Quantifying snowfall from orographic cloud seeding.

Climate change and population growth have increased demand for water in arid regions. For over half a century, cloud seeding has been evaluated as a technology to increase water supply; statistical approaches have compared seeded to nonseeded events through precipitation gauge analyses. Here, a physically based approach to quantify snowfall from cloud seeding in mountain cloud systems is presented. Areas of precipitation unambiguously attributed to cloud seeding are isolated from natural precipitation (<1 mm h-1). Spatial and temporal evolution of precipitation generated by cloud seeding is then quantified using radar observations and snow gauge measurements. This study uses the approach of combining radar technology and precipitation gauge measurements to quantify the spatial and temporal evolution of snowfall generated from glaciogenic cloud seeding of winter mountain cloud systems and its spatial and temporal evolution. The results represent a critical step toward quantifying cloud seeding impact. For the cases presented, precipitation gauges measured increases between 0.05 and 0.3 mm as precipitation generated by cloud seeding passed over the instruments. The total amount of water generated by cloud seeding ranged from 1.2 × 105 m3 (100 ac ft) for 20 min of cloud seeding, 2.4 × 105 m3 (196 ac ft) for 86 min of seeding to 3.4 x 105 m3 (275 ac ft) for 24 min of cloud seeding.

Inappropriate Expression of an NLP Effector in Colletotrichum orbiculare Impairs Infection on Cucurbitaceae Cultivars via Plant Recognition of the C-Terminal Region.

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

Simple method to detect year-to-year variability of blooming phenology of Cerasus × yedoensis by digital camera.

The year-to-year variability of the blooming phenology of cherry trees is important as a proxy climate indicator and strongly affects cultural ecosystem services. Observation of blooming phenology at multiple points requires a simple and flexible approach. We examined changes in the canopy gap fraction extracted from binarized upward images taken periodically beneath a Cerasus × yedoensis 'Somei-yoshino' tree. The gap fraction decreased rapidly after the start of bloom, reached a minimum value at full bloom, and began to increase again, but then decreased rapidly during leaf flush. These changes reflect the phenology of blooming and leaf flush after flower drop of 'Somei-yoshino'. These characteristics allow detection of the year-to-year variability of the bloom and leaf-flush phenology of cherry and other deciduous tree species that show the same patterns.

Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens.

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi.

Hemibiotrophic fungal plant pathogens represent a group of agronomically significant disease-causing agents that grow first on living tissue and then cause host death in later, necrotrophic growth. Among these, Colletotrichum spp. are devastating pathogens of many crops. Identifying expanded classes of genes in the genomes of phytopathogenic Colletotrichum, especially those associated with specific stages of hemibiotrophy, can provide insights on how these pathogens infect a large number of hosts. The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, and C. gloeosporioides, which infects a wide range of crops, were sequenced and analyzed, focusing on features with potential roles in pathogenicity. Regulation of C. orbiculare gene expression was investigated during infection of N. benthamiana using a custom microarray. Genes expanded in both genomes compared to other fungi included sequences encoding small, secreted proteins (SSPs), secondary metabolite synthesis genes, proteases and carbohydrate-degrading enzymes. Many SSP and secondary metabolite synthesis genes were upregulated during initial stages of host colonization, whereas the necrotrophic stage of growth is characterized by upregulation of sequences encoding degradative enzymes. Hemibiotrophy in C. orbiculare is characterized by distinct stage-specific gene expression profiles of expanded classes of potential pathogenicity genes.

Changes in freezing rain occurrence over eastern Canada using convection-permitting climate simulations.

