Chondroitin sulfate is required for follicle epithelial integrity and organ shape maintenance in Drosophila.

Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, we generated mutants for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Our characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed us to analyze the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. Our observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture.

Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

Evaluation of Tungsten Catalysis among Early Transition Metals for N-Aryl-2,3,4,5-tetraarylpyrrole Synthesis: Modular Access to N-Doped π-Conjugated Material Precursors.

Low-valent tungsten species generated from WCl6 and N,N'-bis(trimethylsilyl)-2,5-dimethyldihydropyrazine (Si-Me2-DHP) promotes the catalytic formation of N-phenyl-2,3,4,5-tetraarylpyrroles 3aa-ka from diarylacetylenes 1a-k and azobenzene (2a). An initial catalyst activation process is a three-electron reduction of WCl6 with Si-Me2-DHP to afford transient 'WCl3' species. Catalytically active bis(imido)tungsten(VI) species via successive one-electron reduction and N═N bond cleavage of 2a was revealed by isolating W(═NPh)2Cl2(PMe2Ph)2 from imidotungsten(V) trichloride and 2a in the presence of PMe2Ph. The superior catalytic activity of the tungsten catalyst was clarified by a density functional theory study: activation energies for the key three steps, [2 + 2]-cycloaddition of W═NPh and diarylacetylene to form (iminoalkylidene)tungsten species, enyne metathesis with second diarylacetylene, and C-N bond formation, are reasonable values for the catalytic reaction at 180 °C. In addition, this tungsten catalyst overcame two distinct deactivation processes: α-enediamido formation and aggregation of the low-valent species, both of which were observed for previously developed vanadium and titanium catalysts. We also demonstrated the synthetic utility of pentaarylpyrroles 3aa and 3ba as well as N-(2-bromophenyl)-2,3,4,5-tetraarylpyrrole 3ab by derivatizing their π-conjugated compounds 9aa, 10ba, and 11ab.

The feedback regulator Nord controls Dpp/BMP signaling via extracellular interaction with Dally in the Drosophila wing.

The Drosophila BMP 2/4 homologue Decapentaplegic (Dpp) acts as a morphogen to regulate diverse developmental processes, including wing morphogenesis. Transcriptional feedback regulation of this pathway ensures tightly controlled signaling outputs to generate the precise pattern of the adult wing. Nevertheless, few direct Dpp target genes have been explored and our understanding of feedback regulation remains incomplete. Here we employ transcriptional profiling following dpp conditional knockout to identify nord, a novel Dpp/BMP feedback regulator. nord mutants generated by CRISPR/Cas9 mutagenesis produce a smaller wing and display low penetrance venation defects. At the molecular level, nord encodes a secreted heparin-binding protein, and we show that its overexpression is sufficient to antagonize Dpp/BMP signaling. Mechanistically, we demonstrate that Nord physically interacts with the Dpp/BMP co-receptor Dally and promotes its degradation. In sum, we propose that Nord fine-tunes Dpp/BMP signaling by regulating Dally availability on the cell surface, with implications for both developmental and disease models.

Impact of cilia-related genes on mitochondrial dynamics during Drosophila spermatogenesis.

Spermatogenesis is a dynamic process of cellular differentiation that generates the mature spermatozoa required for reproduction. Errors that arise during this process can lead to sterility due to low sperm counts and malformed or immotile sperm. While it is estimated that 1 out of 7 human couples encounter infertility, the underlying cause of male infertility can only be identified in 50% of cases. Here, we describe and examine the genetic requirements for missing minor mitochondria (mmm), sterile affecting ciliogenesis (sac), and testes of unusual size (tous), three previously uncharacterized genes in Drosophila that are predicted to be components of the flagellar axoneme. Using Drosophila, we demonstrate that these genes are essential for male fertility and that loss of mmm, sac, or tous results in complete immotility of the sperm flagellum. Cytological examination uncovered additional roles for sac and tous during cytokinesis and transmission electron microscopy of developing spermatids in mmm, sac, and tous mutant animals revealed defects associated with mitochondria and the accessory microtubules required for the proper elongation of the mitochondria and flagella during ciliogenesis. This study highlights the complex interactions of cilia-related proteins within the cell body and advances our understanding of male infertility by uncovering novel mitochondrial defects during spermatogenesis.

Syntheses and Biological Activities of Danicalipin A Derivatives.

