direction. At the same time, differentiation of the lamina progresses in the same direction, behind the lamina furrow. This is not just a coincidence: differentiated photoreceptor neurons in the retina sequentially send axons to the developing lamina and trigger differentiation of lamina neurons to ensure the progression of the lamina furrow just like the furrow in the retina. Similarly, development of the medulla accompanies a wave of differentiation called the proneural wave. Thus, the waves of differentiation play important roles in establishing topographic connections throughout the fly visual system. In this article, we review how neuronal differentiation and connectivity are orchestrated in the fly visual system by multiple waves of differentiation.

A temporal mechanism that produces neuronal diversity in the Drosophila visual center.

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf), correspond to types of medulla neurons. Here, we examine the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, we searched for transcription factors that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates (Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)). The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. Our results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequentially expressed in NBs.

Neurogenesis and neuronal circuit formation in the Drosophila visual center.

The Drosophila optic lobe is composed of a wide variety of neurons that form laminar structures and columnar units. The fly optic lobe shares structural features with the mammalian brain, and fly genetics allow precise genetic manipulations. Thus, the Drosophila visual center is an excellent model for studying the mechanisms underlying the establishment of a functional neuronal circuit during brain development. However, little is understood about the developmental mechanisms that produce neuronal diversity and establish neuronal circuits in the medulla, the largest component of the optic lobe. Our recent research revealed key features of medulla development, such as birth-order-dependent specification of neuronal types and the subdivision of the medulla primordium into concentric zones, which is characterized by the expression of four transcription factors. Here, we review recent investigations into the development of the medulla and discuss the mechanisms that establish functional neuronal circuits.

Gestational intermittent hypoxia reduces mandibular growth with decreased Sox9 expression and increased Hif1a expression in male offspring rats.

Introduction: Maternal obstructive sleep apnea (OSA) during pregnancy is the risk factor for impaired fetal growth with low birth weight in the offspring. However, it is unclear whether gestational intermittent hypoxia (IH, a hallmark of maternal OSA) has long-term detrimental consequences on the skeletal development of offspring. This study aimed to investigate postnatal maxillofacial bone growth and cartilage metabolism in male and female offspring that were exposed to gestational IH. Methods: Mother rats underwent IH at 20 cycles/h (nadir, 4% O2; peak, 21% O2; 0% CO2) for 8 h per day during gestational days (GD) 7-20, and their male and female offspring were analyzed postnatally at 5 and 10 weeks of age. All male and female offspring were born and raised under normoxic conditions. Results: There was no significant difference in whole-body weight and tibial length between the IH male/female offspring and their control counterparts. In contrast, the mandibular condylar length was significantly shorter in the IH male offspring than in the control male offspring at 5 and 10 weeks of age, while there was no significant difference in the female offspring. Real-time polymerase chain reaction (PCR) showed that gestational IH significantly downregulated the mRNA level of SOX9 (a chondrogenesis marker) and upregulated the mRNA level of HIF-1α (a hypoxia-inducible factor marker) in the mandibular condylar cartilage of male offspring, but not in female offspring. Conclusion: Gestational IH induced underdeveloped mandibular ramus/condyles and reduced mRNA expression of SOX9, while enhancing mRNA expression of HIF-1α in a sex-dependent manner.

Steroid hormone-dependent changes in trehalose physiology in the silkworm, Bombyx mori.

Holometabolous insects undergo metamorphosis to reconstruct their body to the adult form during pupal period. Since pupae cannot take any diets from the outside because of a hard pupal cuticle, those insects stock up on nutrients sufficient for successful metamorphosis during larval feeding period. Among those nutrients, carbohydrates are stored as glycogen or trehalose, which is the major blood sugar in insects. The hemolymph trehalose is constantly high during the feeding period but suddenly decreases at the beginning of the prepupal period. It is believed that trehalase, which is a trehalose-hydrolyzing enzyme, becomes highly active to reduce hemolymph trehalose level during prepupal period. This change in the hemolymph trehalose level has been interpreted as the physiological shift from storage to utilization of trehalose at that stage. Although this shift in trehalose physiology is indispensable for energy production required for successful metamorphosis, little is known on the regulatory mechanisms of trehalose metabolism in accordance with developmental progress. Here, we show that ecdysone, an insect steroid hormone, plays essential roles in the regulation of soluble trehalase activity and its distribution in the midgut of silkworm, Bombyx mori. In the end of larval period, soluble trehalase was highly activated in the midgut lumen. This activation was disappeared in the absence of ecdysone and also restored by ecdysone administration. Our present results suggest that ecdysone is essentially required for the changes in the function of the midgut on trehalose physiology as development progresses.