Freezing precipitation has major consequences for ground and air transportation, the health of citizens, and power networks. Previous studies using coarse resolution climate models have shown a northward migration of freezing rain in the future. Increased model resolution can better define local topography leading to improved representation of conditions that are favorable for freezing rain. The goal of this study is to examine the climatology and characteristics of future freezing rain events using very-high resolution climate simulations. Historical and pseudo-global warming simulations with a 4-km horizontal grid length were used and compared with available observations. Simulations revealed a northerly shift of freezing rain occurrence, and an increase in the winter. Freezing rain was still shown to occur in the Saint-Lawrence River Valley in a warmer climate, primarily due to stronger wind channeling. Up to 50% of the future freezing rain events also occurred in present day climate within 12 h of each other. In northern Maine, they are typically shorter than 6 h in current climate and longer than 6 h in warmer conditions due to the onset of precipitation during low-pressure systems occurrences. The occurrence of freezing rain also locally increases slightly north of Québec City in a warmer climate because of freezing rain that is produced by warm rain processes. Overall, the study shows that high-resolution regional climate simulations are needed to study freezing rain events in warmer climate conditions, because high horizontal resolutions better define small-scale topographic features and local physical mechanisms that have an influence on these events.

LAC2 encoding a secreted laccase is involved in appressorial melanization and conidial pigmentation in Colletotrichum orbiculare.

Both Colletotrichum and Magnaporthe spp. develop appressoria pigmented with melanin, which is essential for fungal pathogenicity. 1,8-Dihydroxynaphthalene (1,8-DHN) is believed to be polymerized to yield melanin around the appresorial cell wall through the oxidative activity of laccases. However, no 1,8-DHN laccase has yet been identified in either Colletotrichum or Magnaporthe spp. Here, we report a laccase gene, LAC2, that is involved in the appressorial melanization of Colletotrichum orbiculare, which causes cucumber anthracnose. LAC2 encodes a protein with a signal peptide and has high homology to fungal laccases. The conidial color of lac2 mutants is distinct from that of the C. orbiculare wild type, and the mutants are nonpathogenic. Notably, the mutant appressoria are defective in melanization, and a host invasion assay showed that the appressoria are nonfunctional. LAC2 was induced during appressorial melanization. These results suggest that LAC2 oxidizes 1,8-DHN in the appressoria. The LAC2 homologues of other fungi located in the same phylogenetic clade as LAC2 fully complemented the lac2 mutants. Interestingly, a LAC2 homologue, located in a different clade, complemented the conidial pigmentation but not appressorial melanization of the mutants, suggesting that the LAC2 function in appressorial melanization might only be conserved in laccases of the LAC2 clade.

Rice open beak is a negative regulator of class 1 knox genes and a positive regulator of class B floral homeotic gene.

Numerous genes are involved in the regulation of plant development, including those that regulate floral homeotic genes, We identified two recessive allelic rice mutants, open beak-1 (opb-1) and opb-2, which exhibited pleiotropic defects in leaf morphogenesis, inflorescence architecture, and floral organ identity. Abnormal cell proliferation was observed in the leaves and spikelets, and ectopic or overexpression of several class 1 knox genes was detected; thus, the abnormal cell proliferation in opb mutants is probably caused by ectopic class 1 knox gene expression. The opb mutants also had defects in floral organ identity, resulting in the development of mosaic organs, including gluminous lodicules, staminoid lodicules, and pistiloid stamens. These results, together with the reduced expression of a class B gene, indicate that OPB positively regulates the expression of class B genes. Map-based cloning revealed that OPB encodes a transcription factor that is orthologous to the Arabidopsis JAGGED gene and is expressed in leaf primordia, inflorescence meristem, rachis branch meristems, floral meristem, and floral organ primordia. Taken together, our data suggest that the OPB gene affects cellular proliferation and floral organ identity through the regulation of class 1 knox genes and floral homeotic genes.

A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice.