Synthesis of three derivatives of danicalipin A, tetrachloride, trisulfate and a fluorescent probe was achieved through Wittig reaction strategy. Toxicity of the derivatives against brine shrimp (Artemia salina) as also investigated to provide useful information for the biological activity; i) less chloride derivative showed similar toxicity to danicalipin A, ii) the amphiphilic property, a characteristic feature of danicalipin A, was crucial because trisulfate considerably decreased the toxicity and iii) fluorescent derivative kept brine shrimp toxicity of danicalipin A.

Decapentaplegic and growth control in the developing Drosophila wing.

As a central model for morphogen action during animal development, the bone morphogenetic protein 2/4 (BMP2/4)-like ligand Decapentaplegic (Dpp) is proposed to form a long-range signalling gradient that directs both growth and pattern formation during Drosophila wing disc development. While the patterning role of Dpp secreted from a stripe of cells along the anterior-posterior compartmental boundary is well established, the mechanism by which a Dpp gradient directs uniform cell proliferation remains controversial and poorly understood. Here, to determine the precise spatiotemporal requirements for Dpp during wing disc development, we use CRISPR-Cas9-mediated genome editing to generate a flippase recognition target (FRT)-dependent conditional null allele. By genetically removing Dpp from its endogenous stripe domain, we confirm the requirement of Dpp for the activation of a downstream phospho-Mothers against dpp (p-Mad) gradient and the regulation of the patterning targets spalt (sal), optomotor blind (omb; also known as bifid) and brinker (brk). Surprisingly, however, third-instar wing blade primordia devoid of compartmental dpp expression maintain relatively normal rates of cell proliferation and exhibit only mild defects in growth. These results indicate that during the latter half of larval development, the Dpp morphogen gradient emanating from the anterior-posterior compartment boundary is not directly required for wing disc growth.

Long-term colonization exceeding six years from early infancy of Bifidobacterium longum subsp. longum in human gut.

BACKGROUND: The importance of the gut microbiota at the early stage of life and their longitudinal effect on host health have recently been well investigated. In particular, Bifidobacterium longum subsp. longum, a common component of infant gut microbiota, appears in the gut shortly after birth and can be detected there throughout an individual's lifespan. However, it remains unclear whether this species colonizes in the gut over the long term from early infancy. Here, we investigated the long-term colonization of B. longum subsp. longum by comparing the genotypes of isolates obtained at different time points from individual subjects. Strains were isolated over time from the feces of 12 subjects followed from early infancy (the first six months of life) up to childhood (approximately six years of age). We also considered whether the strains were transmitted from their mothers' perinatal samples (prenatal feces and postnatal breast milk). RESULTS: Intra-species diversity of B. longum subsp. longum was observed in some subjects' fecal samples collected in early infancy and childhood, as well as in the prenatal fecal samples of their mothers. Among the highlighted strains, several were confirmed to colonize and persist in single individuals from as early as 90 days of age for more than six years; these were classified as long-term colonizers. One of the long-term colonizers was also detected from the corresponding mother's postnatal breast milk. Quantitative polymerase chain reaction data suggested that these long-term colonizers persisted in the subjects' gut despite the existence of the other predominant species of Bifidobacterium. CONCLUSIONS: Our results showed that several strains belonging to B. longum subsp. longum colonized in the human gut from early infancy through more than six years, confirming the existence of long-term colonizers from this period. Moreover, the results suggested that these strains persisted in the subjects' gut while co-existing with the other predominant bifidobacterial species. Our findings also suggested the importance of microbial-strain colonization in early infancy relative to their succession and showed the possibility that probiotics targeting infants might have longitudinal effects. TRIAL REGISTRATION: TRN: ISRCTN25216339 . Date of registration: 11/03/2016. Prospectively registered.

Genetic variation of major histocompatibility complex genes in the endangered red-crowned crane.