A Reverse-Transcription Loop-Mediated Isothermal Amplification Technique to Detect Tomato Mottle Mosaic Virus, an Emerging Tobamovirus.

Tomato mottle mosaic virus (ToMMV) is an emerging seed-transmissible tobamovirus that infects tomato and pepper. Since the first report in 2013 in Mexico, ToMMV has spread worldwide, posing a serious threat to the production of both crops. To prevent the spread of this virus, early and accurate detection of infection is required. In this study, we developed a detection method for ToMMV based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP). A LAMP primer set was designed to target the genomic region spanning the movement protein and coat protein genes, which is a highly conserved sequence unique to ToMMV. This RT-LAMP detection method achieved 10-fold higher sensitivity than conventional RT-polymerase chain reaction methods and obtained high specificity without false positives for closely related tobamoviruses or healthy tomato plants. This method can detect ToMMV within 30 min of direct sampling of an infected tomato leaf using a toothpick and therefore does not require RNA purification. Given its high sensitivity, specificity, simplicity, and rapidity, the RT-LAMP method developed in this study is expected to be valuable for point-of-care testing in field surveys and for large-scale testing.

A strategy for orthogonal deubiquitination using a bump-and-hole approach.

We have successfully applied a bump-and-hole approach to establish orthogonal deubiquitination in which a ubiquitin substrate variant is specifically targeted by an engineered deubiquitinating enzyme (DUB). This makes it possibe to selectively observe and measure a single type of DUB activity in living cells.

Cloning, monoclonal antibody production, and bodily distribution pattern of a bovine lipocalin.

A bovine lipocalin, previously identified as a putative odorant-binding protein in bovine colostrum (bcOBP), was cloned and expressed, and its monoclonal antibody was established. bcOBP was constantly secreted into milk on day of parturition until at least 10 d postpartum at a concentration of 181±39 µg/L. Besides milk, bcOBP occurred in the nasal mucus, saliva, amniotic fluid, vaginal discharge, and blood plasma. Despite its low concentration, the distribution pattern and the finding that bcOBP harbored a characteristic sequence motif, CxxxC, which is conserved among insect and mammal pheromone binding proteins, suggest that bcOBP functions as a pheromone carrier. The presence of bcOBP in the plasma at varied concentrations depending on the lactation period does not exclude the possibility that bcOBP is secreted into milk from the blood. Cross-reactivity of the monoclonal antibody indicated presence of proteins homologous to bcOBP in the colostrum of farm animals of Cetartiodactyla.

Cutting edge technologies expose the temporal regulation of neurogenesis in the Drosophila nervous system.

During the development of the central nervous system (CNS), extremely large numbers of neurons are produced in a regular fashion to form precise neural circuits. During this process, neural progenitor cells produce different neurons over time due to their intrinsic gene regulatory mechanisms as well as extrinsic mechanisms. The Drosophila CNS has played an important role in elucidating the temporal mechanisms that control neurogenesis over time. It has been shown that a series of temporal transcription factors are sequentially expressed in neural progenitor cells and regulate the temporal specification of neurons in the embryonic CNS. Additionally, similar mechanisms are found in the developing optic lobe and central brain in the larval CNS. However, it is difficult to elucidate the function of numerous molecules in many different cell types solely by molecular genetic approaches. Recently, omics analysis using single-cell RNA-seq and other methods has been used to study the Drosophila nervous system on a large scale and is making a significant contribution to the understanding of the temporal mechanisms of neurogenesis. In this article, recent findings on the temporal patterning of neurogenesis and the contributions of cutting-edge technologies will be reviewed.