The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish.

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Discovery of a Dual Function Cytochrome†P450 that Catalyzes Enyne Formation in Cyclohexanoid Terpenoid Biosynthesis.

The 1,3-enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn (1) is a 1,3-enyne-containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3-enyne biosynthesis in 1, and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1. A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis-diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3-enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five-carbon prenyl chain, rather than a polyprenyl chain, to a p-hydroxybenzoic acid acceptor.

Heterogeneous Nuclear Ribonucleoprotein H1 Coordinates with Phytochrome and the U1 snRNP Complex to Regulate Alternative Splicing in Physcomitrella patens.

Plant photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although gene expression is modulated by photoreceptors at various levels, the regulatory mechanism at the pre-mRNA splicing step remains unclear. Alternative splicing, a widespread mechanism in eukaryotes that generates two or more mRNAs from the same pre-mRNA, is largely controlled by splicing regulators, which recruit spliceosomal components to initiate pre-mRNA splicing. The red/far-red light photoreceptor phytochrome participates in light-mediated splicing regulation, but the detailed mechanism remains unclear. Here, using protein-protein interaction analysis, we demonstrate that in the moss Physcomitrella patens, phytochrome4 physically interacts with the splicing regulator heterogeneous nuclear ribonucleoprotein H1 (PphnRNP-H1) in the nucleus, a process dependent on red light. We show that PphnRNP-H1 is involved in red light-mediated phototropic responses in P. patens and that it binds with higher affinity to the splicing factor pre-mRNA-processing factor39-1 (PpPRP39-1) in the presence of red light-activated phytochromes. Furthermore, PpPRP39-1 associates with the core component of U1 small nuclear RNP in P. patens Genome-wide analyses demonstrated the involvement of both PphnRNP-H1 and PpPRP39-1 in light-mediated splicing regulation. Our results suggest that phytochromes target the early step of spliceosome assembly via a cascade of protein-protein interactions to control pre-mRNA splicing and photomorphogenic responses.

Bioremediation capability evaluation of benzene and sulfolane contaminated groundwater: Determination of bioremediation parameters.

Benzene and sulfolane are commonly used but hazardous chemicals in the petrochemical industry and their leakage and inappropriate disposal certainly causes serious soil and groundwater contamination. In this research, the bioremediation potential of groundwater contaminated with benzene and sulfolane was evaluated, and the operating parameters for bioremediation were established through laboratory batch experiments. Among the various bacterial consortia, the bacterial population of monitoring well c (MWc) contained the highest sulfolane and benzene removal efficiencies. When the dissolved oxygen (DO) level was >1 mg L-1, the bacterial population of MWc showed excellent removal efficiencies toward high and low concentrations of benzene and sulfolane. The C:N:P ratio of 100:10:1 in media facilitated sulfolane and benzene biodegradation, and the degradation time was greatly reduced. Adding additional phosphate into real groundwater could slightly increase benzene removal efficiency. Trace elements only slightly enhanced benzene degradation. On the contrary, additional phosphate and trace elements supplementary did not enhance sulfolane degradation. However, sulfolane removal efficiency could be significantly improved through bioaugmentation of specific sulfolane degrading bacterium and 100% sulfolane removal efficiency was achieved.

Interrupted Morita-Baylis-Hillman-Type Reaction of α-Substituted Activated Olefins.

It was demonstrated that 3-olefinic oxindoles could generate zwitterionic enolate species with tertiary phosphines and undergo C-C bond formation with various electrophiles in an interrupted Morita-Baylis-Hillman-type reaction manner, followed by a dephosphoration process. Although the in situ formation of phosphorus-ylide intermediates was observed, no Wittig reaction was detected, even in the presence of excess formaldehyde. Moreover, excellent enantioselectivity for the construction of quaternary stereogenic centers was induced with chiral phosphines. Deuterium experiments and density functional theory calculations were conducted to elucidate the reaction mechanism.

Design, synthesis and biological evaluation of novel 1,2,4-triazolo [3,4-b][1,3,4] thiadiazines bearing furan and thiophene nucleus.

Twenty-six novel 1,2,4-triazolo [3,4-b][1,3,4] thiadiazines containing furan and thiophene nucleus were designed, synthesized and evaluated for their antiproliferative activities. The results indicated that most of the compounds showed moderate to potent antiproliferative activities against four cancer cell lines, PC-3, HepG2, A549, and MCF-7. Particularly, compound 32 showed eleven-, three-, and two-fold improvement compared to positive control fluorouracil in inhibiting HepG2, PC-3, and A549 cell proliferation with IC50 values of 5.09, 3.70 and 12.74 µM, respectively. Further flow-activated cell sorting analysis revealed that the most promising compound 32 displayed a significant effect on G2/M cell-cycle arrest in a dose-dependent manner in PC-3 cells. These encouraging results should provide important information for the development of new anticancer agents.

Design, Synthesis and Molecular Docking Studies of Novel Indole-Pyrimidine Hybrids as Tubulin Polymerization Inhibitors.