Populations that have drastically decreased in the past often have low genetic variation, which may increase the risk of extinction. The genes of major histocompatibility complex (MHC) play an important role in the adaptive immune response of jawed vertebrates. Maintenance of adaptive genetic diversity such as that of MHC genes is important for wildlife conservation. Here, we determined genotypes of exon 3 of MHC class IA genes (MHCIA) and exon 2 of MHC class IIB genes (MHCIIB) to evaluate genetic variation of the endangered red-crowned crane population on Hokkaido Island, Japan, which experienced severe population decline in the past. We identified 16 and 6 alleles of MHCIA and MHCIIB, respectively, from 152 individuals. We found evidence of a positive selection at the antigen-binding sites in MHCIA exon 3 and MHCIIB exon 2. The phylogenetic analyses indicated evidence of trans-species polymorphism among the crane MHC genes. The genetic variability in both classes of MHC genes at the population level was low. No geographic structure was found based on the genetic diversity of microsatellite and MHC genes. Our study provides useful data for the optimal management of the red-crowned crane population in Hokkaido and can contribute to future studies on MHC genes of the continental populations of the red-crowned crane and other crane species.

Gene duplication and concerted evolution of mitochondrial DNA in crane species.

The gene duplication in mitochondrial DNA (mtDNA) has been reported in diverse bird taxa so far. Although many phylogenetic and population genetic analyses of cranes were carried out based on mtDNA diversity, whether mtDNA contains duplicated regions is unknown. To address the presence or absence of gene duplication in cranes and investigate the molecular evolutionary features of crane mtDNA, we analyzed the gene organization and the molecular phylogeny of mtDNA from 13 crane species. We found that the mtDNA in 13 crane species shared a tandem duplicated region, which consists of duplicated sequence sets including cytochrome b (Cytb), NADH6, control region (CR) and three genes of tRNA. The gene order in the duplicated region was identical among all the 13 crane species, and the nucleotide sequences found within each individual showed high similarities. In addition, phylogenetic trees based on homologous sequences of CR and Cytb indicated the possibility of concerted evolution among the duplicated genes. The results suggested that the duplication event occurred in the common ancestor of crane species or some older ancestors.

Low Genetic Variation of Red-Crowned Cranes on Hokkaido Island, Japan, Over the Hundred Years.

The red-crowned crane (Grus japonensis) is recognized internationally as an endangered species. Migratory populations breed in eastern Russia and northeastern China, whereas the resident population inhabits the island of Hokkaido, Japan. Although the population inhabiting Hokkaido had experienced a severe bottleneck by the end of the 19th century, the population size has recovered to about 1500 and continues to increase now thanks to conservation efforts. A previous study reported that no marked genetic differences were seen in the island population, and that the genetic variation of the whole population on Hokkaido was lower than that of the continental population. However, the precise genetic structure of the island population in the past or near present remains unclear. To better understand the spatiotemporal changes in the genetic structure of the island population, we performed mitochondrial DNA (mtDNA) analyses using stuffed specimens (years 1878-2001) and tissue or blood samples (years 1970-2014). We found three haplotypes in the island population, one of which was a novel mtDNA haplotype in 1997 and 2007 samples. In addition, there was no clear difference in the haplotype frequency through the time span. These results suggest that the low genetic variation of the island population persisted for the last hundred years. It is thus nearly impossible for the island population to recover its genetic variation in isolation. Conservation plans for this species should therefore include the promotion of genetic exchanges between the continental and island populations, such as through artificial introduction to Hokkaido.

Longitudinal Investigation of Carriage Rates, Counts, and Genotypes of Toxigenic Clostridium difficile in Early Infancy.

UNLABELLED: Asymptomatic infant carriers of toxigenic Clostridium difficile are suggested to play a role in the transmission of C. difficile infection (CDI) in adults. However, the mode of C. difficile carriage in infants remains to be fully elucidated. We investigated longitudinal changes in carriage rates, counts, and strain types of toxigenic C. difficile in infants. Stools collected from 111 healthy infants in Belgium periodically from birth until the age of 6 months were examined by quantitative PCR targeting 16S rRNA and toxin genes. Toxigenic C. difficile was detected in 18 of 111 infants (16%) in the period up to the age of 6 months. The carriage rate of toxigenic C. difficile remained below 5% until the age of 3 months. The carriage rate increased to 13% 1 week after weaning (average age, 143 days) and reached 16% at the age of 6 months. Counts of toxigenic C. difficile bacteria ranged from 10(4) to 10(8) cells/g of stool. Notably, two infants retained >10(8) cells/g of stool for at least several weeks. Average counts in the 18 infants hovered around 10(7) cells/g of stool from the age of 3 days until the age of 6 months, showing no age-related trend. Genotyping of toxigenic C. difficile isolates from the 18 infants revealed that 11 infants each retained a particular monophyletic strain for at least a month. The genotype most frequently identified was the same as that frequently identified in symptomatic adult CDI patients. Thus, toxigenic C. difficile strains-potential causes of CDI in adults-colonized the infants' intestines. IMPORTANCE: Our study provides longitudinal data on counts and strain types of toxigenic C. difficile in infants. We found that considerable numbers of toxigenic C. difficile bacteria colonized the infants' intestines. The results of strain typing suggest that toxigenic C. difficile carried by healthy infants could be potentially pathogenic to adults. These results and findings are informative not only for ecological studies but also for efforts to prevent or control the spread of CDI in adults.

NMR Assignment for Diaryl Ether Structures (4-O-5 Structures) in Pine Wood Lignin.

A 4-O-5-tetramer lignin model compound carrying ß-O-4 linkages on each of the side-chain moieties was synthesized, as well as 4-O-5-coupled dehydrodiconiferyl alcohol. By comparison with their NMR data, two cross-signals in the HSQC spectrum of pine milled wood lignin recorded in DMSO-d6 were assigned to H2/C2 and H6/C6 correlations on the aromatic rings of 4-O-5-linked units. Although the H2/C2 correlation peak appeared in the same region as syringyl units, nitrobenzene oxidation of the pine lignin did not yield any syringyl-type product, but did release a 4-O-5-type product.

Isolation and characterization of major histocompatibility complex class II B genes in cranes.

In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution.

Diverse galactooligosaccharides consumption by bifidobacteria: implications of ß-galactosidase--LacS operon.

Galactooligosaccharides (GOS) possess prebiotic properties that specifically increase the number of bifidobacteria in the human intestine, thus giving health benefits to the host. Although the bifidogenic effect of GOS has been demonstrated in numerous studies, the utilization of GOS by specific bifidobacteria remains unclear. The goal of our study was to elucidate GOS consumption by specific bifidobacteria and gain insights into the mechanism. First, we examined GOS consumption by 14 bifidobacterial strains belonging to seven different species by comparing growth rate, carbohydrate consumption, and acid production. We then performed a transcription analysis in the case of one strong GOS consumer, Bifidobacterium adolescentis YIT 4011(T), to predict the operon contributing to GOS use. The study indicated the contribution of an operon consisted of LacS symporter and ß-galactosidase to bifidobacterial GOS consumption.

Computer Simulation and Field Experiment for Downlink Multiuser MIMO in Mobile WiMAX System.

The transmission performance for a downlink mobile WiMAX system with multiuser multiple-input multiple-output (MU-MIMO) systems in a computer simulation and field experiment is described. In computer simulation, a MU-MIMO transmission system can be realized by using the block diagonalization (BD) algorithm, and each user can receive signals without any signal interference from other users. The bit error rate (BER) performance and channel capacity in accordance with modulation schemes and the number of streams were simulated in a spatially correlated multipath fading environment. Furthermore, we propose a method for evaluating the transmission performance for this downlink mobile WiMAX system in this environment by using the computer simulation. In the field experiment, the received power and downlink throughput in the UDP layer were measured on an experimental mobile WiMAX system developed in Azumino City in Japan. In comparison with the simulated and experimented results, the measured maximum throughput performance in the downlink had almost the same performance as the simulated throughput. It was confirmed that the experimental mobile WiMAX system for MU-MIMO transmission successfully increased the total channel capacity of the system.

Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii.

Syringyl lignin, an important component of the secondary cell wall, has traditionally been considered to be a hallmark of angiosperms because ferns and gymnosperms in general lack lignin of this type. Interestingly, syringyl lignin was also detected in Selaginella, a genus that represents an extant lineage of the most basal of the vascular plants, the lycophytes. In angiosperms, syringyl lignin biosynthesis requires the activity of ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). Together, these two enzymes divert metabolic flux from the biosynthesis of guaiacyl lignin, a lignin type common to all vascular plants, toward syringyl lignin. Selaginella has independently evolved an alternative lignin biosynthetic pathway in which syringyl subunits are directly derived from the precursors of p-hydroxyphenyl lignin, through the action of a dual specificity phenylpropanoid meta-hydroxylase, Sm F5H. Here, we report the characterization of an O-methyltransferase from Selaginella moellendorffii, COMT, the coding sequence of which is clustered together with F5H at the adjacent genomic locus. COMT is a bifunctional phenylpropanoid O-methyltransferase that can methylate phenylpropanoid meta-hydroxyls at both the 3- and 5-position and function in concert with F5H in syringyl lignin biosynthesis in S. moellendorffii. Phylogenetic analysis reveals that Sm COMT, like F5H, evolved independently from its angiosperm counterparts.

Bone morphogenetic protein signaling: the pathway and its regulation.

In the mid-1960s, bone morphogenetic proteins (BMPs) were first identified in the extracts of bone to have the remarkable ability to induce heterotopic bone. When the Drosophila gene decapentaplegic (dpp) was first identified to share sequence similarity with mammalian BMP2/BMP4 in the late-1980s, it became clear that secreted BMP ligands can mediate processes other than bone formation. Following this discovery, collaborative efforts between Drosophila geneticists and mammalian biochemists made use of the strengths of their respective model systems to identify BMP signaling components and delineate the pathway. The ability to conduct genetic modifier screens in Drosophila with relative ease was critical in identifying the intracellular signal transducers for BMP signaling and the related transforming growth factor-beta/activin signaling pathway. Such screens also revealed a host of genes that encode other core signaling components and regulators of the pathway. In this review, we provide a historical account of this exciting time of gene discovery and discuss how the field has advanced over the past 30 years. We have learned that while the core BMP pathway is quite simple, composed of 3 components (ligand, receptor, and signal transducer), behind the versatility of this pathway lies multiple layers of regulation that ensures precise tissue-specific signaling output. We provide a sampling of these discoveries and highlight many questions that remain to be answered to fully understand the complexity of BMP signaling.

Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants.

Phenotypic convergence in unrelated lineages arises when different organisms adapt similarly under comparable selective pressures. In an apparent example of this process, syringyl lignin, a fundamental building block of plant cell walls, occurs in two major plant lineages, lycophytes and angiosperms, which diverged from one another more than 400 million years ago. Here, we show that this convergence resulted from independent recruitment of lignin biosynthetic cytochrome P450-dependent monooxygenases that route cell wall monomers through related but distinct pathways in the two lineages. In contrast with angiosperms, in which syringyl lignin biosynthesis requires two phenylpropanoid meta-hydroxylases C3'H and F5H, the lycophyte Selaginella employs one phenylpropanoid dual meta-hydroxylase to bypass several steps of the canonical lignin biosynthetic pathway. Transgenic expression of the Selaginella hydroxylase in Arabidopsis thaliana dramatically reroutes its endogenous lignin biosynthetic pathway, yielding a novel lignin composition not previously identified in nature. Our findings demonstrate a unique case of convergent evolution via distinct biochemical strategies and suggest a new way to genetically reconstruct lignin biosynthesis in higher plants.

Bis(neopentylglycolato)diboron (B2nep2) as a bidentate ligand and a reducing agent for early transition metal chlorides giving MCl4(B2nep2) complexes.

We found that bis(neopentylglycolato)diboron (B2nep2) served as a bidentate ligand and a one-electron reducing agent for early transition metal chlorides to afford MCl4(B2nep2). Treatment of B2nep2 with MCl5 (M = Nb and Mo) produced MCl4(B2nep2) via two successive reactions, coordination of B2nep2 to the metal center and one-electron reduction from M(V) to M(IV), while coordination of B2nep2 to MCl4 (M = Zr, Ti) was observed without reduction of the central metals. DFT studies for the reduction of NbCl5 by B2nep2 clarified the initial formation of seven-coordinated and B2nep2-ligated Nb(V) species, NbCl5(B2nep2), and one chloride on niobium(V) moves to the Lewis acidic boron center to generate NbCl4[(B2nep2)Cl]. The chloride on the boron atom of NbCl4[(B2nep2)Cl] is trapped by the second B2nep2 to give [NbCl4(B2nep2)][ClB2nep2]. After the formation of [ClB2nep2]- as an anionic sp2-sp3 diboron adduct, one-electron reduction of the niobium(V) center produces NbCl4(B2nep2) along with [ClB2nep2]˙ as a plausible diboron species, whose decomposition affords ClBnep and B2nep2. The reduction of metal halides in the presence of B2nep2 was exemplified by green LED irradiation of TiCl4(B2nep2), producing chloride-bridged titanium(III) species, (B2nep2)TiCl2(µ-Cl)2TiCl2(B2nep2).