PURPOSE: To characterize an N-ethyl-N-nitrosourea-induced dominant mouse mutant, M-1156, that exhibits progressive retinal degeneration and to investigate the pathogenesis of the retinal phenotype in the mutant. METHODS: A positional candidate gene approach was used to identify the causative gene in the M-1156 mutant. Funduscopic examination, light microscopy, transmission electron microscopy, and electroretinography were performed to analyze the M-1156 phenotype. Real-time quantitative PCR, immunohistochemistry, and western blotting were also performed. RESULTS: Linkage analysis enabled the mutant gene to be mapped to a region of chromosome 19 containing Rom1, which encodes rod outer segment membrane protein 1. Sequence analysis demonstrated that the mutation consisted of a single base T-->C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed. A putative missense mutation designated Rom1(Rgsc1156) that was identified in the M-1156 mutant mouse causes a Trp to Arg substitution at position 182 in the translated protein. Rom1(Rgsc1156) heterozygotes were found to have a mottled retina and narrowed arteries in the fundus oculi. Photomicrographs of the retina revealed significant differences among the genotypes in the thickness of the outer nuclear layer and in the length of the outer segments of the photoreceptors. The alterations were more marked in the homozygotes than in the heterozygotes. Electron micrographs showed that the diameters of the discs varied in the heterozygotes and that the discs were more compactly stacked than in the wild type. There were significant differences among the genotypes in the amplitude of the a-wave in single-flash electroretinograms, but there were no significant differences among the photopic electroretinograms. Real-time quantitative PCR showed that there were no significant differences among the genotypes in Rom1 or peripherin/rds (Prph2) mRNA levels relative to the rhodopsin (Rho) mRNA level. Rom1 and Prph2 immunoreactivity were decreased in the retinas of the Rom1(Rgsc1156) mutants. Semiquantitative western blot analysis of retinas from 3-week-old Rom1(Rgsc1156) mutants demonstrated significant decreases in Rom1, Prph2, and Rho protein levels in all of the genotypes. CONCLUSIONS: The Trp182Arg substitution in Rom1(Rgsc1156) mutants causes retinal degeneration. The results suggested that Trp182Arg mutant Rom1 causes a decrease in the levels of wild-type Prph2 and Rom1, which in turn cause a reduction in the level of Prph2 containing tetramers in the disc rim region and ultimately result in unstable, disorganized outer segments and photoreceptor degeneration.

Synthesis of new beta-1-C-alkylated imino-L-iditols: A comparative study of their activity as beta-glucocerebrosidase inhibitors.

A short synthesis of new beta-1-C-alkyl-1,5-dideoxy-1,5-imino-l-iditols by means of the diastereoselective addition of Grignard reagents onto a glucopyranosylamine is described. These compounds were evaluated as beta-glucocerebrosidase inhibitors and their activity was compared with that of related iminosugar derivatives in the d-gluco and d-xylo series. The results allowed us to conclude on the influence of the hydroxymethyl moiety and of the piperidine-ring conformation on the inhibitory activity.

2,5-Dideoxy-2,5-imino-d-altritol as a new class of pharmacological chaperone for Fabry disease.

Chromatographic separation of the extract from roots of Adenophora triphylla resulted in the isolation of two pyrrolidines, six piperidines, and two piperidine glycosides. The structures of new iminosugars were elucidated by spectroscopic methods as 2,5-dideoxy-2,5-imino-d-altritol (DIA) (2), beta-1-C-butenyl-1-deoxygalactonojirimycin (8), 2,3-dideoxy-beta-1-C-ethyl-1-deoxygalactonojirimycin (9), and 6-O-beta-d-glucopyranosyl-2,3-dideoxy-beta-1-C-ethyl-1-deoxygalactonojirimycin (10). beta-1-C-Butyl-1-deoxygalactonojirimycin (7) and compound 8 were found to be better inhibitors of alpha-galactosidase than N-butyl-1-deoxygalactonojirimycin. The present work elucidated that DIA was a powerful competitive inhibitor of human lysosome alpha-galactosidase A (alpha-Gal A) with a K(i) value of 0.5muM. Furthermore, DIA improved the thermostability of alpha-Gal A in vitro and increased intracellular alpha-Gal A activity by 9.6-fold in Fabry R301Q lymphoblasts after incubation for 3days. These experimental results suggested that DIA would act as a specific pharmacological chaperone to promote the smooth escape from the endoplasmic reticulum (ER) quality control system and to accelerate transport and maturation of the mutant enzyme.

Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts.

Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-alpha stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-alpha-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-alpha and BMP signaling pathways.

Stereodivergent access to polyhydroxylated 10-azabicyclo[4.3.1]decanes as new calystegine analogues.

A rapid and stereodivergent access to polyhydroxylated 10-azabicyclo[4.3.1]decanes as new calystegine analogues by way of a double benzotriazolyl/carbon nucleophile exchange followed by a ring-closing metathesis was achieved. Preliminary evaluation of the new compounds as glucocerebrosidase inhibitors was also performed.

Synchronicity of genetic variants between primary sites and metastatic lymph nodes, and prognostic impact in nodal metastatic lung adenocarcinoma.

PURPOSE: Nodal positive lung adenocarcinoma includes wide range of survival. Several methods for the classification of nodal-positive lung cancer have been proposed. However, classification considering the impact of targetable genetic variants are lacking. The possibility of genetic variants for the better stratification of nodal positive lung adenocarcinoma was estimated. METHODS: Mutations of 36 genes between primary sites and metastatic lymph nodes (LNs) were compared using next-generation sequencing. Subsequently, mutations in EGFR and BRAF, rearrangements in ALK and ROS1 were evaluated in 69 resected pN1-2M0 adenocarcinoma cases. Recurrence-free survival (RFS), post-recurrence survival (PRS), and overall survival (OS) were evaluated with respect to targetable variants and tyrosine kinase inhibitor (TKI) therapy after recurrence. RESULTS: About 90% of variants were shared and allele frequencies were similar between primary and metastatic sites. In 69 pN1-2M0 cases, EGFR/ALK were positive in primary sites of 39 cases and same EGFR/ALK variants were confirmed in metastatic LNs of 96.7% tissue-available cases. Multivariate analyses indicated positive EGFR/ALK status was associated with worse RFS (HR 2.366; 95% CI 1.244-4.500; P = 0.009), and PRS was prolonged in cases receiving TKI therapy (no post-recurrence TKI therapies, HR 3.740; 95% CI 1.449-9.650; P = 0.006). OS did not differ with respect to targetable variants or TKI therapy. CONCLUSIONS: Cases harbouring targetable genetic variants had a higher risk of recurrence, but PRS was prolonged by TKI therapy. Classification according to the targetable genetic status provides a basis for predicting recurrence and determining treatment strategies after recurrence.

Iminosugars from Baphia nitida Lodd.

Chromatographic separation of the 50% aqueous EtOH extract of the leaves of the African medicinal tree Baphia nitida resulted in isolation of 10 iminosugars. The plant contained 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) as a major alkaloid. The structure of a new alkaloid was also elucidated by spectroscopic methods as the 1-O-beta-D-fructofuranoside of DMDP, and this plant produced 3-O-beta-D-glucopyranosyl-DMDP as well. DMDP is a potent inhibitor of beta-glucosidase and beta-galactosidase, whereas the other two derivatives lowered inhibition toward both of these enzymes and improved inhibitory activities toward rice alpha-glucosidase and rat intestinal maltase.

Effect of five-membered sugar mimics on mammalian glycogen-degrading enzymes and various glucosidases.

We investigated inhibitory activities of five-membered sugar mimics toward glycogen-degrading enzymes and a variety of glucosidases. 1,4-Dideoxy-1,4-imino-D-arabinitol (D-AB1) is known to be a potent inhibitor of glycogen phosphorylase. However, the structural modification of D-AB1, such as its enantiomerization, epimerization at C-2 and/or C-3, introduction of a substituent to C-1, and replacement of the ring nitrogen by sulfur, markedly lowered or abolished its inhibition toward the enzyme. The present work elucidated that d-AB1 was also a good inhibitor of the de-branching enzyme of glycogen, amylo-1,6-glucosidase, with a IC(50) value of 8.4 microM. In the present work, the de-sulfonated derivative of salacinol was isolated from the roots of Salacia oblonga and found to be a potent inhibitor of rat intestinal isomaltase with an IC(50) value of 0.64 microM. On the other hand, salacinol showed a much more potent inhibitory activity toward maltase in Caco-2 cell model system than its de-sulfonated derivative, with an IC(50) value of 0.5 microM, and was further a stronger inhibitor of human lysosomal alpha-glucosidase than the derivative (IC(50)=0.34 microM). This indicates that the sulfate in the side chain plays an important role in the specificity of enzyme inhibition.