A series of novel hybrids of indole-pyrimidine-containing piperazine moiety were designed, synthesized and evaluated for their antiproliferative and tubulin polymerization inhibitory activities. The results indicated that most of these compounds possessed significant cytotoxic potency against four cancer cell lines, HT-29, A549, MDA-MB-231 and MCF-7. Particularly, the most promising compound 34 showed more potent and broad-spectrum cytotoxic activities with the IC50 values ranged from 5.01 to 14.36 µm against A549, MDA-MB-231 and MCF-7 cell lines. Meanwhile, 34 also displayed the most potent tubulin polymerization inhibitory activity with IC50 value of 11.2 µm. Furthermore, molecular docking analysis demonstrated 34 interacts and binds efficiently with the tubulin protein at the colchicine-binding site. It was worth noting that the compound did not affect the normal human embryonic kidney cells, HEK-293. These results suggest that this novel class of indole-pyrimidine hybrids may have potential to be developed as new a class of tubulin polymerization inhibitors.

Genome-wide analysis of light-regulated alternative splicing mediated by photoreceptors in Physcomitrella patens.

BACKGROUND: Light is one of the most important factors regulating plant growth and development. Light-sensing photoreceptors tightly regulate gene expression to control photomorphogenic responses. Although many levels of gene expression are modulated by photoreceptors, regulation at the mRNA splicing step remains unclear. RESULTS: We performed high-throughput mRNA sequencing to analyze light-responsive changes in alternative splicing in the moss Physcomitrella patens, and found that a large number of alternative splicing events were induced by light in the moss protonema. Light-responsive intron retention preferentially occurred in transcripts involved in photosynthesis and translation. Many of the alternatively spliced transcripts were expressed from genes with a function relating to splicing or light signaling, suggesting a potential impact on pre-mRNA splicing and photomorphogenic gene regulation in response to light. Moreover, most light-regulated intron retention was induced immediately upon light exposure, while motif analysis identified a repetitive GAA motif that may function as an exonic regulatory cis element in light-mediated alternative splicing. Further analysis in gene-disrupted mutants was consistent with a function for multiple red-light photoreceptors in the upstream regulation of light-responsive alternative splicing. CONCLUSIONS: Our results indicate that intensive alternative splicing occurs in non-vascular plants and that, during photomorphogenesis, light regulates alternative splicing with transcript selectivity. We further suggest that alternative splicing is rapidly fine-tuned by light to modulate gene expression and reorganize metabolic processes, and that pre-mRNA cis elements are involved in photoreceptor-mediated splicing regulation.

Distinct phytochrome actions in nonvascular plants revealed by targeted inactivation of phytobilin biosynthesis.

The red/far-red light photoreceptor phytochrome mediates photomorphological responses in plants. For light sensing and signaling, phytochromes need to associate with open-chain tetrapyrrole molecules as the chromophore. Biosynthesis of tetrapyrrole chromophores requires members of ferredoxin-dependent bilin reductases (FDBRs). It was shown that LONG HYPOCOTYL 2 (HY2) is the only FDBR in flowering plants producing the phytochromobilin (PΦB) for phytochromes. However, in the moss Physcomitrella patens, we found a second FDBR that catalyzes the formation of phycourobilin (PUB), a tetrapyrrole pigment usually found as the protein-bound form in cyanobacteria and red algae. Thus, we named the enzyme PUB synthase (PUBS). Severe photomorphogenic phenotypes, including the defect of phytochrome-mediated phototropism, were observed in Physcomitrella patens when both HY2 and PUBS were disrupted by gene targeting. This indicates HY2 and PUBS function redundantly in phytochrome-mediated responses of nonvascular plants. Our studies also show that functional PUBS orthologs are found in selected lycopod and chlorophyte genomes. Using mRNA sequencing for transcriptome profiling, we demonstrate that expression of the majority of red-light-responsive genes are misregulated in the pubs hy2 double mutant. These studies showed that moss phytochromes rapidly repress expression of genes involved in cell wall organization, transcription, hormone responses, and protein phosphorylation but activate genes involved in photosynthesis and stress signaling during deetiolation. We propose that, in nonvascular plants, HY2 and PUBS produce structurally different but functionally similar chromophore precursors for phytochromes. Holophytochromes regulate biological processes through light signaling to efficiently reprogram gene expression for vegetative growth in the light.

[Monitoring of organic pollutants in river based on polarimetric SAR].

The rivers with the distinct gradient of water quality in the southern region of China were selected as a case study. The objective of this study was to develop the monitoring and evaluating technology of the water quality based on C-band polarimetric synthetic aperture radar (POLSAR). The random rough surface scattering model to describe the electromagnetic scattering characteristics of polluted water was briefly introduced. The potential effect of organic pollutants to the scattering model and backscattering coefficient were explored. The simultaneously obtained POLSAR data and the measured water quality indexes were analyzed. By comparing the POLSAR data and the water quality indexes, it could be observed that the chemical oxygen demand (COD) was in proportional to the ratio between HH and VV backscattering coefficients, which matched the analysis based on electromagnetic scattering theory. A fitting model was proposed to retrieve the chemical oxygen demand by ratio between HH and VV channel backscattering coefficients using least square method, with the fit coefficient of 0.90. In this study, the model using the ratio between HH and VV backscattering coefficients was established, which was mainly based on the analysis of experimental results, and was also supported by theoretical interpretation.

Electrostatic interaction of phytochromobilin synthase and ferredoxin for biosynthesis of phytochrome chromophore.

In plants, phytochromobilin synthase (HY2) synthesize the open chain tetrapyrrole chromophore for light-sensing phytochromes. It catalyzes the double bond reduction of a heme-derived tetrapyrrole intermediate biliverdin IXalpha (BV) at the A-ring diene system. HY2 is a member of ferredoxin-dependent bilin reductases (FDBRs), which require ferredoxins (Fds) as the electron donors for double bond reductions. In this study, we investigated the interaction mechanism of FDBRs and Fds by using HY2 and Fd from Arabidopsis thaliana as model proteins. We found that one of the six Arabidopsis Fds, AtFd2, was the preferred electron donor for HY2. HY2 and AtFd2 formed a heterodimeric complex that was stabilized by chemical cross-linking. Surface-charged residues on HY2 and AtFd2 were important in the protein-protein interaction as well as BV reduction activity of HY2. These surface residues are close to the iron-sulfur center of Fd and the HY2 active site, implying that the interaction promotes direct electron transfer from the Fd to HY2-bound BV. In addition, the C12 propionate group of BV is important for HY2-catalyzed BV reduction. A possible role for this functional group is to mediate the electron transfer by interacting directly with AtFd2. Together, our biochemical data suggest a docking mechanism for HY2:BV and AtFd2.

Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea.

Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi.

Identification of proteins that interact with a TcMago-TcY14 heterodimer complex in Taiwania crytomerioides.

The Mago-Y14 heterodimer, which is highly conserved among eukaryotes, is the core component of the exon-exon junction complex (EJC) and regulates oogenesis, embryogenesis and germ-line sex determination in Drosophila and the nematode Caenorhabditis elegans. To further survey these proteins in plants and to identify plant developmental processes with which the Mago-Y14 heterodimer is associated, we used the TcMago-TcY14 heterodimer as the bait protein in a yeast two-hybrid screen and searched for its binding partners in a cDNA library from a 3-year-old Taiwania (Taiwania cryptomerioides Hayata) seedling. We found three clones, an unknown protein (Tc61), a pectin methylesterase-like protein (Tc62) and a TPR (tetratricopeptide repeat)-like protein (Tc72). The Tc61 protein interacted strongly with the TcMago-TcY14 heterodimer, and its transcript was expressed in stems and leaves of 3-year-old Taiwania seedlings and in microsporangiate and ovulate cones. The pectin methylesterase-like protein was expressed abundantly in the roots of 3-year-old Taiwania seedlings and in pollinated ovulate cones, indicating that binding with the TcMago-TcY14 heterodimer may be involved in root development and growth of pollen tubes during pollination. The Tc72 protein encoded a complete TPR-like protein that is highly conserved among plants, with 10 alpha-helices and three conserved TPR motifs containing five consensus residues necessary for stable inter-helix packing. A tissue expression assay and light/dark treatments showed that the Tc72 gene was expressed abundantly in the leaves of seedlings and was sensitive to light and dark, as are the Tcmago and TcY14 genes, implying that the TcMago-TcY14 heterodimer interaction with Tc72 may be related to chloroplast protein transport.

Molecular identification and characterization of Tcmago and TcY14 in Taiwania (Taiwania cryptomerioides).

Mago nashi (Mago) and Y14 proteins are highly conserved among eukaryotes, and not only play important roles in oogenesis, embryogenesis and germ-line determination during animal development, but also participate in mRNA localization and splicing in cell growth. We identified mago (Tcmago) and Y14 (TcY14) homologues derived from expressed sequence tags of Taiwania cryptomerioides Hayata seedlings. Tcmago and TcY14 contain coding regions encoding 149 and 216 amino acids, respectively. Multiple amino acid sequence alignments as well as secondary and tertiary structure all predicted that TcMago and TcY14 possessed similar protein structures to the crystal structures of Drosophila melanogaster and human Mago and Y14 proteins. We demonstrated by yeast two-hybrid analysis and a GST pull-down assay that TcMago and TcY14 interacted in vivo and in vitro, confirming structural predictions of their interaction. TcMago and TcY14 were predominately localized in the nucleus. Whole mount in situ hybridization and immunolocalization showed that Tcmago and TcY14 were both detected in root hairs at the levels of transcription and protein expression. Overexpression of Tcmago in transgenic tobacco plants resulted in longer roots and a more complex root system. TcMago and TcY14 may have cellular functions similar to the Mago and Y14 proteins in animals and may be involved in root development in plants.