In vitro inhibition of glycogen-degrading enzymes and glycosidases by six-membered sugar mimics and their evaluation in cell cultures.

We investigated in vitro inhibition of mammalian carbohydrate-degrading enzymes by six-membered sugar mimics and their evaluation in cell cultures. 1-Deoxynojirimycin (DNJ) showed no significant inhibition toward glycogen phosphorylase (GP) but was a potent inhibitor of another glycogen-degrading enzyme, amylo-1,6-glucosidase (1,6-GL), with an IC(50) value of 0.16 microM. In primary rat hepatocytes, the inhibition of glycogen breakdown by DNJ reached plateau at 100 microM with 25% inhibition and then remained unchanged. The potent GP inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) inhibited hepatic glucose production with an IC(50) value of about 9 microM and the inhibition by D-AB1 was further enhanced in the presence of DNJ. DNJ and alpha-homonojirimycin (HNJ) are very potent inhibitors of rat intestinal maltase, with IC(50) values of 0.13 and 0.08 microM, respectively, and also showed a similar strong inhibition toward maltase in Caco-2 cell model system, with IC(50) value of 0.05 and 0.10 microM, respectively. D-Isofagomine (D-IFG) and L-IFG are competitive and noncompetitive inhibitors of human lysosomal beta-glucosidase (beta-GL), respectively, with K(i) values of 8.4 nM and 6.9 microM. D-IFG increased intracellular beta-GL activity by twofold at 10 microM in Gaucher N370S cell line as an 'active-site-specific' chaperone, and surprisingly a noncompetitive inhibitor L-IFG also increased intracellular beta-GL activity by 1.6-fold at 500 microM.

Low-intensity pulsed ultrasound stimulates osteogenic differentiation in ROS 17/2.8 cells.

There have been no studies investigating the effects of the mechanical stimulation provided by Low-intensity pulsed ultrasound (LIPUS) treatment on periodontal disease accompanying bone loss. LIPUS is known to accelerate mineralization and bone regeneration, but the precise cellular mechanism is unclear. Here, we investigated the effect of LIPUS on osteogenesis by examining the effect of LIPUS stimulation on cell proliferation, alkaline phosphatase (ALPase) activity, osteogenesis-related gene expression, and mineralized nodule formation in a rat osteosarcoma cell line. The cells were cultured in medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 30 mW/cm(2) for 20 min for all cultures. LIPUS stimulation did not affect the rate of cell proliferation. ALPase activity was increased at day 7 of culture after LIPUS stimulation. Real-time PCR analysis indicated that LIPUS significantly increased the expression of mRNA for the transcription factors Runx2, Msx2, Dlx5, and Osterix and for bone sialoprotein, whereas the mRNA expression of AJ18 was significantly reduced. The mineralized nodule formation and the calcium content in mineralized nodules were markedly increased on day 14 of culture after LIPUS stimulation. Our study demonstrates that LIPUS stimulation directly affects osteogenic cells, leading to mineralized nodule formation. In view of the widespread use of LIPUS for the clinical therapy of periodontal disease, it is likely that LIPUS has an important influence on key functional activities of osteoblasts in alveolar bone.

Synthesis of 4-O-glycosylated 1-deoxynojirimycin derivatives as disaccharide mimics-based inhibitors of human beta-glucocerebrosidase.

Examples of a new type of inhibitor of human beta-glucocerebrosidase based on imino-disaccharides as glycosylceramide mimetics have been synthesized by way of the glycosylation of 1-deoxynojirimycin derivatives with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide.